E Book - Journal of Experimental Biology and Agricultural Sciences

ISSN: 2320-8694

Journal of Experimental Biology and Agricultural Sciences Volume 4 | Issue (Spl.4.EHIDZ)| Dec, 2016

Special Issue on: EQUINE HEALTH, INFECTIOUS DISEASES AND ZOONOSIS (EHIDZ)

Lead Guest Editor Sandip Kumar Khurana

Principal Scientist, National Research Centre on Equines, India – 125001

Guest Editor

Amarpal

Principal Scientist, Indian Veterinary Research Institute, India - 243122

Yashpal S. Malik


Kuldeep Dhama


K. Karthik

Assistant Professor, Tamil Nadu Veterinary & Animal Sciences University, India

Minakshi Prasad

Professor, College of Veterinary Sciences, India - 125001

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JEBAS ISSN No. 2320 – 8694 Peer Reviewed - open access journal Common Creative Licence - NC 4.0 Volume No – 4 Issue No – Spl-4-EHIDZ December, 2016 Journal of Experimental Biology and Agricultural Sciences Journal of Experimental Biology and Agricultural Sciences (JEBAS) is an online platform for the advancement and rapid dissemination of scientific knowledge generated by the highly motivated researchers in the field of biological sciences. JEBAS publishes high-quality original research and critical up-to-date review articles covering all the aspects of biological sciences. Every year, it publishes six issues. The JEBAS is an open access journal. Anyone interested can download full text PDF without any registration. JEBAS has been accepted by EMERGING SOURCES CITATION INDEX (Thomson Reuters – Web of Science database), DOAJ, CABI, INDEX COPERNICUS INTERNATIONAL (Poland), AGRICOLA (USA), CAS (ACS, USA), CABI – Full Text (UK), AGORA (FAO-UN), OARE (UNEP), HINARI (WHO), J gate, EIJASR, DRIJ and Indian Science Abstracts (ISA, NISCAIR) like well reputed indexing database.

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JEBAS From the Desk of Guest Editors _____________________________________________________________________________ Dear Authors, Equines include horses, donkeys, mules, zebra, and their hybrids. The utility of equines exists for transportation, agricultural, riding, sports, racing, equestrian events, army, police services, brick kilns, companions and ceremonial purposes. Equines are very sensitive and vulnerable to infectious diseases and require very good managemental practices in comparison to other animal species. There are several existing and emerging equine pathogens having zoonotic potential, which are threat to public health. Latest information relating to equine health management and infectious diseases with emphasis on diseases prevalence, surveillance, monitoring and strategic approaches in diagnosis, prevention, control and treatment of equine infectious diseases and their zoonotic aspects, is essentially required. Novel molecular diagnostics techniques, development of improved therapeutics and immunomodulatory strategies, vaccines and package of practices for prevention, control and eradication of infectious diseases are relevant today for effective prevention and control of equine infectious diseases. This issue is aimed for the benefit of equine veterinarians, equine researchers, other equine health care specialists and all other professionals and persons related with animal health in general. This special issue is published with nine articles including eight review articles and one research article. The review on ‚Leptospirosis in horses: special reference to equine recurrent uveitis‛ provides an insight in to Leptospirosis, a bacterial zoonotic disease affecting several domestic and wild animal species. Special emphasis is on ocular and systemic manifestations in horses. The ocular manifestation of leptospirosis in equines, the equine recurrent uveitis (ERU) has been dealt in detail. Another review on ‚Emergence of equine herpes virus 1 myeloencephalopathy: A brief review‛ elaborates the nervous form of EHV1, equine herpes myeloencephalopathy (EHM). The review describes the genetics, epidemiology, pathogenesis, treatment, prevention and control of EHM along with host, agent and environmental factors which play a role in the development of EHM. The review on ‚Equine ocular setariasis and its management‛ enriches the knowledge regarding etiology, diagnosis and management of ocular setariasis in equine species. The common post operative complications along with their management are also discussed. The research article ‚Parasitological, biochemical and clinical observations in ponies experimentally infected with Trypanosoma evansi‛ describes the observations on experimentally infected ponies with Trypanosoma evansi. Serum urea, uric acid, triglyceride, cholesterol, bilirubin indirect (BID) and total bilirubin (BIT) contents increased, while albumin contents significantly decreased indicating impairment of liver and kidney functions.

JEBAS The review on ‚Equine brucellosis: Review on epidemiology, pathogenesis, clinical signs, prevention and control‛ describes equine brucellosis, its epidemiology, pathogenesis, clinical signs along with appropriate prevention and control strategies with special mention of two important conditions in equines namely poll evil and fistulous withers. The review on ‚Biotechnological tools for diagnosis of equine infectious diseases‛ focuses on biotechnological tools available for equine diseases diagnosis and its applications which hold great promise for improving the speed and accuracy of diagnostics for equine infectious diseases needed to promote optimal clinical outcomes and general public health. The review ‚Evolving views on enteric viral infections of equines: an appraisal of key pathogens‛ discusses the current status of key enteric viruses that cause diarrheic disorders in foals and horses and their public health aspects. The review ‚Lyme borreliosis in the horse: A mini-review‛ discusses beautifully the Lyme borreliosis which is a multisystemic tick borne disease and converses with the disease etiology, pathobiology, disease management and public health aspects. The review ‚An overview of ozone therapy in equine- an emerging healthcare solution‛ unfolds the significance of ozone therapy in equine medicine providing an insight into the mechanism of action of ozone therapy and various conditions where ozone therapy could be used in equines. The information compiled in this special issue will be useful for equine researchers, equine veterinary professionals, equine industry persons, students/scholars, public health experts to equip them with latest armour to develop disease diagnostics and therapeutics for prevention, control of equine diseases including zoonotic aspect. This special issue will help in formulating various strategies related to equine health and also provide a solution on zoonotic aspects. The gain in knowledge on various aspects through this special issue will help in dealing equine health issues in a better way.

Thank you

Sandip Kumar Khurana Amarpal Yashpal S. Malik Kuldeep Dhama K. Karthik Minakshi Prasad

JEBAS Guest Editors Lead Guest Editor Sandip Kumar Khurana (MVSc, Ph.D) Principal Scientist National Research Centre on Equines (NRCE) Sirsa Road, Hisar – 125 001, Haryana, India Email: [email protected]

Guest Editors Amarpal (MVSc, PhD, FISACP, FNAVS, FISVS) Principal Scientist and Head, Division of Surgery Indian Veterinary Research Institute Izatnagar-243122 (UP), India Email: [email protected] Yashpal S. Malik (MVSc., Ph D, Post-Doc USA) National Fellow, Principal Scientist Division of Biological Standardization Indian Veterinary Research Institute (IVRI), Izatnagar 243122, India E-mail: [email protected] Kuldeep Dhama (MVSc, Ph.D) Principal Scientist & NAAS Associate Division of Pathology, ICAR-Indian Veterinary Research Institute (IVRI) Izatnagar-243 122, Bareilly, Uttar Pradesh, India Email: [email protected] K. Karthik (MVSc) Assistant Professor, Central University Laboratory, Tamil Nadu Veterinary & Animal Sciences University, Madhavaram Milk Colony Chennai-51, Tamil Nadu, India E mail: [email protected] Minakshi Prasad (PhD), Fellow NAAS, Fellow NAVS Professor and Head, Department of Animal Biotechnology College of Veterinary Sciences LALRUVAS, Hisar 125001, Haryana Email: [email protected]

JEBAS Welcome Message - Managing Editor (Dr Kamal Kishore Chaudhary, M.Sc, Ph.D)

_____________________________________________________________________________ Dear Authors, It is with much joy and anticipation that we celebrate the launch of special issue - Spl-4-EHIDZ (Volume 4) of Journal of Experimental Biology and Agricultural Sciences (JEBAS). On behalf of the JEBAS Editorial Team, I would like to extend a very warm welcome to the readership of JEBAS. I take this opportunity to thank our authors, editors and anonymous reviewers, all of whom have volunteered to contribute to the success of the journal. I am also grateful to the staff at Horizon Publisher India [HPI] for making JEBAS a reality. JEBAS is dedicated to the rapid dissemination of high quality research papers on how advances in Biotechnology, Agricultural sciences along with computational algorithm can help us meet the challenges of the 21st century, and to capitalize on the promises ahead. We welcome contributions that can demonstrate near-term practical usefulness, particularly contributions that take a multidisciplinary / convergent approach because many real world problems are complex in nature. JEBAS provides an ideal forum for exchange of information on all of the above topics and more, in various formats: full length and letter length research papers, survey papers, work-in-progress reports on promising developments, case studies and best practice articles written by industry experts. Finally, we wish to encourage more contributions from the scientific community and industry practitioners to ensure a continued success of the journal. Authors, reviewers and guest editors are always welcome. We also welcome comments and suggestions that could improve the quality of the journal.

Thank you. We hope you will find JEBAS informative.

Dr. Kamal K Chaudhary Managing Editor - JEBAS December 2016

JEBAS INDEX _____________________________________________________________________________

Leptospirosis in horses: special reference to equine recurrent uveitis doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S123.S131 Emergence of equine herpes virus 1 myeloencephalopathy: A brief review doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S132.S138 Equine ocular setariasis and its management doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S139.S143 Parasitological, biochemical and clinical observations in ponies experimentally infected with Trypanosoma evansi doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S144.S150 Equine brucellosis: Review on epidemiology, pathogenesis, clinical signs, prevention and control doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S151.S160 Biotechnological tools for diagnosis of equine infectious doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S161.S181

diseases

Evolving views on enteric viral infections of equines: an appraisal of key pathogens doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S182.S195 Lyme borreliosis in the horse: A mini-review doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S196.S202 An overview of ozone therapy in equine- an emerging healthcare solution doi: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S203.S210

Journal of Experimental Biology and Agricultural Sciences, December - 2016; Volume – 4(Spl-4-EHIDZ)

Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

ISSN No. 2320 – 8694

LEPTOSPIROSIS IN HORSES: SPECIAL REFERENCE TO EQUINE RECURRENT UVEITIS Sandip Kumar Khurana1,*, Kuldeep Dhama2, Minakshi P3, Baldev Gulati1, Yashpal Singh Malik2 and Kumaragurubaran Karthik4 1

NRCE, Hisar, Haryana, India Indian Veterinary Research Institute, Izatnagar, Barrielly, U.P., India 3 Department of Animal Biotechnology, LUVAS, Hisar, Haryana, India 4 Tamil Nadu University of Veterinary and Animal Sciences, Chennai, Tamil Nadu, India 2

Received – August 27, 2016; Revision – September 04, 2016; Accepted – October 03, 2016 Available Online – December 04, 2016 DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S123.S131

KEYWORDS Equine Leptospirosis ERU

ABSTRACT Leptospirosis is a bacterial zoonotic disease with worldwide distribution. The disease affects several domestic and wild animals. Leptospirosis has seasonal nature with high incidence in hot rainy season especially in tropical regions. The disease in horses has ocular and systemic manifestations. However, stillbirths and neonatal mortality due to this disease is also common. The ocular manifestation is equine recurrent uveitis (ERU), also known as periodic ophthalmia or moon blindness, where autoimmune mechanisms also play an important role. Several diagnostic assays are employed and microscopic agglutination test has been commonly employed in several parts of the world though isolation is the gold standard test. Recent diagnostic advances like PCR, real time PCR, LAMP also aid in early diagnosis of the disease so that the spread of disease to other animals and human can be prevented.

* Corresponding author E-mail: [email protected] (Sandip Kumar Khurana) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.

Production and Hosting by Horizon Publisher India [HPI] (http://www.horizonpublisherindia.in/). All_________________________________________________________ rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org

All the article published by Journal of Experimental Biology and Agricultural Sciences is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License Based on a work at www.jebas.org.

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1 Introduction

2 Equine Leptospirosis

Leptospirosis is a major animal and human health problem worldwide. Leptospires belong to family Leptospiraceae, order spirochaetales. Leptospires are about 0.1 µm in diameter and 6-20 µm in length. Their major antigenic component is lipopolysaccharide (LPS). The disease is prevalent in many parts of the world both in urban and rural settings (Vinetz, 1997; Bharti et al., 2003; Verma et al., 2013; Khurana et al., 2015). The leptospirosis primarily causes chronic kidney infection in several domestic and wild animals. The leptospira colonize in renal tubules and are shed in urine. These bacteria survive for prolonged periods in moist conditions and thus transmit the infection.

2.1 Disease occurrence

Rats and other rodents are natural reservoirs of leptospira with no apparent signs of disease. They clear infections from their bodies except the kidney tubules. Other animals which are not natural carriers of infection have mild to severe infection and even death. The leptospires may cause reproductive problems mainly abortions (Coghlan & Bain, 1969; Faine et al., 1984; Faine et al., 1999; Bharti et al., 2003; Hamond et al., 2015; Hamond et al., 2016). Mother to foetus transmission is also common (Vinetz, 1997; Bharti et al., 2003; Alder & de la Pena Moctezuma, 2010; Verma et al., 2013; Hamond et al., 2014). Animal handlers and waste water/ recycle workers are highly susceptible (Campagnolo et al., 2000). Leptospira spp. are endemic to several tropical and subtropical areas affecting military personnel, aid workers, tourists and general public (Ko et al., 1999; Bharti et al., 2003). Leptospirosis is less common in temperate regions. There are different reservoirs of infection in rural and urban areas, domestic and wild animals act as reservoirs in rural settings, dogs and rats are reservoirs in urban areas (Vinetz et al., 1996; Levett, 2001; Meites et al., 2004). Natural disasters, like floods may be followed by leptospirosis outbreaks (Fuortes & Nettleman, 1994). The leptospirosis has been described as “an occupational disease of soldiers” as the soldiers fighting in adverse terrains and conditions during wars are at a greater risk of acquiring leptospirosis infection (Johnston et al., 1983). Symptoms in human beings may vary, but commonly include fever, headache, muscular pain, uneasiness, vomiting conjunctivitis, uveitis, meningitis and jaundice. About 5 and 10% of patients progress to icteric phase. Fatality rates at this stage may be more than 20%. Mortality is primarily due to acute renal failure, pulmonary haemorrhages, intracerebral haemorhage and multisystem organ failure (Vinetz, 1997; Faine et al., 1999; Ko et al., 1999; Levett, 2001; Bharti et al., 2003). Leptospirosis in horses has been considered a relatively uncommon infection. Most of the infections are asymptomatic. A specific outcome of equine leptospirosis is recurrent uveitis which appears to be mediated by autoimmune mechanisms.

_________________________________________________________


The incidence of leptospirosis in horses remains uncertain as systematic studies on leptospirosis in horses are scanty. Its importance and economic impact in equines is also not as accurately distinguished as for other species of animals. Most epidemiological studies are based on serology with highly variable incidence in different geographical regions. There is also variability in the serovars. A sero-prevalence of only 1.5% was reported in central Italy, for serovars Icterohaemorrhagiae, Bratislava or Pomona (Ebani et al., 2012). However, a Brazilian study of 119 race horses showed a much higher seropositivity rate of 71% against serovar Copenhageni (Hamond et al., 2012b). All these horses were apparently healthy with no clinical signs of leptospirosis. In another study by the same group, seropositivity of 48% was reported and 35% of urine samples were detected positive by PCR, however these were culturally negative (Hamond et al., 2013). Recently a prevalence study conducted in Brazil in 38 mares having reproductive problems examined for leptospira by examining the serum, urine and vaginal fluid by isolation and PCR. Seventeen serum samples were positive (44.7%) for leptospira and of which sero groups Australis accounts for 76.4% and Pomona 23.6%. PCR results were positive for 17 vaginal fluid samples and 10 urine samples and the PCR products were sequenced which showed that the samples belonged to L. interrogans (sv Bratislava and Pomona) and L. borgpertersenii. Thus this study implies the presence of leptospira in reproductive tract (Hamond et al., 2015). Sixty two cart horse samples were screened in Curitiba, southern Brazil by microscopic agglutination test (MAT) and real time PCR of which 80.8% of the samples were positive for Icterohaemorrhagiae serovar (Finger et al., 2014). Sero-positivity of 25% in Korea with serovars Sejroe and Bratislava being prominent (Jung et al., 2010), 79% in The Netherlands, serovars Copenhegi and Bratislava (Houwers et al., 2011) and 25% in Sweden (Baverud et al., 2009) show marked differences in prevalence and varied serovars according to geographical region. It was also indicated that the majority of equine infections were asymptomatic. A North American report in aborting mares revealed 20 out of 21 as due to serovar Pomona subtype Kennewicki (Timoney et al., 2011), which is correlated to the presence of this serovar and subtype in local wildlife mainly raccoons. Serovar Bratislava has been implicated in horses in Northern Ireland, based on culture and serology (Ellis et al., 1983). Pikalo et al. (2016) examined the presence of leptospiral antibodies in horses in middle Germany by MAT. 54 out of 314 (17.2%) horses were positive for one or more of eight leptospiral serovars analysed. Icterohaemorrhagiae (11.1%) was most prevalent followed by Bratislava (9.6%) and Grippotyphosa (1.9%). Dorrego-Keiter et al. (2016) detected 57.5% (127/221) horses with ERU having antibodies against leptospira by MAT in Germany.

Leptospirosis in horses: special reference to equine recurrent uveitis.

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Figure 1Transmission and clinical signs of equine leptospirosis. The most frequent antibodies were against Grippotyphosa (79/127), followed by Icterohaemorrhagiae (34/127) and Bratislava (29/127). Tsegay et al. (2016) detected significant antibody titres in 184 of 418 carthorses to at least one of 16 serovars of Leptospira species in central and southern Ethiopia. Serovar Bratislava (34.5%) was found to be most prevalent.

apparently healthy animals suggest either a subclinical form of the disease in these animals or previous infection. Region-wise sero-prevalence of leptospiral antibodies showed that studs in southern and western part of India country had higher seropositivity irrespective of the group. 2.2 Clinical signs, symptoms and disease manifestations

National Reference Centre for Leptospirosis (NRCL) of Italy along with other units evaluated the occurrence and distribution of leptospira in Italy. Analysis of the data for the one year (2010-2011) revealed that Australis was common among horses in Italy (Tagliabue et al., 2016). Khurana et al. (2003) studied the sero-prevalence of leptospira in 436 equines in India by ELISA for detection of antibodies against six leptospiral serovars including Leptospira interrogans serovars Canicola, Pomona, Australis, Autumnalis, Icterohaemorrhagiae and Grippotyphosa. These samples included 379 serum samples from apparently healthy equines, 12 from cases of abortion and 45 from equines in contact with aborted animals. Out of the 379 apparently healthy horses, 64 (16.89%) harboured antibodies against Leptospira spp. They further reported the mares that came into contact with aborted animals, 66.7% had positive titres indicative of leptospiral infection. Animals in contact with aborted animals had also showed higher sero-prevalence of 64.4%. Antibody titres in _________________________________________________________


Equine leptospirosis is accompanied with mild fever. Loss of appetite and lethargy are common in mild form of disease. Jaundice, haemorrhages on the mucosa and depression are predominant signs in the severe form. Renal failure is more common in foals in comparison to old horses. Classic icteric leptospirosis occurs mainly in foals and is comparatively rare in adult horses. Leptospirosis may cause placentitis, abortions and stillbirths in pregnant mares (Figure 1) (Timoney et al., 2011). The letospires could be seen in foetal and maternal tissues, microscopically (Poonacha et al., 1993). Leptospiral abortions in the late stage of gestation, with no apparent clinical signs are common. Weak and icteric foals are also born (Donahue et al., 1991; Donahue et al., 1995). Infected mares shed leptospires in the urine for prolonged periods and transmit the infection (Donahue & Williums, 2000; Newman & Donahue, 2007).

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Microscopically placental lesions include vasculities, thrombosis, inflammatory cells in the stroma and villi, cystic adenomatous hyperplasia of allantoic epithelium. Foetal liver and kidneys are enlarged. Microscopic lesions in foetus include suppurative and nonsuppurative nephritis, leukocytic infiltration of the portal triads, giant cell hepatopathy, pulmonary haemorrhages, pneumonia and myocarditis (Wilkie et al., 1988; Poonacha et al., 1993). Histopathological findings in young horses are marked with petechiae and lymphoytic infiltration in renal proximal tubules and glomeruli (Bernard, 1993; Faine et al., 1999). Equine recurrent uveitis is a common sequel in equines, which is dealt separately in this review. The pulmonary haemorrhage is not common in equines (Bharti et al., 2003). Recently this syndrome is being reported more commonly than previous information (Broux et al., 2012) and endoscopy revealing pulmonary haemorrhage in 35% of seropositive adult horses (Hamond et al., 2012a). 2.3 Diagnosis of leptospirosis in equines The diagnosis of leptospirosis in horses is similar to that for other species. The gold standard is the culture and identification of leptospira. PCR is a more convenient and rapid (Alder & de la Pena Moctezuma, 2010). Real time PCR assay was recently compared with fluorescent antibody test (FAT) and microscopic agglutination test (MAT) for effective diagnosis of equine leptospirosis from foetal specimens like placenta, kidney, liver and heart blood. Out of the 21 confirmed cases of equine abortion real time PCR could detect all the positives correctly while MAT and FAT detected only 19 and 18 samples respectively. Thus qPCR is a better assay compared to MAT and FAT for diagnosis of leptopsiral abortion in equines (Erol et al., 2015). Silver staining and FAT may be used to demonstrate leptospires in the placenta or foetal kidney. FAT is more sensitive than silver staining and more specific than MAT (Donahue & Williams, 2000; Szeredi & Haake, 2006; Newman & Donahue, 2007). Enzyme-linked immunosorbent assay (ELISA) has also been developed using different proteins of leptospira and its efficacy has been assessed time to time. Recently a cocktail of recombinant proteins namely rLipL21, rLoa22, rLipL32, and rLigACon4-8 of Leptospira interrogans were analyzed for its potential as a diagnostic marker through ELISA. The assay was tested with 130 serum samples and the results were compared with MAT and it was found that ELISA was sensitive and specific yielding similar results with MAT assay (Ye et al., 2014). MAT is the test of choice for serological diagnosis. In an endemic area the value of a single positive specimen is limited. The four-fold rise in titre in paired sera is important for accurate diagnosis. In cases of leptospiral abortion, MAT on foetal fluids and maternal serum gives a very high titre indicative of positive diagnosis (Donahue & Williams, 2000). Serological tests in different areas should include prevalent serovars of that area as test antigens. Commercial enzyme immunoassays incorporating locally prevalent serovars are available. _________________________________________________________


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2.4 Prevention and control Treatment regimens for horses have mostly been derived by extrapolation from other species, due to non availability of specific information for horses. Streptomycin and penicillin are most common antibiotics of choice. Tetracyclines are used as an alternative. The penicillin dose is related to titre of leptospiral antibodies. The streptomycin is has severe toxic effects in horses (Bernard 1993; Newman & Donahue, 2007). Till now there were no leptospirosis vaccine for horses. Cattle vaccines were being occasionally used in horses, which is not advisable. In leptospiral uveitis molecular mimicry occurs between leptospiral proteins and ocular tissues, in this situation vaccination with whole-cell bacterin may prime equines with cross-reacting antigens resulting in stronger immunological responses and development of eye inflammation in subsequent exposures. Ideally, proposed leptospirosis vaccine should be free of cross-reacting antigens. Several leptospiral antigens have been tested for protective efficacy (Alder & de la Pena Moctezuma, 2010; Murray et al., 2013). No antigen has been tested in horses. Recently, Zoetis has introduced a licensed equine leptospiral vaccine for prevention of leptospirosis caused by Leptospira Pomona. Prevention must therefore revolve around normal husbandry and hygiene practices, vaccination of other animals on the farm, minimizing contact with rodents and other wildlife carriers and other infected horses. 3 Equine Recurrent Uveitis (ERU) A major consequence of leptospirosis in horses is uveitis or moon blindness also called periodic ophthalmia (Verma et al., 2013; Malalana et al., 2015). The uvea consists of three components, the iris, ciliary body (anterior uvea) and choroid (posterior uvea). The uveal tract is highly vascular, usually pigmented (Samuelson, 2007; Gilger & Deeg, 2011: Hollingsworth, 2011). Direct proximity to the peripheral vasculature, makes the uveal tract vulnerable to any disease of the systemic circulation (Hughes, 2010; Leiva et al., 2010; Gilger & Deeg, 2011). A blood-ocular barrier exists between the peripheral vasculature and the inner structures of the eye, divided into the blood-aqueous barrier (iris and ciliary body) and blood-retinal barrier (choroid). These barriers make the eye a protected or immune-privileged site. Disruption of this barrier allows the leakage of blood products and cells into the eye and the activation of several immune responses. Leptospira-associated uveitis forms an important part of ERU cases (Halliwell et al., 1985; Hartskeerl et al., 2004; Witkowski et al., 2016). ERU is inflammation of uvea which occurs recurring episodes (Cook & Harling, 1983). It is reported to have a worldwide prevalence of around 10% and thought to be a major cause of blindness in horses (Schwink, 1992; Hartskeerl et al., 2004). Pathogenesis of ERU is not exactly elucidated though several possible ways has been reported. Eye of horses affected with ERU shows infiltration of macrophages, lymphocytes and


plasma cells into the ciliary body and also the iris. This shows that immunologically privileged sites wall has been breached by the organism. There is huge flow of CD4+ T lymphocytes in the anterior uveal tract (Romeike et al., 1998). In these affected horses T cell response is mainly of Th1 based (Gilger et al., 1999). Kalsow et al. (1994) reported that T- and B-cells are highly organized in the germinal centres in the horses affected with ERU. This highly organized structure shows the antibody response towards leptospira antigen in the anterior uvea (Kalsow et al., 1994). Leptospira can directly cause damage to the eye leading to ERU but mainly it is caused by the autoimmune response due to the antigen (Verma et al., 2005). Two leptopsiral proteins namely LruA and LruB were suggested to play a major role in ERU since IgG and IgA specific for these proteins has been identified in the fluid from the eye (Verma et al., 2005). Hence these proteins can also be used as a diagnostic marker for detection of leptospiral infection in equines. An evidence of a antigenic relationship between Leptospira and equine eye is established further cross reactivity as a mechanism of disease progression has been proposed (Parma et al., 1985; Parma et al., 1987; Parma et al., 1997). Verma et al. (2005) described that intraocular expression of two leptospiral proteins, LruA and LruB antibodies was significantly higher than in the sera, indicating local production and antibodies in uveitic eyes. The lens proteins cross-reacting with LruA antiserum were identified as crystalline B and vimentin, and cross reacting retinal protein was identified as crystalline B2 (Verma et al., 2010). Therefore cross reactivity between leptospiral and ocular proteins may be responsible for immunopathogenesis of ERU of leptospirai origin. ERU has three distinct clinical forms: classic, insidious and posterior (Gilger & Michau, 2004; Gilger & Deeg, 2011; Malalana et al., 2015). Classic ERU is characterised by active intraocular inflammation followed by quiet periods, where subsequent inflammatory phases show increased severity. Insidious ERU is characterised by low grade persistent inflammation. The Vitreous, choroid and retina are primarily affected in posterior uveitis. The signs associated with an acute episode of anterior uveitis are varied including ocular pain, blepharospasm, lacrimation, chemosis, photohobia, oedema of the eyelid, swollen conjunctiva and corneal oedema, aqueous flare, hypopyon, hyphaema, miosis, iris colour changes and low intraocular pressure (Cook et al., 1983: Wada, 2006; Gilger & Deeg, 2011). Posterior uveitis is characterised by vititis with liquefaction of the vitreous and retinal changes. Changes associated with previous episodes of uveitis in an otherwise quiescent eye may give clues to previous episodes. Some of these changes may include depigmentation, corneal scarring, atrophy and fibrosis of iris, abnormalities of the iris margin, cataract, glaucoma and fundic changes etc. (Williams et al., 1971) but are not specifically pathognomonic (Cook et al., 1983; Barnett, 1987; Spiess, 2010; Mathes et al., 2012). The prognosis is dependent on an early diagnosis and _________________________________________________________


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treatment. A period of low inflammation follows the acute phase (Cook & Harling, 1983). Secondary cataract, anterior or posterior attachment of iris, lens luxation, vitreous exudates and retinal detachment are also witnessed due to severe inflammatory reaction (Rebhun, 1979; Cook & Harling, 1983; Gilger et al., 2000; Gilger & Michau, 2004). A pathognomonic sign of ERU is thick hyaline membrane near posterior aspect of iris and eosinophilic linear cytoplasmic inclusion bodies in nonpigmented ciliary epithelial cells (Cooley et al., 1990; Dubielzig et al., 1997). ERU has been associated with sulphonamides administration or vaccination (Matthews & Handscombe, 1983; Whitcup, 2010). Higher prevalence in geldings compared to mares and stallions has been reported (Szemes & Gerhards, 2000). No particular sex related differences in prevalence have been reported (Gilger & Deeg, 2011; Kulbrock et al., 2013). Age of presentation has been reported to vary in different studies (Dwyer et al., 1995; Szemes & Gerhards, 2000). Diagnosis of Leptospira associated ERU is based on the presence of classical signs of uveitis, history of recurrence and seropositivity by MAT. No specific test is available for the diagnosis of leptospiral uveitis. Negative MAT titres are not always indicative of absence of leptospiral infection. Reducing inflammation is of primary concern in ERU therapy. An intraocular device containing cyclosporine A is found effective in treatment of leptospiral ERU (Werry & Gerhards, 1991; Gilger & Michau, 2004). However, usefulness of antibiotics in treating ERU has not been fully explored. A recent review conducted in the North Carolina State University Veterinary Health Complex with the medical records of ERU from 1999 to 2014 showed that most cases had blindness, eye globe loss and loss of eye function. Several owners opted for euthanasia and some opted to sell the animals due to recurrent eye problem (Gerding & Gilger, 2016). Thus this problem of ERU has caused great financial loss to the horse owners. Thus leptospiral infections result in reproductive and respiratory problems in equines. However, the most important manifestation of leptospiral infection in equines is ERU, which affects equine population by causing blindness, thus rendering them useless. Therefore more researches are needed to explore the pathogenesis and mechanism of occurrence of ERU with an aim of its prevention and control. Conflict of interest Authors would hereby like to declare that there is no conflict of interests that could possibly arise. References Adler B, de la Pena Moctezuma A (2010) Leptospira and leptospirosis. Veterinary Microbiology 140: 287-296. doi: 10.1016/j.vetmic.2009.03.012.

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Barnett KC (1987) Equine periodic opthalmia: A continuing aetiological riddle. Equine Veterinary Journal 19: 90-91. Baverud V, Gunnarsson A, Engvall EO, Franzen P, Egenvall A (2009) Leptospira seroprevalence and associations between seropositivity, clinical disease and host factors in horses. Acta Veterinaria Scandinavica 51:15. doi: 10.1186/1751-0147-5115 Bernard WV (1993) Leptospirosis. The Veterinary Clinics of North America, Equine Practice 9: 435-444. Bharti AR, Nally JE, Ricaldi JN, Matthias MA, Diaz MM, Lovett MA, Levett PN, Gilman RH, Wiling MR, Gotuzzo E, Vinetz JM (2003) Leptospirosis: a zoonotic disease of global importance. The Lancet, Infectious Diseases 3: 757-771. Broux B, Torfs S, Wegge B, Deprez P, van Loon G, (2012) Acute respiratory failure caused by Leptospira spp. in 5 foals. Journal of Veterinary Internal Medicine 26: 684-687. Campagnolo E, Warwick M, Marx H, Cowart R, Donnell H, Bajani M, Bragg S, Esteban J, Alt D, Tappero J, Bolin C, Ashford D (2000) Analysis of the 1998 outbreak of leptospirosis in Missouri in humans exposed to Infected swine. Journal of American Veterinary Medical Association 216: 676682. Coghlan JD, Bain AD (1969) Leptospirosis in human pregnancy followed by death of the foetus. British Medical Journal 1: 228-230. Cook CS, Harling DE (1983) Equine recurrent uveitis, Equine Veterinary Journal 2: 2-15. Cook CS, Peiffer RL, Harling DE (1983) Equine recurrent uveitis. Equine Veterinary Journal 15: 57-60. Cooley PL, Wyman M, Kindig O (1990). Pars plicata in equine recurrent uveitis. Veterinary Pathology 27: 138-140. doi:10.1177/030098589002700215 Donahue JM, Smith BJ, Poonacha KB, Donahoe JK, Rigsby CL (1995) Prevalence and serovars of Leptospira involved in equine abortions in central Kentucky during the 1991-1993 foaling seasons. Journal of Veterinary Diagnostic Investigation 7: 87-91. doi: 10.1177/104063879500700114 Donahue JM, Smith BJ, Redmon KJ, Donahue JK (1991) Diagnosis and prevalence of leptospira infection in aborted and stillborn horses. Journal of Veterinary Diagnostic Investigation 3: 148-151. Donahue JM, Williams NM (2000) Emergent causes of placentitis and abortions. The Veterinary Clinics of North America, Equine Practice 16: 443-456.

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Dorrego-Keiter E, Toth J, Dikker L, Sielhorst J, Schusser GF (2016) Detection of leptospira by culture of vitreous humor and detection of antibodies against leptospira in vitreous humor and serum of 225 horses with equine recurrent uveitis. Berl Munch Tierarztl Wochenschr. 129 : 209-215. Dubielzig RR, Render JA, Morreale RJ (1997) Distinctive morphologic features of the ciliary body in equine recurrent uveitis. Veterinary and Comparative Opthalmology 7: 163167. Dwyer AE, Crockett RS, Kalsow CM (1995) Association of leptospiral seroreactivity and breed with uveitis and blindness in horses-372 cases (1986-1993). Journal of the American Veterinary Medical Association 207: 1327-1331. Ebani VV, Bertelloni F, Pinzauti P, Cerri D (2012) Seroprevalence of Leptospira spp. and Borrelia burgdorferi sensu lato in Italian horses. Annals of Agricultural and Environmental Medicine 19: 237-240. Ellis WA, O’Brien JJ, Cassells JA, Montgomery J (1983) Leptospiral infection in horses in Northern Ireland: serological and microbiological findings. Equine Veterinary Journal 15: 317-320. Erol E, Jackson CB, Steinman M, Meares K, Donahoe J, Kelly N, Locke S, Smith JL, Carter CN (2015) A diagnostic evaluation of real-time PCR, fluorescent antibody and microscopic agglutination tests in cases of equine leptospiral abortion. Equine Veterinary Journal 47 : 171-4. Faine S, Adler B, Bolin C, Perolat P (1999) Leptospira and leptospirosis 2nd edition. Melbourne, Australia: MedSci. ISBN 0 9586326 0 X. Faine S, Adler B, Christopher W, Valentine R (1984) Fatal congenital human leptospirosis. Zentralblatt fur Bakteriologie Mikrobiologie und Hygiene I Abteilung Originale A 257.548. Finger MA, de Barros Filho IR, Leutenegger C, Estrada M, Ullmann LS, Langoni H, Kikuti M, Dornbush PT, Deconto I, Biondo AW (2014). Serological and molecular survey of Leptospira spp. among cart horses from an endemic area of humanleptospirosis in Curitiba, southern Brazil. Revista do Instituto de Medicina Tropical de Sao Paulo 56 : 473-6. Fuortes L, Nettleman M (1994) Leptospirosis: a consequence of the lowa flood. lowa Medicine 84: 449-450. Gerding JC, Gilger BC (2016) Prognosis and impact of equine recurrent uveitis. Equine Veterinary Journal 48 : 290-8. Gilger BC, Malok E, Cutter KV, Stewart T, Horohov DW, Allen JB (1999) Characterization of T-lymphocytes in the anterior uvea of eyes with chronic equine recurrent uveitis. Veterinary Immunology and Immunopathology 77: 17–28.


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ISSN No. 2320 – 8694

EMERGENCE OF EQUINE HERPES VIRUS 1 MYELOENCEPHALOPATHY: A BRIEF REVIEW Baldev Raj Gulati1,*, Gayathri Anagha2, Thachamvally Riyesh1 and Sandip Kumar Khurana1 1 2

ICAR-National Research Centre on Equines, Hisar, Haryana-125001, India ICAR- Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh-243122, India

Received – October 15, 2016; Revision – October 26, 2016; Accepted – November 20, 2016 Available Online – December 04, 2016 DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S132.S138

KEYWORDS Equine herpesvirus 1 Equine herpesvirus myeloencephalopathy Neuropathogenicity ORF30

ABSTRACT Equine herpesvirus 1 (EHV1) is an economically important viral pathogen of equines and causes respiratory disease, neonatal foal mortality, late-term abortion and sporadic encephalomyelitis aka equine herpes myeloencephalopathy (EHM) in affected horses. The nervous form of EHV1 (EHM) has been recognized as early as 1950s in horse population; however, many aspects of this disease remained poorly understood. In recent years, there has been much progress in our understanding of genetics, epidemiology and pathogenesis of EHM through close monitoring of field outbreaks in different parts of the world. Various host, agent and environmental factors have been found to a play a role in the development of EHM, the most significant being the identification of a single nucleotide polymorphism in DNA polymerase gene (A2254 to G2254), which imparts neuropathogenic potential to the virus. EHM affects horses of all ages, including un-weaned foals and produces clinical symptoms that are indistinguishable from other viral encephalitis/ central nervous system (CNS) disorders. EHM treatment includes supportive therapy, and reducing inflammation of CNS. Diagnosis of affected horses and monitoring of in-contact animals is the best measures to prevent EHM outbreaks. This review in brief discusses about progress made in epidemiology, pathogenesis, treatment, prevention and control of EHM.

* Corresponding author E-mail: [email protected] (Baldev R. Gulati) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.



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1 Introduction Equine herpesvirus 1 (EHV1) is a highly contagious respiratory pathogen associated with a variety of disease conditions in horses. It is estimated that 80 to 90% of horses have been exposed to EHV1 infections by two years of age (Allen, 2008). EHV1 infection causes upper respiratory tract infection in young horses, abortion in pregnant mares, neonatal foal mortality and neurological disorders. Abortion is the most economically crippling outcome of EHV1 infection with 95% of EHV1 associated abortions occurring in the last four months of pregnancy. Respiratory disease associated with EHV1 is most commonly seen in young animals at the time of weaning (Allen, 2008). Neurological disease associated with EHV1 is called equine herpesvirus myeloencephalopathy (EHM). Although clinical form of EHM is less frequently observed, it can cause serious economic losses in breeding horses and has very negative impact on equine industry (Friday et al., 2000; van Mannen et al., 2001; Henninger et al., 2007; Pronost et al., 2010). During past decade, incidence of abortion and rhinopneumonitis due to EHV1 has been declining, possibly due to widespread vaccination practices. At the same time, there has been rise in incidence of EHM in many parts of world viz., Europe, North America, South America, Africa and Oceania (Perkins et al., 2009; Vissani et al., 2009; Pronost et al., 2010; Smith et al., 2010; Fritsche & Borchers 2011; Tsujimura et al., 2011, Cuxson et al., 2014; Negussie et al., 2015; McFadden et al., 2016). The neuropathogenic strains of EHV1 (causing EHM) have also been reported from Asian countries such as Japan (Tsujimura et al., 2011) and India (Unpublished data). This article discusses the aetiology, pathogenesis, epidemiology of EHM and gives an overview of prevention, control and treatment of EHM. 2 Etiology EHV1 is an enveloped, double-stranded DNA virus belonging to the genus Varicellovirus of the subfamily Alphaherpesvirinae within family Herpesviridae (Davison et al., 2009). As many as nine equine herpesviruses (EHV 1-9) species have been known to infect equines. Among these only five (viz., EHV1, 2, 3, 4 and 5) have the ability to produce diseases in horses. EHV3 is responsible for equine coital exanthema while EHV1 and 4 are the economically important viruses affecting the horses globally (Davison et al., 2009) with EHV1 capable of even causing abortion and neurological disorders as compared to EHV4 (Patel & Heldens, 2005; Lunn et al., 2009). EHV2 and 5 do not cause any specific diseases but remain associated with upper respiratory tract diseases, immunosupression, general malaise and poor performance (Thein 1978; Belak et al., 1980). EHV1 genome is 150 kbp linear double-stranded DNA composed of a unique long region and unique short region flanked by inverted repeat regions, the terminal repeat and the _________________________________________________________


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internal repeat regions. The EHV-1 genome encodes for 76 open reading frames (ORFs). EHV1 isolates have special virulence markers, which are thought to induce EHM. 3 Equine herpesvirus myeloencephalopathy EHM was present in equine population as early as 1950s, however, its importance came to limelight in the last decade after large outbreaks of EHM occurred in Europe and America (Perkins et al., 2009; Vissani et al., 2009; Pronost et al., 2010; Smith et al., 2010; Fritsche & Borchers 2011; Pusterla et al., 2012; Damiani et al., 2014; Stasiak et al., 2015). Neurological disease can affect horses of all ages, including un-weaned foals, and often requires euthanasia of affected animal Horses exhibiting neurologic diseases can shed the virus in their nasal secretions and transmit the disease to in-contact animals (Henninger et al., 2007).) The ORF30 spanning the nucleotide region 51522-55184 (3662 nt) in EHV1 genome encodes for a protein referred to as Pol, the putative DNA polymerase catalytic subunit which possesses DNA synthesis activity. This gene is highly conserved throughout its length. Recently, a single nucleotide polymorphism (SNP) of guanine (G) for adenine (A) at 2254 nucleotide position of the ORF30 region resulting in an amino acid variation, from asparagine to aspartic acid (N/D752) have been proven to be associated with the neuropathogenic potential of the EHV1 strain (Nugent et al., 2006). This DNA polymerase enzyme of EHV1 has two sets of identical protein subunits each of which contains two catalytic pockets (Liu et al., 2006), serving as site for polymerase activity and the site for 3’- 5’ exonuclease activity. In EHV1, neuropathogenic strains, the point mutation results in a switch from no charge to a negative charge and induces a conformational change within the viral polymerase structure and thereby increases the replicative capacity of the virus and produce significantly higher viral loads (Nugent et al., 2006; Liu et al., 2006). 4 Prevalence of neuropathogenicty Increased numbers of EHM cases have been reported from various parts of the world during the last decade with majority of them from Europe and North America. Europian countries viz., France (Pronost et al., 2010; van Galen et al., 2015), Germany (Fritsche & Borchers, 2011; Damiani et al., 2014), Belgium (van der Meulen et al., 2003; Gryspeerdt et al., 2011), Poland (Stasiak et al., 2015), Netherlands (Goehring et al., 2006) and Croatia (Barbic et al., 2012); North American countries viz., Canada (Burgess et al., 2012) and U.S.A (Nugent et al., 2006; Henninger et al., 2007; Perkins et al., 2009; Smith et al., 2010; Pusterla et al., 2012); South American countries viz., Brazil (Mori et al., 2011) and Argentina (Vissani et al., 2009); Asian countries viz., Turkey (Yilmaz et al., 2012); Japan (Tsujimura et al., 2011) and India (Unpublished data); Islands viz ., Australia (Cuxson et al., 2014) and Newzealand (McFadden et al., 2016); African countries viz., Ethiopia (Negussie et al., 2015) experienced

Emergence of equine herpes virus 1 myeloencephalopathy: A brief review.

outbreaks of EHV1 infection by neuropathogenic strains of EHV1. The incidence of neuropathogenic genotype from cases of neurological illness reported from different countries varies between 20% and 86% (Perkins et al., 2009; Vissani et al., 2009; Pronost et al., 2010; Fritsche & Borchers, 2011; Cuxson et al., 2014). The prevalence of neuropathogenic strains in abortion outbreaks varies between 1.5% and 25.8%. The percentage prevalence was highest (25.8%) in France (Pronost et al., 2010) followed by 19.4% in U.S.A (Smith et al., 2010), 10.6% in Germany (Fritsche & Borchers, 2011), 7% in Argentina (Vissani et al., 2009), 3.1% in Poland (Stasiak et al., 2015), 2.7% in Japan (Tsujimura et al., 2011) and 1.5% in Australia (Cuxson et al., 2014). 5 Pathogenesis of EHM Upon entry into the animal body, virus multiplies in the epithelial cells of upper respiratory tract. Following initial replication, the virus spreads to the cells of lamina propria and underlying tissues within 12-24 h, after crossing the basement membrane. By 1-2 days post-infection (dpi), the virus reaches in the local lymph nodes draining the respiratory tract where further replication and infection of leukocytes occurs. Leukocytes harbouring the virus are released to the blood stream (Leukocyte-associated viremia) between 4-10 dpi which enables the virus to reach internal organs including CNS (Kydd et al., 1994; Gryspeerdt et al., 2011). Secondary replication occurs in endothelial cells of CNS-associated arterioles (in particular the vessels of the spinal cord), which may result in nervous system disorders 9-13 dpi. As a consequence, vasculitis, thrombosis, perivascular cuffing of lymphocytes at sites of endothelial infection occurs, probably caused by direct interaction of the host's immune system and infectious agent (Edington et al., 1986). This vascular damage leads to ischaemia and re-perfusion injury of the CNS. Neuropathogenic strains are capable of exhibiting longer and higher level viremia. This high level viremia, interfere the blood flow to CNS and resulting in development of neurological diseases (Fritsche & Borchers, 2011). The exact mechanism by which leukocyte-associated viremia leads to myeloencephalopathy is not known. 6 Clinical Signs of EHM Onset of clinical signs of EHM usually occur 6-10 dpi following the onset of viremia. Clinical signs depend on number and size of affected sites, as well as relevance and location of affected nervous tissue (caudal spinal cord is most affected). Clinical signs usually include fever, ataxia, paresis/paralysis of hind limbs, bladder dysfunction, urinary incontinence and sensory deficit in the perineal area. In addition ventral oedema, scrotal or preputial oedema in male horses, and limb oedema are also noticed. In severe cases of EHM, paralysis may advance to tetraplegia and death of animal is observed (van Mannen, 2002; Pusterla & Hussey, 2014). EHM affected horse that remains in standing posture may have good prognosis. However, horses with severe neurologic disease may take more than a year for complete _________________________________________________________


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recovery, although some horses may be left with permanent neurologic sequalae. 7 Factors affecting EHM Mechanism behind EHM is poorly understood. Studies on the evaluation of the risk factors associated with the development of EHM have been performed in Europe and in North America. Various factors viz., season, age, breed, sex, immunological status and latency have been found to be associated with EHM. A study in the Netherlands revealed a strong association between season and outbreaks of EHV neurological disease with all outbreaks occurring between mid-November and midMay. However, this season specificity has not been observed in all countries. Paillot et al. (2008) reported that neurological signs due to EHM were seen at an increased frequency in standard breeds, Hispanic breeds and draught breeds, with no cases of EHV-induced myeloencephalopathy in archetypical ponies, Haflinger, Fjord and Icelandic horses. Experimental infection proved that older horses are more predisposed to the development of neurological disease as compared to young to young/middle aged horses. Adult horses may develop viremia 100 times higher than young horses and they are 8 times more likely to develop the disease (Allen, 2008). Latency by alpha herpes viruses is an important epidemiological strategy ensuring survival and spread within the natural host population (Whitley & Gnann, 1993). EHV1 latency has been demonstrated in lymphoid as well as in neural tissues (Baxi et al., 1995; Borchers et al., 1999). Following reactivation, latently infected carriers may shed the virus in their nasal secretion and also may result in EHM following invasion of nervous system (Allen & Timoney, 2007). 8 Laboratory Diagnoses Laboratory diagnosis of EHM is currently based on at least one of the following criteria: clinical symptoms, cerebrospinal fluid examination, serological testing, virus isolation, molecular detection methods and post-mortem examination. Differential diagnosis should also be made from other viral cause of encephalitis, rabies, protozoal myeloencephalitis and non infectious conditions like neuritis of the cauda equina, central nervous system (CNS) trauma and different plant/chemical intoxications (Pusterla et al., 2009; Pusterla & Hussey, 2014). Horses presented with clinical symptoms as explained elsewhere may be suspected for EHM. An increased protein concentration and albumin quotient may be noticed in CSF of affected horses. Serological examination suggesting a 4-fold or greater increase in serum antibody titer between acute and convalescent samples in the clinically affected horses, along with antibodies in CSF, is strongly suggestive of EHM (Friday et al., 2000; van Maanen et al., 2001). However, many horses with EHM do not exhibit a 4-fold rise in SN titer, since the antibody titers rise rapidly and may have peaked by the time neurological signs appear (Friday et al., 2000; van Maanen et al., 2001). Virus

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isolation from nasal or nasopharyngeal swabs or buffy coat samples is considered as the ‘gold standard’ test for a laboratory diagnosis of EHV1 infection. However, EHM cases might not yield virus isolation, as virus shedding may stop by the time neurological signs appear (Pusterla et al., 2009). Many of the published conventional PCR detection protocols (Ballagi- Pordany et al., 1990; Sharma et al., 1992; Wagner et al., 1992; Borchers & Slater, 1993; Kirisawa et al., 1993; Lawrence et al., 1994., Wang et al., 2007) are unable to differentiate between neuropathogenic and nonneuropathogenic viruses. Hence, PCR assays based on ORF30 followed by sequence analysis can be used to differentiate neuropathogenic and non-neuropathogenic EHV1 isolates (Nugent et al., 2006; Allen, 2007; Leutenegger et al., 2008; Pusterla & Hussey, 2014). Use of novel PCR platforms, such as real-time PCR assays based on ORF30 enable the differentiation of neuropathogenic and non-neuropathogenic viruses (Allen, 2007; Leutenegger et al., 2008). Single nucleotide polymorphism (SNP)-real-time PCR (Smith et al., 2012) and primer-probe energy transfer method (Malik et al., 2010) have been used for diagnosis of EHM. A SNP-based real-time PCR has been developed in our laboratory that is able to differentiate neuropathogenic and non-neuropathogenic EHV1 strains. Using this assay, we observed circulation of neuropathogenic EHV1 among Indian equine population (unpublished data). 9 Treatment of EHM There is no specific treatment for EHM and the line of treatment is aimed at supportive medication to reduce CNS inflammation. Antiviral drugs for reducing viremia, nonsteroidal anti-inflammatory drugs (NSAID) for countering inflammation and anti-thrombotic drugs for preventing clot formation are commonly used for treatment (Lunn et al., 2009; Pusterla & Hussey, 2014). Treatment with corticosteroids, such as prednisolone acetate or dexamethasone for 2 to 3 days, is frequently recommended for severely affected animals as their use could aid in reducing the incidence of vasculitis, thrombosis, and the resultant neural injury. Flunixin meglumine (nonsteroidal anti-inflammatory drug), which is commonly used for the treatment of CNS vasculitis can be used as these drugs suppress cellular interactions between infected lymphocytes and endothelial cells (Pusterla et al., 2009). Drugs like dimethyl sulfoxide, acetylsalicylic acid and pentoxifylline have also been used for thromboembolic events associated with vasculitis. Antiviral drugs such as acyclovir have been found effective in in-vitro studies, however, limited data is available on the in vivo efficacy of acyclovir. Administration of broad-spectrum antimicrobials is also found effective to combat the risk of development of cystitis in affected horses (Pusterla & Hussey, 2014). 10 Control of EHM There is no specific method for prevention and control of EHM. However, routine management practices aimed at _________________________________________________________


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reducing the likelihood of introduction and dissemination of EHV1 infection can prevent EHM in herd. The control measures are mainly focused around quarantine and vaccination (Lunn et al., 2009; Pusterla et al., 2009). Affected or suspected horses must be removed from the stable immediately and placed in strict isolation. Once EHV1 infection is confirmed, horses should remain in strict quarantine until they are fully recovered and are asymptomatic for 21 days. Horses from farms experiencing EHM infection should be maintained in their existing stable and segregated from other horses. There should be total movement restriction of animals from such farms (Pusterla et al., 2009; Pusterla & Hussey, 2014). The currently used EHV1 vaccines are not able to provide protection against EHM. However, regular use of commercially available EHV1 vaccines enhances herd immunity, reduce viral shedding at the event of exposure and hence reduce EHM risk (Pusterla & Hussey, 2014). 11 Conclusions and future perspectives The development of neurological disease due to EHV1 infection is likely to be multi-factorial. Potential horse-specific risk factors for EHM include advanced age, breed, postexposure viraemic load, low cytotoxic-T lymphocyte precursors and environmental factors. Antemortem diagnosis of EHM relies mainly on real time-PCR detection of EHV1 in nasal secretions and blood. Although several vaccines are commercially available to prevent respiratory and abortigenic form of EHV1 infections, they do not provide protection from neurologic form of the disease. Even though there is a strong association between EHM and the G2254 mutation, this nucleotide substitution is not the only determinant of neurological disease. EHV1 isolates with A2254 genotypes have been associated with a number of cases of neurological disease. On the other hand, G2254 genotype EHV1 isolates have been recovered from horses with no evidence of neurological symptoms. One of the possible reasons for this observation could be the fact that besides A2254→G2254 substitution, other nonsynonymous nucleotide substitutions in ORF30 region can also have an effect on the production of neurological disease by either enhancing/attenuating the capability of viral replication rates in vivo. Furthermore, DNA polymerase is only one out of six proteins involved in ‘elongation complex’ of DNA replication machinery Substitutions occurring in the ORF of any one of these proteins could have a considerable impact on viral replication rates, which will in turn have an effect on neuropathogenicity. This is an area of research that needs further investigation. Comparative whole genome sequencing of neuropathogenic EHV1 strains from different geographical location might decipher other markers related to neuropathogenicity. There is also need to understand the role of host factors in the pathogenesis of EHM, including host immunopathological mechanisms in response to EHV1 infection and latency.


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ISSN No. 2320 – 8694

EQUINE OCULAR SETARIASIS AND ITS MANAGEMENT

Malik Abu Rafee* and Amarpal Division of Surgery and Radiology, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P India-243122 Received – October 15, 2016; Revision – November 02, 2016; Accepted – November 21, 2016 Available Online – December 04, 2016 DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S139.S143

KEYWORDS Ocular setariasis Eye worm Equine Surgery

ABSTRACT Ocular setariasis is a common vision threatening ophthalmic condition in equine resulting from ectopic parasitism by Setaria digitata, Setaria equina and Thelazia lacrymalis. The disease occurs mostly in summer and autumn seasons and it displays signs of lacrimation, photophobia, corneal opacity, conjunctivitis and loss of vision. Close inspection of the eye reveals a moving worm in the anterior chamber of the eye. B-mode (brightness mode) ultrasonography helps in the diagnosis in case of complete opacity. The best treatment is the surgical removal of the parasite under regional/ general anesthesia. Needle paracentesis at 3 O’ clock and nick incision at 12 O’ clock position are most commonly used surgical procedure. Both the techniques give good results. A slightly modified technique of using a 21 gauge needle attached with the syringe to aspirate the worm into the syringe also gives satisfactory results. In medicinal therapy ivermectin is the most advocated drug for ocular equine setariasis, but long term tying of medicinal should be avoided and surgery should be advocated. Corneal opacity is the most common post operative complication reported. Post surgical use of placentrex has also been advocated to enhance healing and to resolve corneal opacity. The present review is aimed at etiology, diagnosis and management of ocular setariasis in equine species.

* Corresponding author E-mail: [email protected] (Malik Abu Rafee) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.



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1 Introduction

2 Diagnosis

Among the most common surgical conditions of equine ocular setariasis is a vision threatening disease of equine resulting from ectopic parasitism caused by Setaria spp, a genus of filaroid worms (Gangwar et al., 2008; Radwan et al., 2016). In India equine ocular setariosis, an important cause of corneal opacity is commonly caused by Setaria digitata, Setaria equina and Thelazia lacrymalis (Sathu, 1974; Ladoucer & Kazacos, 1981; Parrah et al., 2004; Sellon & Long, 2013). S. digitata is a parasite of cattle and hoofed animals and is found mainly in Asia. S. equina infects horses and other equids worldwide. The usual predilection site of adult Setaria worms is the peritoneal cavity. Occasionally they can get into the central nervous system or the eyes (Yadav et al., 2006). Microfilariae (immature larvae) are found in the blood. The parasite is transmitted by mosquitoes (Anopheles peditaneniatus and Culex nilgiricus) through the blood stream. Adult female worms release microfilariae in the abdominal cavity of their hosts. These microfilariae get into the blood stream and reach the capillaries in the skin. Mosquitoes become infected with microfilariae when they feed blood of infected hosts that contains microfilariae. These microfilariae develop to infective larvae inside the mosquitoes in 2 to 3 weeks. The infected mosquitoes then transmit these infective larvae to other susceptible hosts during their blood meals.

Lacrimation, photophobia, blepharospasm and corneal opacity are the common signs seen in horses with eye worm. Keen inspection of the eye usually reveals moving worm in the anterior chamber of the eye. The affected eye reacts to bright flash stimulus and fluorescein staining test is usually negative, whereas slit-lamp biomicroscopic examination reveals corneal edema (Tuntivanich et al., 2011). In eyes with complete corneal opacity B-mode ultrasonography (12 MHz, corneal contact technique) can be performed to visualize the anterior chamber and other intraocular structures (Patil et al., 2012). Though CBC (complete blood count) does not show major changes but a decrease in erythrocyte count, haemoglobin and haematocrit , together with leucocytosis and an accelerated erythrocyte sedimentation rate (ESR) has been reported in previous studies (Muhammad & Saquib, 2007). Microscopic examination of wet blood films is also recommended as it sometimes reveals motile microfilariae. Knott’s test (a technique for the detection of microfilariae by haemolysis and concentration of blood samples) can be performed to detect the microfilariae of the Setaria species (Slim & Fouad, 1965).

The ocular setariasis spreads mostly in summer and autumn when the mosquito vectors are most prevalent (Mritunjay et al., 2011; Al-Azawi et al., 2012). The parasite exhibits migratory behavior in unusual hosts such as horses, donkeys or human beings and can be found in various organs such as heart, lung, spleen, kidney, uterus, oviduct, ovary, and urinary bladder (Varma et al., 1971). All equines are generally more prone for ocular worm (Pratap, et al., 2005; Jayakumar et al., 2012; Radwan et al., 2016).The immature worm can also invade eye (Sreedevi et al., 2002; Tuntivanich et al., 2011) through the vascular system (Townsend, 2013). The eye infection occurs when the adult worm meanders through intraocular tissue, thus it is also called as eye worm. The infected animals usually display signs of photophobia and lacrimation (Basak et al., 2007). The serrated cuticle of the worm and lashing movements within the anterior chamber of the eye caused severe trauma and inflammation to the cornea which then results into corneal opacity, which eventually results into blindness (Jaiswal et al., 2006). Basak et al. (2007) has reported corneal edema caused by dead filarial worm attachment to the endothelium in the anterior chamber. The dead worm possibly liberates toxins into the anterior chamber, which may be lethal to the endothelium and resulting into corneal edema. It may lead to devastating sequel like synechia, cataract, and retinal detachment (Paglia et al., 2004). Though, the involvement of the eye is commonly unilateral but bilateral occurrence has also been reported (Shin et al., 2002; Buchoo et al., 2005). _________________________________________________________


3 Surgical treatments Although both medical and surgical treatments have been advocated for the equine ocular filariasis (Muhammad & Saquib, 2007), the best treatment is the surgical removal of the parasite (Tuntivanich et al., 2011) that can be performed under general anesthesia or regional nerve blocks with or without sedation. Regional nerve blocks like supraorbital, auriculopalpebral and retrobulbar can be performed using 2% lidocaine as per the standard methods described in literature (Lumb & Jones, 2001). Akinesis of the eyelids can further be achieved by blockade of the ventral and dorsal branches of the palpebral nerve (Facial VII) (Skarda, 1996). The supraorbital nerve is desensitised as it emerges from the supraorbital foramen, which is easily palpated 1 cm caudal to the upper orbital rim, 5–7 cm dorsal to the medial canthus. By using a 23–25 gauge needle, 1–3 ml lidocaine can be injected subcutaneously and into the foramen. This desensitises the forehead and the middle two-thirds of the upper eyelid. Motor paralysis of the auriculo-palpabral nerve (VII) is achieved by perineural administration of local anesthetics to this nerve at the most dorsal point of the zygomatic arch or just caudal to the vertical ramus of the mandible, just ventral to the zygomatic arch. The retrobulbar block may be achieved using a 19 gauge 80 mm long spinal needle passed over the zygomatic arch in a ventro-medial direction until it encounters the medial wall of the bony orbit (Fletcher, 2004; Labelle & Clark-Price, 2013). The cornea and sclera may be desensitised most effectively spraying topical application of 1% solution of amethocaine (Durham et al., 1992) or 1% tropicamide (McMullen et al., 2014).

Equine Ocular Setariasis and Its Management

Surgical interventions used for the treatment of ocular setariasis include needle paracentesis at 3 O’ clock (Sreedevi et al., 2002; Vadalia, 2013) and nick incision at 12 O’ clock (Buchoo et al., 2005). Prior to surgery, it is better that horses should receive topical non-steroidal anti-inflammatory agent (0.3% flurbiprofen) along with systemic non-steroidal antiinflammatory agents (flunixin meglumine or ketoprofen) and antibiotics. For preparation of the eye for surgery topical antiseptic (like 0.5% betadine) can be used (Patil et al., 2012). The head is held in still position with a twitch. Eye lids are retracted with the Castroviejo eye speculum and a stab incision is made at 12 O’ clock with BP blade No. 11 (Buchoo et al., 2005). The parasite usually gets ejected along with the aqueous humor; however, sometimes the parasite gets stuck in the incision. In such cases the worm is removed with the help of forceps. The incision is left unsutured. Dorsal and lateral approaches allow monitoring of the incision postoperatively and at the same time does not create the potential for possible suture trauma associated with excursions of the nictitating membrane (Kalpravidh et al., 1992). However, when additional protection of wound by nictitating membrane is required a stab incision at the ventral margin of limbus is preferred (Patil et al., 2012). The use of viscoelastic substance like hypromellose is injected into the anterior chamber to decelerate the vigorous movement of the worm to facilitate the removal of the worm (Patil et al., 2012). In the second method, a 16 gauge needle is inserted into the anterior chamber of the eye at 3 O’ clock position (Sreedavi et al., 2002) or at 6-8 O’ clock (Gopinathan et al., 2013) position of the cornea, approximately 1 mm away from the limbus, as soon as the worm appear near this site. Due to the aqueous humor pressure, the eye worm usually escapes through the hub of the needle or it appears at the puncture site thereby facilitating removal. Aqueous humor leakage is minimal as the needle puncture hole is very small (Gopinathan et al., 2013). In a slightly modified needle technique a 21 gauge needle attached with the syringe is inserted through the conjunctiva into the anterior chamber and directed carefully towards the worm to aspirate the worm into the syringe (Yang et al., 2014). The puncture site is left without suturing. Needle stabbing technique, is economical, time saving and recommended for the removal of parasite (Singh et al., 1976). Postoperatively sub-conjunctival injection of dexamethasone (2 mg)gentamicin (20 mg) may be given. Topical application of ofloxacin or other eye ointment is considered. Corneal opacity at the site of stab incision is the most common postoperative complication reported (Sharma et al., 2005). Sometimes it diffuses to involve the whole upper quadrant (Patil et al., 2012). This takes days to 3 to 8 weeks to get resolved (Buchoo et al., 2005; Jaiswal et al., 2006; Patil et al., 2012). Human placenta extract has anti-inflammatory and analgesic effects and enhance wound healing (Piyali & Debasish, 2012; Changole et al., 2015; Shukla et al., 2016).

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Placentrex facilitate post surgical healing at the insertion site in equine ocular setariasis (Mritunjay et al., 2011). 4 Medicinal therapies Taking in consideration complications of surgical treatment like phthisis bulbi, corneal oedema, and scarring and prolapse of the iris (Lavach, 1990), various medicinal therapies have been advocated. However, Medical treatment has not been considered suitable because of the slow absorption of dead parasites and the attendant antigenicity (Moore et al., 1983; Lavach, 1990). The standard antifilarial drug, diethylcarbamazine citrate (DEC) has given inconsistent results (Perumal & Seneviratna, 1954; Ahmad & Gupta, 1965). Also, an inconveniently large number of repeat treatments (for example, 32 treatments over 45 days) (Razig, 1989) has precluded DEC as a practical chemotherapeutic agent for equine setariasis. Muhammad & Saquib (2007) have advocated a medicinal therapy for ocular equine microfilariasis using ivermectin and death of the parasite in the eye took 15 days after administration of ivermectin. These suggested that in situations in which surgical intervention is difficult, the offlabel use of ivermectin would be appropriate to treat ocular equine setariasis. Conclusion Ocular setariasis commonly known as eye worm is a common surgical condition of equine eye affecting horse, donkey and pony equally. The condition can be easily diagnosed on the basis of clinical symptoms like lacrimation, photophobia, blepharospasm, corneal opacity and visible worm in the anterior chamber of the eye. Surgical treatment under regional/ general anesthesia is an effective treatment of the condition. Though, medicinal therapy with ivermectin is advocated, however relying on the medicinal treatment for too long should be avoided. Ophthalmic ointments decreasing inflammation and chances of infection and/or enhancing the healing can be used to reduce the chances of postsurgical complication. Conflict of interest Authors would hereby like to declare that there is no conflict of interests that could possibly arise. References Ahmad SA, Gupta BN (1965) Filaria oculi in equines: a therapeutic trial with the filaricidal drug Hetrazan (diethylcarbamazine citrate) Lederle. Indian Veterinary Journal 42:140-142. Al-Azawi AK, Fadhl AR, Fadhl SR (2012) Epidemiological study of Setaria equina infection in donkeys. Iraq Veterinary Journal 36 : 93-97.

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Basak SK, Hazra TK, Bhattacharya D (2007) Persistent corneal edema secondary to presumed dead adult filarial worm in the anteriorchamber. Indian Journal of Ophthalmology 55: 67-69. DOI: 10.4103/0301-4738.29501.

McMullen RJ Jr1, Davidson MG, Gilger BC (2014) The effect of 1% tropicamide-induced mydriasis and cycloplegia on spherical refraction of the adult horse. Veterinary Ophthalmology 17 : 120–125. doi: 10.1111/vop.12055.

Buchoo BA, Pandit BA, Shahardar RA, Parrah JD, Darzi, MM (2005) Surgical management and prevalence of ocular filariasis in equines. Indian Veterinary journal 82: 81-82.

Moore CP, Sarazan RD, Whitley RD, Jackson WF (1983) Equine ocular parasites: a review. Equine Veterinary Journal Supplement 2:76-85. DOI: 10.1111/j.20423306.1983.tb04565.x.

Changole S, Gupta B, Nandagawali V, Palyekar A, Chipde H (2015) Comparative Study of Efficacy of Human Placental Extract Over Beta Glucan Collagen Sheets in Partial Thickness Burn Patients. Bombay Hospital Journal 57 : 279-284. Durham RA, Sawyer DC, Keller WF, Wheeler CA (1992) Topical ocular anesthetics in ocular irritancy testing: a review. Laboratory animal Sciences 42: 535-541. Fletcher BW (2004) How to perform effective equine dental nerve blocks. Proceeding of American Association of equine Practioners 50: 233-239. Gangwar AK, Devi S, Singh HN, Singh A (2008) Ocular filariasis in equines. Indian Veterinary Journal 85: 547-548. Gopinathan A, Singh K, Saxena AC, Khurana KL, Amarpal (2013) Evaluation of two techniques for management of ocular Setariasis in horses. Research Opinion in Animal and Veterinary Sciences 3 : 407-411. Jaiswal S, Singh SU, Singh B, Singh HN (2006) Ocular setariosis in a horse. Intas Polivet 7: 67-68. Jayakumar K, Dharmaceelan S, Rajendran N, Senthilkumar S, Kathirvel S, Nagarajan L, Kumaresan A (2012) Ocular Setariasis in a Pony. Indian Veterinary Journal 89 : 64 – 66. Kalpravidh M, Bramasa A, Kalpravidth C (1992) Surgical removals of intraocular parasites from the anterior chambers of the horse eyes. Thailand Journal of Veterinary Medicine 22: 13-20. Labelle AL, Clark-Price SC (2013) Anesthesia for Ophthalmic Procedures in the Standing Horse. Veterinary Clinics of North America: Equine Practice 29 : 179–191. doi: 10.1016/j.cveq.2012.12.001. Ladoucer CA, Kazacos KR (1981) Thelazia lacrimalis in horses in India. Journal of American Veterinary Medical Association 178: 301-302. Lavach JD (1990) Parasitic diseases. In Large Animal Ophthalmology. Vol 1. Philadelphia, Mosby pp 260-263. Lumb WV, Jones EW (2001) Local and regional anesthetic and analgesic techniques. In: Veterinary anesthesia, 3rd ed. Lea and Febiger, Philahelphia pp 449.

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Mritunjay K, Monsang SW, Pawde AM, Singh SK, Madhu DN, Zama MMS (2011) Post surgical healing effect of placentrex in equine (Equus cabalus) ocular setariasis: A review of 22 cases. The Indian Journal of Field Veterinarians 6: 71-73. Muhammad G, Saqib M (2007) Successful treatment of ocular equine microfilariasis (Setaria species) with ivermectin. Veterinary Record 160: 25-26. Paglia DT, Miller PE, Dubielzig RR (2004) James Wardrop and equine recurrent uveitis. Archives of ophthalmology 122:1218–1223. doi:10.1001/archopht.122.8.1218. Parrah JD, Buchoo BA, Moulvi BA (2004) Ocular filariasis in equines. A study of 9 cases. Centaur 4: 70-71. Patil DB, Parikh PV, Nisha J, Jhala SK, Din DMU,Tiwari DK (2012) Equine eye worm: a review of 50 cases. Indian Journal of Veterinary Surgery 33: 61-62. Perumal PC, Seneviratna P (1954) Kumri (Syn: Kamri) in horses associated with ocular setariasis with a short note on attempted treatment. Ceylon Veterinary Journal 2:92-94. Piyali DC, Debasish B (2012) Aqueous Extract of Human Placenta. In: Zheng J (Ed.), Recent Advances in Research on the Human Placenta, InTech Publisher ISBN: 978-953-510194-9. Pratap K, Amarpal A, Aithal HP, Pawde AM (2005) Survey of eye disorders in domestic animals. The Indian Journal of Animal Science 75:33–34. Radwan AM, Ahmed NE, Elakabawy LM, Ramadan MY, Elmadawy RS (2016) Prevalence and pathogenesis of some filarial nematodes infecting donkeys in Egypt. Veterinary World 9 : 888-892. doi: 10.14202/vetworld.2016.888-892. Razig SA (1989) A preliminary clinical trial on the use of diethylcarbamazine citrate for the treatment of equine filariasis. Acta Veterinaria (Belgrade) 38 : 145-151. Sathu S (1974) Intraocular parasites in horses. A report of five cases. Indian Veterianary Journal 5: 225. Sellon CD, Long M (2013) Equine Infectious Diseases. 2nd ed. Science Direct publication, St. Louis, MI.

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Sharma R, Shrivastava HK, Chauhan S, Kumar R (2005) Equine keratouveitis by Setaria spp. microfilaria and its management- A review of 14 cases. Intas Polivet 6: 260-261. Shin S, Cho K, Wee SH (2002) Ocular infection of cattle with Setaria digitata. Journal of Veterinary Medical Science 64: 710. Shukla AD, Kamath AT, Kudva A, Pai D, Patel N (2016) Our Experience in the Management of Traumatic Wound Myiasis: Report of 3 Cases and Review of the Literature. Case Reports in Dentistry. DOI:10.1155/2016/7030925. Singh H, Chaudhuri PC, Kumar A (1976) Paracentesis oculi: a preferred technique for removal of intra-ocular parasites in horses. Indian Veterinary Journal 53: 467-468. Skarda S (1996) Regional anaesthetic techniques In: Thurnon JC, Tranquilli WJ, Benson JG (Eds) Lumb and Jones’ Veterinary Anesthesia, 3rd edn., Lea and Febiger, Baltimore. pp 448-477. Slim MK, Fouad KA (1965) Incidence of equine filariasis in Egypt. Veterinary Medical Journal 10:113-118. Sreedavi C, Sudhakar K, Murthy PR, Prasad V (2002) Clinical microfilariasis in a horse:a case report. Indian Veterinary Journal 79: 487-488.

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Townsend WM (2013) Food and fiber-producing animal ophthalmology. In: Gelatt KN (Ed). Essentials of veterinary ophthalmology, 2nd ed., Wiley-Blackwell publication, UK, pp: 532. DOI: 10.1002/9781118910337 Tuntivanich N, Tiawsirisup S, Tuntivanich P (2011) Success of Anterior Chamber Paracentesis as a treatment for Ocular Setariasis in Equine Eye: Case Report. Journal of Equine Veterinary Science 31: 8-12. DOI: http://dx.doi.org/10.1016/j.jevs.2010.11.017. Vadalia JV (2013) Surgical Treatment of Ocular Setariosis in a Stallion. The Indian Journal of Veterinary Science 13:11. Varma AK, Sahai BN, Singh SP, Lakra P, Shrivastava VK (1971) On Setaria digitata, its specific characters, incidence and development in Aedes vittatus and Armigeres obturbans in India with a note on its ectopic occurrence. Zeitschrift für Parasitenkunde 36: 62-72. DOI: 10.1007/BF00328975. Yadav A, Kumar A, Bhadwal MS, Khajuria JK, Gupta A (2006) Ocular setariosis in horses: A case study. Journal of Veterinary Parasitology 20 : 1-10. Yang YJ, Cho YJ, Choi SK, Cho GJ (2014) Modified Needle Aspiration Technique for Extracting Live Eye Worm in a Thoroughbred Horse. Journal of Animal and Veterinary Advances 13: 998-1001. DOI: 10.3923/javaa.2014.998.1001.



ISSN No. 2320 – 8694

PARASITOLOGICAL, BIOCHEMICAL AND CLINICAL OBSERVATIONS IN PONIES EXPERIMENTALLY INFECTED WITH Trypanosoma evansi Yadav SC1,*, Jaideep Kumar2, Gupta AK1, Jerome A4, Prabhat Kumar3, Rajender Kumar1, Kanika Tehri3 and Ritesh Kumar3 1

ICAR-National Research Centre on Equines, Sirsa Road, Hisar, India Research Scholar, Guru Jambheshwar University of Science and Technology, Hisar, India 3 Research Associate, cJunior Research Fellow, ICAR-National Research Centre on Equines, Sirsa Road, Hisar, India 4 ICAR-Central Institute for Research on Buffaloes, Sirsa Road, Hisar, India 2

Received – October 25, 2016; Revision – November 08, 2016; Accepted – November 25, 2016 Available Online – December 04, 2016 DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S144.S150

KEYWORDS Trypanosoma evansi Ponies Surra Biochemical changes Clinical signs Haematology Parasitaemia

ABSTRACT The present investigation aimed to study the parasitological, biochemical and clinical alterations in ponies during the course of Trypanosoma evansi experimental infection. Six female ponies were experimentally infected sub-cutaneously with mice adapted 2x106 T. evansi parasites, isolated from naturally infected horse, while two ponies were maintained as uninfected healthy controls. All six ponies became parasitologically positive between 5-7 days post infection (DPI) tested by standard parasitological detection method (SPDM) by blood smear examination showing varying degree of parasitaemia and two prominent peaks during the course of infection. The main clinical signs observed were intermittent fever, weakness, emaciation, anaemia, anorexia and incoordination in hind quarters leading to significant weight loss at terminal stage of infection. All the infected ponies developed subacute to acute disease within 56 days and reached to recumbency stage. Of them, four ponies died at different stages of infection and few of them showing neurological signs at terminal stage of infection. The present investigation also revealed that horse ponies are more susceptible than donkeys in experimental infection of T. evansi. Haematological studies showed a gradual fall in the levels of haemoglobin (Hb), hematocrit (HCT) and red blood cell (RBC) count from 10.57 to 4.83 (g/dl), 32.81 to 16.33 (%) and 8.53 to 3.33 (x1012 cells/l) respectively, in infected animals over the study period. Serum

* Corresponding author E-mail: yadavsc@rediffmail com (S. C. Yadav) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.



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urea, uric acid, triglyceride, cholesterol, bilirubin indirect (BID) and total bilirubin (BIT) contents increased, while albumin contents significantly decreased in T. evansi infected ponies at different stages indicating impairment of liver and kidney functions. However, no changes in parasitological and biochemical responses were observed in the healthy controls. 1 Introduction Animal trypanosomosis is caused by different species of trypanosomes, including Trypanosoma vivax, T. evansi, T. congolense, T. simiae and T. equiperdum in different parts of the world. Of them, T. evansi is the most widely spread organism with the greatest range of hosts (Hoare, 1972), thus making it one of the most significant animal health problems in the world. T. evansi is transmitted mechanically and noncyclically by haematophagus flies such as horseflies (Tabanus) and stable flies (Stomoxys) which act both as vector and host reservoir. This parasite is responsible for the disease 'surra' in domestic as well as wild animals, causing severe constraints to agriculture development and leading to significant impact on livestock in Asia, sub-Saharan Africa, and Latin America. In India, trypanosomosis caused by T. evansi is enzootic, since it affects many species of domestic and wild animals. Several sporadic outbreaks of equine trypanosomosis have been reported from different states (Yadav et al., 2012; Kumar et al., 2013). The course of the disease lasts from one week to six months (Woo, 1977) and usually results in emaciation and death. The pre-patent period varies from 4 to 13 days and parasitaemia displays an undulating course (Ramirez et al., 1979). Anaemia is commonly found in horses, donkeys, dogs and coatis experimentally infected with T. evansi (Soodan et al., 1996; Marques et al., 2000; Aquino et al., 2002; Herrera et al., 2002). The disease manifests itself in different forms: acute, sub-acute, chronic and in-apparent. Clinical signs are only indicative of surra, which include progressive weakness, emaciation, fever, anaemia and death of affected animal. More recently rising trends of neurological cases due to T. evansi have also been reported, showing marked ataxia, hyperexcitability, circling, depression, gradual onset of paralysis of hind quarters (Rodrigues et al., 2009; Berlin et al., 2009; Ranjith kumar et al., 2013). The disease progresses in two phases; an acute phase, characterized by high levels of parasitaemia and noticeable clinical symptoms, and a chronic phase, characterized by low parasitaemia, which can either lead to emaciation or become clinically unapparent with undetectable changes in variables such as body temperature and haematocrit count (Fernández et al., 2009). The chronic form is most common and is likely to present an association with secondary infection due to immune-suppression caused by T. evansi infection (Ahmed, 2008). Some alterations in blood biochemistry, including decrease in blood albumin and increase in globulin levels, hypoglycemia and increase in icterus index, have been reported in donkeys and horses (Soodan et al., 1996; Marques et al., 2000). The aim _________________________________________________________


of the present study was to record the characteristic clinical course of the disease as well as parasitological, haematological and biochemical aspects in ponies experimentally infected with Indian isolate of T. evansi. 2 Material and Methods 2.1 Source and maintenance of T. evansi T. evansi was isolated at National Research Centre on Equines at Hisar, Haryana during 2009 from infected horse and was maintained in mice by in vivo propagation. Briefly, the blood was collected from the parasitological positive horse in ethylene diamine tetra acetic acid (EDTA) and injected intraperitoneally (i/p) in mice. The level of parasitaemia was checked microscopically by collecting blood daily from tail of the mice. After attaining parasitaemic peak, T. evansi isolate was maintained in the laboratory by inoculating the 104 parasites in naive mice, through i/p route. The parasites were also kept in cryopreserved form in liquid nitrogen for further use, as and when required. 2.2 Experimental infection in ponies Eight female ponies, aged 9-12 months, procured from local market, were divided in two groups (infected and healthy groups), comprising of six (P1 to P6) and two ponies (P7 & P8), respectively. All the animals were examined for the presence of helminth ova/oocyst and treated with albendazol@10 mg/kg body weight six weeks prior to experiment. Further these animals were also confirmed for abesence of T. evansi and Theilaria equi antibodies prior to infection by antibody ELISA. These animals were housed in fly proof stable and maintained throughout the experiment under intensive system of management. They were fed on balanced diet consisting of water and green fodder ad libitum, during the experiment. The experimental infection was set up in six ponies by inoculating 2×106 parasites sub-cutaneously (s/c) / pony. Another group of two ponies was kept as uninfected control during the course of experiment. The serum/blood samples were collected initially, at short intervals i.e. on day 0, 3, 5, 7, 10 and 14 from both groups. Thereafter, weekly blood samples were collected for parasitological, biochemical and clinical observations till 56 days post-infection. During the period, three ponies which became terminally ill reached in recumbent stage and were euthanized. The remaining three ponies at the end of experiment were treated with quinapyrimine sulphate @ 3 mg/kg body weight and monitored clinically after treatment.

Parasitological, Biochemical and Clinical observations in ponies experimentally infected with Trypanosoma evansi.

The animal experimentation was carried out according to the rules and regulations set forth by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) Animal Welfare Division, Ministry of Environment, Government of India. The research protocol for the experimentation was duly approved by the Institute Animal Ethics Committee of the National Research Centre on Equines, Hisar, Haryana.

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2.5 Statistical Analysis Statistical analysis was performed using SPSS (version 16), using repeated measures ANOVA. Data represented as mean I S E and considered significant at p < 0.05 3 Results 3.1 Parasitological observations

2.3 Parasitological observations During the course of experiment, the parasites were observed by wet blood smear examination and further counted in 50 microscopic fields at 400x magnification in thin blood smears stained with Giemsa (Cadioli et al., 2006). 2.4 Haematological and biochemical indices Erythrocyte count, packed cell volume and haemoglobin content were obtained from HM5 Vet Scan haematology analyzer (Abaxis, Pvt. Ltd, USA) as per standard procedure using the Vet Scan haematology kit, while biochemical metabolites related to liver and kidney functions mainly [Urea, Triglyceride (Tgl), Cholesterol (Chol), Albumin (Alb), Total Serum Protein (Prot), Bilirubin indirect (BID) and Total bilirubin (BIT), Creatinine (Cre), Uric acid (UA), Calcium (Cal)] were evaluated at different intervals in serum samples of both healthy and infected animals using XL system packs for each metabolite in a Clinical Chemistry Analyzer (ERBAEM200).

All six ponies inoculated sub-cutaneously with 2x106 T. evansi became parasitologically positive by 5-7 DPI as tested by standard parasitological detection method (SPDM) using wet/ thin blood smear examination. Parasitaemia was consistent in all ponies (low to moderate) and parasites were regularly observed in blood examination during the course of experiment except 10th and 35th DPI (only P3 showed high count). The first peak of parasitaemia occurred on day 7 with an average of 416.17 parasites followed by next peak by 28 DPI with an average parasite count of 324.67 (Figure. 1). It was interesting to note that all the individual ponies on day 10 had nil parasite count except pony P-3 which got high parasite count at 35 DPI. Thereafter, all the ponies showed relapsing or undulating parasitaemia ranging from only few trypanosomes in blood to maximum densities 1-6 x107 parasites / ml. Body temperature increased up to 102-107°F but was intermittent in nature throughout the experiment. The first rise in temperature occurred on 5th day after the appearance of trypanosomes in the blood, followed by a succession of peaks.

Figure 1 Parasite count in ponies infected with T. evansi at different intervals during infection. *Only pony P3 was recorded with high parasite count of 439* parasites at 35 DPI, while all other five infected ponies had nil count

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with unsteady and irregular steps. Ponies subjected to physical exertion readily fell down and were unable to support weight on the hind limbs by 56 DPI. The experiment was terminated at 61 DPI, as three ponies (P1, P2 & P5) died/ euthanized during 58-61 DPI while rest three of the debilitated ponies, which showed sub-acute disease (unable to stand, walk and anorexic) were treated with quinapyrimine sulphate (3 mg/kg body weight). After treatment, ponies were monitored for parasitaemia and found negative by 48 hrs using SPDM methods. These animals recovered well at 10 weeks post-treatment, showing normal haematological indices and maintained good health, except for pony- 4 that showed acute neurological signs, tilting of head, circling motion, hyper-excitability and was finally recumbent and euthanized. 3.3 Haematological observations Haematological studies showed that there was a continuous and sharp fall (p < 0.05) in the levels of haemoglobin (Hb), hematocrit (HCT) and red blood cell (RBC) count from 10.57 to 4.83 (g/dl), 32.81 to 16.33 (%) and 8.53 to 3.33 (x1012cells/l), respectively in infected animals (Figure. 2) while in healthy ponies, no significant change in these parameters was observed. Moreover, no clinical, parasitological and biochemical changes were detected in two uninfected control ponies. 3.4 Changes in biochemical indices

Figure 2 Changes in haemoglobin, RBC and hematocrit values in T. Evansi infected and uninfected ponies. 3.2 Clinical signs Apart from increased body temperature, there were few more symptoms of disease during the first 4 weeks of infection. The main clinical signs observed in most of ponies were intermittent fever, fast breathing, lacrimation from eyes and became anorexic by 6 DPI. All the infected ponies displayed progressive emaciation, jaundice, mucosal pallor and in few cases sub-mandibular oedema also. Besides this, motor incoordination of hind limbs was observed (pony 4 and 5), reduced appetite and lateral recumbency (pony 1, 2 & 5) while ponies 3 & 4 showed oedema in brisket, abdominal regions, staggering gait and incoordination in hind quarters were also observed during the course of infection. At terminal stage of disease, all the ponies were reluctant to move, revealed pronounced hind quarters weakness with ataxia and incoordination of the hind limbs, demonstrating staggering gait

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Blood urea contents ranged from 20.52 to 57.37 mg/dl and 14.65 to 22.05 mg/dl, while uric acid content ranged from 0.84 to 1.96 and 0.66 to 0.89 mg/dl in infected and healthy ponies, respectively (Figure 3). In infected ponies, urea content increased significantly 7 DPI onward and remained high throughout the experiment duration as compared to healthy ponies and zero days of infected ponies. Further a very sharp increase in this index was observed after 56 DPI in three ponies (P1, P2 and P5) which died on 61DPI. In rest of the three infected ponies (P3, P4 and P6), which survived on 61 DPI, urea content either remained static or decreased slowly. Uric acid content also showed a significant increase after 21 DPI and followed similar pattern between 56 to 61 DPI. No significant increase was observed in creatinine and calcium contents of infected ponies as compared to healthy ones. Triglyceride content showed a continuous increase in infected ponies 7 DPI. Further cholesterol, protein BID and BIT contents increased significantly about one to two weeks before death of infected ponies (P1, P2 and P5) while albumin content decreased sharply in later one as compared to healthy ponies. Among infected ponies, three ponies which remained alive, changes in levels of various metabolites were appreciably high between 7 to 35 DPI but during later stages (56 DPI onward), no significant increase was observed.


Figure 3 Changes in the level of various metabolites in T. evansi infected (I) and healthy (H) ponies during the study period.

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Conclusion and Discussion In the present study, six healthy ponies inoculated subcutaneously with T. evansi, became positive within 5-7 DPI as detected by wet blood smear examination, however, prepatent period has been reported to vary from 4 to 13 days and parasitaemia displays an undulating course during the infection (Ramirez et al., 1979). In present investigation parasitaemia was consistent, throughout the experiment and all ponies developed acute disease within 56 days post-infection. Three ponies died exhibiting symptoms of acute trypanosomosis and fourth died due to neurological signs which appeared 10 weeks post-treatment with quinapyramine sulphate. Contrary to above observations, the experimentally infected donkeys showed comparatively low parasitaemia and the disease persisted years together in sub-clinical stage without mortality (Kumar et al., 2013). These observations clearly indicated that ponies are susceptible of T. evansi infection, while donkeys are resistant with a long course of disease. The parasite count, body temperature and blood indices of the infected ponies underwent significant changes. The high temperature was observed in all infected ponies 8-15 DPI and was intermittent throughout the experimental period and there was no definitive correlation with peak parasitaemia. The increase in the temperature of infected animals is a characteristic that has been previously described and related to the waves of parasitaemia in experimental infections with T. evansi (Oshiro et al., 1989; Uche & Jones 1992; Aquino et al., 1999; Marques et al., 2000; Dargantes et al., 2005). Thereafter, similar observations of intermittent fever, weakness, emaciation, anaemia, anorexia and incoordination in hind quarters were recorded as reported in experimental infection in equines (Marques et al., 2000; Wernery et al., 2001). Motorial disturbance, a frequent symptom of equine Surra, usually reported to affect mainly the hind legs (Curasson, 1943; Horchner et al., 1983) was also observed during this investigation. The alterations in the haematological indices observed during the course of infection are consistent with the findings of previous workers in horses infected with T. evansi, wherein fall in haematocrit, erythrocyte counts and haemoglobin content was described. Anaemia has been a consistent finding in the infected animals throughout the study period. Marques et al. (2000) reported about 35% decrease in red cells count, packed cell volume and haemoglobin concentration in the experimentally infected horses in three weeks of infection and thereafter small variations were observed. However, the animals remained anaemic until the end of observation period. Despite being a significant feature of trypanosomosis, the origin of anaemia in surra is not completely elucidated. Evidences suggest that its aetiology is multifactorial and haemolysis, haemodilution or/ and noncompensatory erythropoiesis are some of the mechanisms proposed (Jenkins & Facer 1985). In conformity with our findings, death of T. evansi infected animals without clear previous indications has been also reported earlier (Horchner et al., 1983). _________________________________________________________


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Among biochemical indices, both liver and kidney functions were observed to be affected in T evansi infected ponies as their serum urea, uric acid, triglyceride, cholesterol, albumin, BID, BIT contents varied and increased significantly at different stages during experimental period. Similar observations mainly in terms of serum albumin and globulin have also been reported in donkeys (Cadioli et al., 2006). Acknowledgements The authors are thankful to Director, National Research Centre on Equines, Hisar for providing facilities to conduct the work. The authors also wish to acknowledge Department of Biotechnology (DBT), Government of India for partial funding through research Grant no. BT/PRi4499/ADV/57/107/2010 in terms of manpower support. The authors also wish to acknowledge and thank Mr. R.K. Dayal for technical support during the investigation. Conflict of interest statement All authors disclose that they have no financial and personal relationships with other people or organization that could inappropriately influence (bias) their work, including employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registration, and grants or other funding. References Ahmed A (2008) Epidemiological studies (parasitological, serological and molecular techniques) of T. evansi infection in camels in Egypt. Veterinary World Journal 1: 325-328. Aquino LPCT, Machado RZ, Alessi AC, Marques LC, de Castro MB, Malheiros EB (1999) Clinical, parasitological and immunological aspects of experimental infection with Trypanosoma evansi in dogs. Memorias do Instituto Oswaldo Cruz 94: 255-260. Aquino LPCT, Machado RZ, Alessi AC, Santana AE, Castro MB, Marques LC, Malheiros EB (2002) Hematological, biochemical and anatomopathological aspects of experimental infection with Trypanosoma evansi in dogs. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 54: 8-18. Berlin D, Loeb E, Baneth G (2009) Disseminated central nervous system disease caused by Trypanosoma evansi in a horse. Veterinary Parasitology 161: 316-319. Cadioli FA, Marques LC, Machado RZ, Alessi AC, Aquino LPCT, Barnabé PA (2006) Experimental Trypanosoma evansi infection in donkeys: hematological, biochemical and histopathological changes. Arquivo Brasileiro de Medicina Veterinária e Zootecnia 58: 749-756.


Curasson G (1943) Trypanosoma vivax et variétés. In Traité de protozoologie vétérinaire et comparée Tome 1 Trypanosomes. Paris: Vigot Frères : 270-278. Dargantes AP, Reid SA, Copeman DB (2005) Experimental Trypanosoma evansi Infection in the Goat. I. Clinical Signs and Clinical Pathology. Journal of Comparative Pathology 133: 261-266. Fernández D, González-Baradat B, Eleizalde M, GonzálezMarcano E, Perrone T, Mendoza M (2009) Trypanosoma evansi: A comparison of PCR and parasitological diagnostic tests in experimentally infected mice. Experimental Parasitology 121: 1-7. Herrera HM, Alessi AC, Marques LC, Santana AE, Aquino LP, Menezes RF, Moraes MA, Machado RZ (2002) Trypanosoma evansi experimental infection in the South American coati (Nausa nausa): hematological, biochemical and histopatological changes. Acta Tropica 81: 203-210. Hoare CA (1972) The Trypanosomes of Mammals: A Zoological Monograph. Blackwell Scientific Publications. Oxford, UK. Horchner F, Schonefeld A, Wust B (1983) Experimental infection of horses with Trypanosoma evansi I. Parasitological and clinical results. Annales de la Societe Belge de Medecine Tropicale 63: 127-135. Jenkins GC, Facer CA (1985) Hematology of African trypanosomes. In Tizard, I. Immunology and Pathogenesis of Trypanosomiasis. CRC Press, Boca Raton pp13-44. Kumar R, Kumar S, Khurana SK, Yadav SC (2013) Development of an antibody-ELISA for seroprevalence of Trypanosoma evansi in equids of North and North-western regions of India. Veterinary Parasitology 196: 251-257. Marques LC, Machado RZ, Alessi AC, Aquino LPCT, Pereira GT (2000) Experimental infection with Trypanosoma evansi in horses: clinical and haematological observations. Brazil Journal of Veterinary Parasitology 9: 11-15.

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Oshiro ET, Rodrigues M, Nunes VLB, Ribeiro OC (1989). Trypanosoma (Trypanozoon) evansi (Steel, 1885) Balbiani, 1888, infecco experimental emequino com amostraisolada de capivara, Hydrochaeris hidrochaeris Linnaeus, 1766 (Rodentia: hydrochacridae). Semina 10: 51-55 Ramirez LE, Wells EA, Betancourt A (1979) La Tripanosomiases en los Animales Domisticos en Clolumbia. Centro Internacional de Agricultura Tropical pp 71. Ranjithkumar M, Saravanan BC, Yadav SC, Kumar R, Singh R, Malik TA, Dey S (2013) Neurological trypanosomiasis in quinapyramine sulfate-treated horses – A breach of the bloodbrain barrier? Tropical Animal Health and Production 46: 371377. Rodrigues A, Fighera A, Souza TM, Schild AL, Barros CSL (2009) Neuropathology of Naturally Occurring Trypanosoma evansi Infection of Horses. Veterinary Pathology 46: 251-258. Soodan JS, Sood NK, Khahra SS (1996) Clinic-pathological studies in donkeys experimentally infected with Trypanosoma evansi. Indian Journal of Animal Science 66: 443-448. Uche UE, Jones TW (1992) Pathology of experimental Trypanosoma evansi infection in rabbits. Journal of Comparative Pathology 106: 299-309. Wernery U, Zachariah R, Mumford JA, Luckins T (2001) Preliminary evaluation of diagnostic tests using horses experimentally infected with Trypanosoma evansi. The Veterinary Journal 161: 287–300. Woo PTK (1977) 7-Salivarian trypanosomes producing disease in livestock outside of sub-saharan Africa. In: Julius Kreir (Ed) Taxonomy Kinetoplastids and Flagellates of Fish. Academic Press, New York, pp 269-296. Yadav SC, Kumar R, Manuja A, Goyal L, Gupta AK (2012) Early detection of Trypanosoma evansi infection and monitoring of antibody levels by ELISA following treatment. Journal of Parasitic Disease 38 : 124-127.



ISSN No. 2320 – 8694

EQUINE BRUCELLOSIS: REVIEW ON EPIDEMIOLOGY, PATHOGENESIS, CLINICAL SIGNS, PREVENTION AND CONTROL Kumaragurubaran Karthik1,*, Govinthasamy Prabakar2, Ramasamy Bharathi1, Sandip Kumar Khurana3 and Kuldeep Dhama2 1

Tamil Nadu Veterinary and Animal Sciences University, Chennai- 51, India Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P., India 3 NRCE, Hisar, Haryana, India 2

Received – November 03, 2016; Revision – November 25, 2016; Accepted – December 02, 2016 Available Online – December 04, 2016 DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S151.S160

KEYWORDS

ABSTRACT

Brucellosis

Brucellosis is one of the major zoonotic diseases that affect several domestic animals, wild animals and also marine mammals. Though there is no specific Brucella sp. that can affect horses, B. abortus and B. suis can affect horses naturally and B. canis experimental infection has also been reported in equines. Brucellosis in equines is characterized by two conditions namely Poll evil and fistulous withers. Organism has its predilection for joints, ligaments and tendons in case of equines and causes inflammatory conditions leading to formation of fistula. Equine brucellosis has been documented from several parts of the world and prevalence has been reported time to time mostly based on serological diagnosis. Diagnosis of brucellosis mainly depends on serological methods though isolation of the organism is the gold standard. Due to hazardous nature of the pathogen, tests like Rose Bengal plate agglutination test, Standard tube agglutination test and other serological assays are commonly employed. Isothermal amplification assay like LAMP are gaining momentum these years due to swiftness in diagnosis of the pathogen. LAMP with high specificity and sensitivity for detection of Brucella spp. and also B. abortus has been developed in the recent years. Prevention and control of brucellosis is of utmost important to halt the spread of the organism to other animals and human. Trauma is a major reason for predisposition of poll evil and fistulous withers hence proper fitting of saddle will help to prevent the disease. Housing and feeding the horses separately can prevent spread of disease from cattle. The present review discusses equine brucellosis, its epidemiology, pathogenesis, clinical signs along with appropriate prevention and control strategies to be adapted.

Equine B. abortus Poll evil Fistulous withers LAMP

* Corresponding author E-mail: [email protected] (Kumaragurubaran Karthik) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.



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1 Introduction Brucellosis is a major zoonotic disease caused by the bacterium of the genus Brucella of which several species has been listed to cause disease in domestic and wild animals. History shows that the disease was first identified in Malta in the year 1887 by Sir David Bruce who isolated Brucella melitensis, the then Micrococcus melitensis from a soldier died due to Maltese fever (Godfroid et al., 2005). Recent years has seen this bacterium as a pathogen causing disease in sea mammals thus showing the long journey of this pathogen from Malta to Marine. There are eleven Brucella species namely B. abortus (cattle), B. melitensis (sheep and goats), B. ovis (sheep), B. suis (hogs), B. canis (dogs), B. neotomae (wood rats), B. microti, B. maris, B. ceti and B. pinnipedialis (marine mammals) and B. inopinata (isolated recently from women breast implant) (Verger et al., 1987; Foster et al., 2007; Scholz et al., 2008). These eleven Brucella spp. do not follow the strict rule towards host specificity as they can infect other animals and also human beings hence this pathogen is considered as an important zoonotic organism. B. abortus and B. suis affects wild animals and reports regarding B. melitensis infection in wild animals are less (Rhyan, 2000; Godfroid & Kasbohrer, 2002). There is no specific Brucella sp. that can cause disease particularly in horses. All the documented reports of brucellosis in horse are mainly by the cattle pathogen, B. abortus and to a milder extent B. suis biovar 3 (Lucero et al., 2008; Colavita et al., 2016). Members of the genus Brucella are facultative intracellular gram negative cocco-bacilli or short rods measuring around 0.6 to 1.5 μm long and 0.5 to 0.7 μm width. Brucella genus neither form capsules nor spores nor motile though carry the genes for motility (except the chemotactic system that helps in assembling a functional flagellum) (Fretin et al., 2005). Brucella genus is classified phylogenetically within the α2 subdivision of Proteobacteria which comprises of Agrobacterium, Bartonella, Ochrobactrum, Rhizobium, Rhodobacter, and Rickettsia (Moreno et al., 1990). Genome of B. abortus has two circular chromosomes of 2.1 Mb and 1.5 Mb size without plasmids (Michaux et al., 1993; MichauxCharachon et al., 1997). Recent years has seen the completion of genome of B. abortus (Sanchez et al., 2001), B. melitensis (GenBank NC 003317 and NC 003318) (DelVecchio et al., 2002) and B. suis (GenBank NC 002969) thus increasing the opportunity for understanding the pathogenicity of Brucella. Brucellosis mainly causes reproductive diseases in various animals, and in horses the clinical manifestation is termed as "poll-evil" or "fistulous withers" due to the inflammation of supraspinous bursa and connective tissue, leading to abscess formation and fistulation in the affected region and occasionally abortions and other reproductive problems are also reported (Denny, 1972). B. abortus has been reported worldwide causing infection in domestic animals though some _________________________________________________________


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countries like Australia, Canada, Japan, Israel and New Zealand have controlled the disease (Nicoletti, 2007). Reports shows that age, breed and sex specificity does not exist for brucellosis in equine though more cases are documented in horses above 3 years of age (Nicoletti, 2007). 2 Epidemiology Three species of Brucella namely B. abortus, B. suis and B. canis have been incriminated to infect horses, of which B. abortus and B. suis have been reported with natural infection and B. canis infection has been reported with experimental infection (Hagler et al., 1982). Brucella antibodies have been reported from several parts of the world at various time frames showing that horses get infected from other animals. A study in Brazil was conducted where 123 crossbred cart horse serum samples were collected from a period April 2005 to June 2006. These samples were subjected to Rose Bengal Plate agglutination Test (RBPT) which showed eight animals (6.5%) to be positive for brucella antibodies. This study did not report isolation of the pathogen and the seropositive animals did not show any clinical manifestation of the disease (Antunes et al., 2013). A multispecies farm in Nigeria comprising of different livestock species like cattle, horse, goat and sheep was investigated for B. abortus infection status. This study revealed that all the seven horses under the study were found positive by RBPT. Vaginal samples from 6 horses were subjected for isolation of B. abortus which showed that two samples were positive for B. abortus. This study is the first report of isolation of the pathogen from non clinical cases from horse (Bertu et al., 2015). The report of this study warrants attention as disease free horses can also play role in transmission of the pathogen to other animals and also to human. Several other studies have been conducted earlier in Nigeria to investigate the presence of brucellosis in horses which documented 8.4% (14 positive of 166 animals) and 14.7% (11 samples out of 75 horses) which were lesser compared to the recent survey documenting 100% positivity (Bale & Kwanashie, 1984; Ehizibolo et al., 2011). Multispecies housing can increase the possibility of disease spread. Already there are established reports that horse to horse or other animals is a less likely event and these animals do not excrete the organism (Corbel & Henry, 1983; Macmillan & Cockrem, 1985). Sadiq et al. (2013) reported 5.5% seropositivity of brucellosis by RBPT and Microtiter Serum Agglutination Test in donkeys from Borno and Yobe states of Nigeria. A very recent study conducted on the Mambilla plateau of Taraba state, Nigeria showed 16% prevalence of brucellosis and adult horses were affected more that young animals (Ardo & Abubakar, 2016). Another study conducted in the same Taraba state of Jalingo region showed 7 animals out of 90 adult horses screened by RBPT (Ardo et al., 2016).

Equine Brucellosis: Review on epidemiology, pathogenesis, clinical signs, prevention and control.

Studies conducted in Iran also reported isolation of B. abortus from infected horses and serological diagnosis by RBPT, standard tube agglutination test (STAT) and 2-mercapto ethanol (2ME) showed 2.5% to 12% prevalence (Tahamtan et al., 2010). Another study in the Hamadan region of Iran showed 0.5% prevalence by RBPT and STAT (Ghobadi & Salehi, 2013). Positive animal in this study was also in close contact with other animals hence there was a higher chance of infection. Earlier study in the Mashhad region of Iran also showed 2.5% positive cases (Tahamtan et al., 2010). An epidemiological survey was conducted in Peshawar district of Pakistan employing 500 serum samples from horses (n= 196), donkeys (n= 267) and mules (n= 37) collected during a time period of January to December, 2012. Higher prevalence (71.93%) was reported in horses than donkeys (63.67%) and mules (5.4%). Study also reported females and animals of 5-11 years were affected most (Safirullah et al., 2014). Younger animals tend to clear the pathogen faster though there is possibility of latent infection which may be the reason that the study had higher infection rate among older animals (Quinn et al., 2004). Sex hormone and erythritol tend to increase as the age advances and this may be another reason brucella infection is seen in older animals after sexual maturity (Radostits et al., 2000). Serum samples of 227 equines (178 donkeys, 43 horses and 6 mules) collected in the Mossoró, Rio Grande do Norte, Brazil were subjected to RBPT, STAT and 2 ME showed 1.76% prevalence of brucellosis in horse serum (Dorneles et al., 2013). A massive survey was conducted recently employing serum samples of 6,439 animals comprising samples from 5,292 horses, 110 donkeys and 1,037 mules from 1936 herds of Minas Gerais State, Brazil over a period of September 2003 and March 2004 (Junqueira et al., 2015). RBPT and STAT was performed which showed that 70 horses out of 5,292 animals to be positive (1.32%), 1 out of 110 donkeys (0.91%) and 14 out of 1,037 mules (1.35%). This study documented that males (52 out of 4106 males) had higher sero-positivity compared to female (33 out of 2333 females) (Junqueira et al., 2015). A study in Western Sudan employing serum samples from 346 horses and 28 donkeys showed 4.9% and 3.6% prevalence of brucellosis in horses and donkey (Musa, 2004). A total of 1954 serum samples (horses 782 and donkeys 1172) were collected from Sanliurfa and Diyarbakir provinces of South-East Turkey and were subjected to RBPT. Prevalence was higher in horses (13.68%) than donkeys (6.05%) (Tel et al., 2011). Several other workers have earlier studied the prevalence of brucellosis in horse in various regions of Turkey. Solmaz et al. (2004) reported 60.59% prevalence of brucellosis by RBPT in horses in the Van province of Turkey. Göz et al. (2007) reported 9.5% horses to be positive by STAT in Hakkari region of Turkey. Brucella organism is to an extent resilient which has been recovered from manure and fetal samples even after 2 months when kept in cool weather condition. Exposure to sunlight for

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few hours kills the bacteria and they are also readily killed by commonly used disinfectants (Glynn & Lynn 2008). 3 Pathogenesis The major route of entry of the pathogen into the host (equines) is through the consumption of feed contaminated with B. abortus and contact infection from cattle has been reported by several workers (Denny 1973; O’Sullivan 1981; Ocholi et al., 2004). There prevails mystery regarding the role played by horse in the transmission of infection to other horses or to other animals/ humans. Hence there is no clear evidence that supports that horse’s act as a ware house/ reservoir for transmission of brucellosis in endemic areas (Acosta-Gonzalez et al., 2006). Being a facultative intracellular pathogen, Brucella survives, multiplies and evades host immune mechanism simultaneously found developing inside phagocytic cells (Gorvel & Moreno, 2006). Ecological niche inside the phagosomal compartment of host macrophages is conducive for the survival of brucellae and maintaining chronic infections depends upon the ability of surviving and replicating within these phagocytic cells (Roop et al., 2004; Neta et al., 2010) (Figure 1). Brucella infection occurs through ingestion and the organism enters the oral and pharyngeal cavities (Brinley et al., 1990). The bacteria are transported following penetration of the mucosal epithelium either free or within phagocytic cells to the regional lymph nodes through macrophages leading to spread and multiplication of organism in lymph nodes, spleen, liver, bone marrow, reproductive organs, tendon sheath and bursae occur (Canning et al., 1986; Riley et al., 1984; Memish et al., 2000; Adams, 2002). The capacity of Brucella to hide inside the macrophages makes it difficult to diagnose the disease and also hinder in the treatment of the disease (Glynn & Lynn 2008). 4 Clinical signs In equines, the clinical signs due to brucellosis are mostly noticed in the musculoskeletal system mainly as the organism localise in the bursae (causing septic bursitis), joints (causing septic arthritis) and tendon sheaths (causing septic tenosynovitis) (Denny, 1972, Denny,1973; Carrigan et al., 1987; Ocholi et al., 2004). Few reports regarding abortion, vertebral osteomyelitis and infertility in male horses have also been documented (Collins et al., 1971; Denny 1973). Most classical clinical signs observed in horses due to brucellosis are poll evil (septic supra-atlantal bursitis) and fistulous withers (septic supraspinatous bursitis). Draining sinuses are seen as these problems are chronic in case of equines (Crawford et al., 1990). An experimental infection of B. abortus instillation into the conjunctival sac was carried out in a horse to know the clinical signs. Serum antibody level was evident after 7 to 12 days of infection and intermittent bacteraemia was observed for 2 months (MacMillan et al., 1982).

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Figure 1 Pathogenesis and clinical signs of equine brucellosis. Lesions are of granulomatous type were observed in lung, liver, testes and metatarsophalangeal synovial membranes (Megid et al., 2010). No apparent clinical signs were noted and female animals bred normally yielding negative results to isolation of organism (MacMillan & Cockrem, 1986). B. abortus induced abortion in equines are not common though some documented reports state that mid to late term abortion may occur (McNutt & Murray 1924; McCaughey & Kerr 1967; Shortridge, 1967; Robertson et al., 1973; Hinton et al., 1977). Organism in the vaginal excretions does not last longer as compared to cattle. Some authors reported the isolation of B. suis from aborted equine fetuses (McNutt & Murray, 1924), horses affected with septic bursitis (Portugal et al., 1971) and also from the reproductive organs of mares without apparent clinical signs (Cvetnic et al., 2005). 5 Diagnosis The gold standard test for diagnosis of Brucellosis is isolation and identification of the organism which needs 5-10% carbon dioxide for its growth. Even with all the conditions conducive for the growth of Brucella, it will take around 3-5 days for a _________________________________________________________


colony to appear. At times it may even take longer; hence culturing work is laborious and time consuming. The major setback in culturing is that Brucellosis is zoonotic and the handlers are always at the risk of infection (Karthik et al., 2014a). Hence it needs level 3 biocontainment facilities and highly skilled technical personnel for handling live culture (Alton et al., 1988). Serological tests like Rose Bengal Plate Test (RBPT), Standard Tube Agglutination Test (STAT) and Enzyme Linked Immunosorbent Assay (ELISA) are commonly employed (Nicoletti, 2007). Other serological tests include complement fixation test (CFTs), 2-mercaptoethanol (2ME), buffered Brucella antigen tests (BBAT), Milk ring test (MRT), etc. (Acha & Szyfres, 2003; Godfroid et al., 2010). These tests are inexpensive, fast and sensitive but not necessarily highly specific, antibodies may cross react with Yersinia enterocolitica serotype O:9, Escherichia coli O: 157, Francisella tularensis, Salmonella urbana O: 30, Vibrio cholerae, and others (Radostits et al., 2000). Molecular techniques like PCR has been employed with various samples like blood, serum tissues from aborted foetus, semen and milk for diagnosis of brucellosis (Fekete et al.,


1992; Leal-Klevezas et al., 1995; Queipo-Ortuno et al., 1997; Amin et al., 2001; Kanani, 2007). More recently, real-time PCR has been used for detection of Brucella, offering improvement in detection times and specificity (QueipoOrtuno et al., 2005). Real-time PCR is the latest method used in which hybridization probes are used to increase specificity (Bricker, 2002; Probert et al., 2004; Elfaki et al., 2005). Isothermal amplification assay has the advantage of employing a set temperature for amplification of DNA, reducing the time for amplification and also there is no need for post amplification protocol for result visualization (Dhama et al., 2013). LAMP for the Brucella was developed against Brucella cell surface protein (bcsp)31 gene and omp25 gene and sensitivity of both LAMP assay was higher than PCR (Ohtsuki et al., 2008; Lin et al., 2011). LAMP with visual detection based on calcein has also been developed targeting the same omp25 (Pan et al., 2011). Reports regarding development of LAMP targeting IS711 genes and real time quantitative LAMP are available (Pérez-Sancho et al., 2013). Visual LAMP targeting omp25 gene including loop primers was developed recently that can detect all Brucella spp. Developed LAMP assay with loop primers was 10 fold more sensitive than commonly employed PCR (Karthik et al., 2016). LAMP assay has the intrinsic property of product carry over contamination hence a novel closed tube LAMP assay was also developed for detection of Brucella spp. (Karthik et al., 2014b). LAMP assay for specific diagnosis of B. abortus was also developed which was 100 fold more sensitive than the commonly employed PCR (Karthik et al., 2014c). Severity of bone damage in cases of poll evil and fistulous withers can be identified by radiography.

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material gets discharged through one or more opening. These fistulas may heal slowly and there are always chances that they reappear (Cohen et al., 1992). Few years back researchers have found an Iron Age horse cranium from Tuva Republic, Central Asia and the cranium had occipital lesions, cavities around the nuchal ligament attachment site was noted in the skull which may be due to inflammation followed by necrosis (Bendrey et al., 2011). The researchers concluded that condition is poll evil originating due to bacterial cause. Authors discussed that this case may be due to B. abortus the common cause of poll evil in horses. Thus this study shows that the pathogen existed long back causing poll evil in horses (Bendrey et al., 2011). Drainage of the infected tissue (poll evil and fistulous withers) and treatment with systemic antibiotics can be employed to treat brucellosis in horse. Chloramphenicol, tetracyclines, streptomycin and some sulphonamides are commonly used for treatment of Brucellosis but these antimicrobials cannot penetrate the infected tissues (Nicoletti, 2007). Clofazimine has been reported to have good effect in the treatment of brucellosis in equines (Knottenbelt et al., 1989). B. abortus S19 vaccine has also be used with good effect for treatment of brucellosis with the regimen ranging from one dose to three doses at 10 days interval (Denny, 1973; Gardner et al., 1983; Cohen et al., 1992; Nicoletti, 2007). Use of this vaccine for treatment also involves local and systemic reactions and death has been reported in a horse that received intra venous injection (Denny, 1973; Cohen et al., 1992). Periodical drainage, cleaning the region with antiseptics and dimethyl sulphoxide will aid to control further complications of poll evil and fistulous withers (Cohen et al., 1992). 7 Prevention and control

6 Poll evil and fistulous withers a. Poll evil and fistulous withers are chronic inflammatory conditions affecting supra-atlantal bursa and supraspinatus bursa and its associated tissues respectively (Gaughan et al., 1988; Rashmir-Raven et al., 1990; Cohen et al., 1992). The term fistula refers to draining wound from a normally closed structure, through the skin and fistulous withers is an infection of the bursa overlying the spines of the withers by usually caused by Brucella sp. in horse. Though B. abortus has been incriminated as the major cause for this condition, there are other pathogens/ wound which also plays role in causing this condition in horses. Other pathogens like Streptococcus zooepidemicus, Streptococcus equi, Staphylococcus aureus, Staphylococcus epidermidis, Corynebacterium spp. Actinomyces bovis, Bacteroides fragilis, Proteus mirablis, Escherichia coli and Pasteurella spp. have also been isolated from horses suffering from fistulous withers (Cohen et al., 1992; Hawkins & Fessler, 2000). Signs of fistulous withers include single or multiple draining tracts and in some case disseminated swelling in the wither region without drainage. Walls of the bursa gets thickened carrying clear, thick fluid which is of straw coloured (Megid et al., 2010). Initial signs include pain, swelling, heat at the bursal region leading to stiffness of the neck. Later stages the bursa ruptures and pus _________________________________________________________


b.

c.

d.

In most of the instances horse gets brucellosis when they are housed or allowed for grazing together with cattle. Hence horse should be housed or allowed for grazing away from cattle suspected for brucellosis (Cramlet & Bernhanu, 1979). Trauma is a major cause of predisposition of fistulous withers in horses hence proper fitting of saddle has to be taken care. Parasitic problem Onchocerca spp. can also cause fistulous withers hence minimizing the parasitic load by proper hygienic measures needs to be practiced (Cramlet & Bernhanu, 1979). Many of the countries follow testing and quarantine the horses or euthanize the horse since brucellosis is zoonotic. Hence measures needs to be followed for early diagnosis of the disease so that horses can be segregated or culled to prevent further spread of the disease.

8 Conclusion Brucellosis, an age old disease is one of the important zoonotic disease that can infect several domestic animals, wild animals and also marine mammals. Eleven brucella species have been

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described till date of which many are host restricted though it can infect other animals and also human. There is no single species of brucella that can cause brucellosis in equines. B. abortus and B. suis have been implicated to cause natural disease in equines. Mostly brucellosis in domestic animals are oriented to reproductive tract infection but in equines it causes mainly two important conditions affecting the musculoskeletal system termed as Poll evil and Fistulous withers. Abortions are also noticed in some cases but shedding of bacteria through the vaginal discharge is not documented yet. Though the disease has been reported for several years in horses there are still several questions that need to be clarified like the pathogenesis of bacterium in horse, predilection of bacterium towards musculoskeletal system in horse while mainly reproductive tract in other domestic animals. Diagnosis of brucellosis at the early stage is important to identify the diseased animals so that prior segregation of the animals can be done to minimize its spread to other animals. Advances in the field of diagnostics have made it possible to identify the agent early though proper sampling procedures needs to be practiced to achieve the same. Similarly treatment and vaccination aspects needs to be strengthened in order to control and eliminate the disease from the animal population so that its spread to humans can also be prevented. Conflict of interest Authors would hereby like to declare that there is no conflict of interests that could possibly arise. References Acha NP, Szyfres B (2003) Zoonoses and Communicable Diseases Common toe Man and Animals, third ed. Vol.1 Pan American Health Organization (PAHO), Washington, DC. Acosta-Gonzalez RI, Gonzalez-Reyes I, Flores-Gutierrez GH (2006) Prevalence of Brucella abortus antibodies in equines of a tropical region of Mexico. Canadian Journal of veterinary Research 70: 302-304. Adams LG (2002) The pathology of brucellosis reflects the outcome of the battle between the host genome and the Brucella genome. Veterinary Microbiology 90 : 553-61. DOI: 10.1016/S0378-1135(02)00235-3 Alton GG, Jones LM, Angus RD, Verger JM (1988) Techniques for the Brucellosis Laboratory. Institute National de la Recherche Agronomique, Paris, France. INRA. ISBN 27380-0042-8. Amin AS, Hamdy ME, Ibrahim AK (2001) Detection of Brucella melitensis in semen using the polymerase chain reaction assay. Veterinary Microbiology 83: 37-44. DOI: 10.1016/S0378-1135(01)00401-1 Antunes JMAP, Allendorf SD, Appolinário CM, Peres MG, Perotta JH, Neves TB, Deconto I, Filho IRB, Biondo AW, _________________________________________________________


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ISSN No. 2320 – 8694

BIOTECHNOLOGICAL TOOLS FOR DIAGNOSIS OF EQUINE INFECTIOUS DISEASES Minakshi Prasad1,*, Basanti Brar1, Ikbal1, Koushlesh Ranjan2, Upendra Lalmbe1, J. Manimegalai1, Bhavya Vashisht1, Sandip Kumar Khurana4 and Gaya Prasad3 1

Department of Animal Biotechnology, LLR University of Veterinary and Animal Sciences, Hisar, Haryana, India, 125004 Department of Veterinary Physiology and Biochemistry, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, India, 250110 3 Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, Uttar Pradesh, India, 250110 4 NRCE, Hisar, Haryana, India, 125001 2


KEYWORDS Biotechnology Immunoassay Equine Infectious disease

ABSTRACT Rapid diagnosis of infectious diseases and appropriate treatment with in time are important steps that promote optimal clinical outcomes and general public health. Today there is large number of new technologies such as nanotechnology, biosensors, and microarray techniques, are being developed and used as diagnostic tools for equine infectious diseases. Nucleic acid based techniques such as polymerase chain reaction (PCR) have become conventional tools in veterinary research and plays an important role in specific typing determinations as well as for rapid screening of ample numbers of samples at the time of equine disease outbreaks. Other biotechnological techniques are populous to be used in the coming times as they can enhance diagnostic efficacy in less time and cost as compared to conventional techniques. This review focuses on biotechnological tools available for equine diseases diagnosis and its applications hold great promise for improving the speed and accuracy of diagnostics for equine infectious diseases.

* Corresponding author E-mail: [email protected] (Minakshi Prasad) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.



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1 Introduction

2 Serological assays

Modern molecular biology provides us newer technology to diagnose and control several equine diseases. It is also used for the development of novel diagnostic tools for equine infectious disease control (Pusterla et al., 2006; Amaya, 2014). Several diagnostic tools such as nucleic acid probes, monoclonal antibodies, restriction fragment length polymorphisms, real time PCR, proteomics, biosensors and nanotechnology have increased the livestock productivity. These methods have been commonly used for equine disease diagnosis and control (Yeh et al., 2010; Johnson et al., 2010; Rakhshandehroo et al., 2014). Several viral and bacterial pathogens such as Japanese encephalitis virus, West Nile virus, Hendra virus, borna virus, equine rabies, Rhodococcus equi, Bacillus anthracis etc., are causes several serious diseases in equines and induce economic to human population and these are zoonotic in nature (Yeh et al., 2010; Booth et al., 2010; Priestnall et al., 2011; Khurana, 2015).

Protein based assays are based on antibody and antigen interaction. These types of several assays such as enzyme linked immunosorbent assay (ELISA), falcon assay screening test –ELISA, indirect or direct immunofluroscencent antibody tests, immunoblotting dot-ELISA, peptide based-ELISA, complement fixation test, agar gel immunodiffusion and neutralization test are used for equine infectious disease diagnosis. These serological assays are highly sensitive and specific than other techniques like microscopy and it allow clearance of post-therapeutic pathogen.

On various occasion equines are used for various purposes such as ceremonies, riding, sports, draught racing, transport and antitoxin/antibody production, throughout the world (Burnouf et al., 2004). There is possibility of disease transmission and spread at the time of equines movement from one country to another. Therefore, OIE (World Organisation for Animal Health) has enlisted several diagnostic tests for international movement of equines (Table 1) (OIE, 2016). Biotechnology may play an important role in prevention of disease caused by these pathogens. The correct knowledge of molecular biology of infectious agents and their hosts is very important for controlling the disease (Tavares et al., 2011). Biotechnological and protein based assays can play a main role in equine disease control due to its everlasting developments with the use of developed anti pathogenic drugs and diagnostic chemicals. Even though conventional techniques are still used commonly, recent biotechnological assays have widened the scope of equine diseases detection and give us powerful new techniques for quick and specific identification of equine diseases. This manuscript reviews the current and potential uses of biotechnology tools for equine infectious disease diagnostics.

2.1 Enzyme-linked immunosorbant assay (ELISA) Components of immune system used for detection of immune response against infection in ELISA test. For detection of specific immune response, ELISA assay involves antigen, antibody and enzymes. The antigens are adhered to surface of microtitre plate and antibody specific to the antigen is applied over the surface for binding. It was followed by the conjugation of antibody with an enzyme-Horseradish peroxidise. Further, substrate was added to the plate for producing visible colour change in a reaction mixture. Based on use or not of a secondary antibody, the ELISA test may be either direct or indirect (Figure 1). This test is successfully used for diagnosis of various diseases in equines. Singha et al. (2014) have reported an indirect ELISA using truncated TssB protein for serodiagnosis of glanders. A sensitive antigen capture ELISA was developed for the detection of secreted NS1 from infected equines with West Nile virus (Macdonald et al., 2005; Chung & Diamond, 2008). Similarly, ELISA has been developed for the detection of EHV-1, EHV-4 (Yasunaga et al., 2000), equine rhinitis virus A (ERAV) (Kriegshauser et al., 2009) and equine rhinitis virus B (ERBV) (Kriegshauser et al., 2008). In the recent studies, ELISA targeting antibodies to the spike (S) of equine corona virus was developed and validated to detect antibodies to EqCoV in infected horses (Kooijman et al., 2016).

Table 1 Prescribed test for equine diseases according to OIE, 2016. Disease name African horse sickness Contagious equine metritis Dourine Equine infectious anaemia Equine piroplasmosis Equine viral arteritis Glanders

OIE prescribed tests CF, ELISA Agent identification. CF AGID ELISA, IFA Agent identification (semen only), Virus Neutralization Complement Fixation

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Biotechnological tools for diagnosis of equine infectious diseases.

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Figure 1 Principle of ELISA test a) Direct ELISA b) Indirect ELISA 2.2 Dot-ELISA Dot-ELISA works on the basis of attachment of small amount of antigen on to a nitrocellulose membrane. Specific antibody is incubated with antigen containing dotted membrane followed by adding of enzyme conjugated anti-antibody. A substrate is added in the last which causes precipitation of a detectable coloured dot on the membrane (Svobodova et al., 2013). It was reported that the dot-ELISA is simple, quick, specific, sensitive, low cost field test that detects minute levels of antibodies much faster than complement fixation test and indirect hemagglutination antibody test (Verma & Misra, 1989; Verma et al., 1990). Dot-ELISA has been used for the serodiagnosis of glanders (John et al., 2010). By the use of nitrocellulose membrane in this test makes it applicable in the field. This assay is quick and specific in detection of various diseases. It gives us low background as compared to ELISA assay that can easily differentiate between the positive and negative samples. 2.3 Fluorescent Antibody Test (FAT) In Fat assay, antibody is labelled with fluorescent dye, is used in visualization of antigen in a clinical specimens. The antibody conjugated with fluorescent dye and antigen-antibody complex gives a visible glow sign when examined under a fluorescent microscope. The fluorescent dye can be tagged directly with primary antibody which is known as direct fluorescent antibody test or with a secondary anti-antibody known as Indirect Fluorescent Antibody Test (Figure 2). The FAT is used in diagnosis of several equine diseases. This assay was recently investigated for diagnosis of equine leptospiral abortion in mare (Erol et al., 2015). The sarcocystis neurona causes a dreadful disease, equine protozoal myeloencephalitis (EPM) in equines. The IFAT was successfully validated for CSF testing for confirmation of EPM in equines (Duarte et al., 2006; Johnson et al., 2013).

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The overall specificity and accuracy of IFAT was shown to be better than that of the western blot and modified western blot, which showed its potential to use as a diagnostic assay for detection of EPM caused by Sarcocystis neurona (Duarte et al., 2003). The FAT and immune-histo-chemistry (IHC) assay confirmed the presence of Australian bat lyssa virus (ABLV) antigen in horse brain tissues (Shinwari et al., 2014). A FAT assay has been used for the direct identification of bacterial Helicobacter on the equine gastric mucosa (Perkins et al., 2012). This technique has been used to describe the spatial distribution of Helicobacter species in the stomach of healthy horses to demonstrate the microbiota of normal appearing squamous and glandular mucosa (Burton et al., 2007). 2.4 Complement Fixation Test Complement fixation test (CFT) is an immunological test used for detection of presence of either antigen or antibody in the serum sample. It was generally used for microorganisms which are not easily cultured in research laboratory (Figure 3). Although, several studies have revealed its low specificity and sensitivity for virus detection, it is still used for many equine viral disease diagnoses. CFT is the OIE recommended test for glanders. Due to low prevalence of glanders in equine population it is important to use test with high specificity and sensitivity. CFT was found reproducible and reliable assay for clinical investigation and detection of latent infection of Equine herpes virus 1 (EHV1) (Hartley et al., 2005). A CFT assay has some limitations such as laborious, time consuming and often cross reactivity in nature. The non-specific hemolysis of RBC can be prevented using Potassium Periodate (KIO4). The KIO4 treatment to horse sera prevented the non-specific hemolysis which helped in determination of precise titers during CF test for EHV-1 diagnosis (Bannai et al., 2013). The CFT and virus neutralisation assays were used for determination of sero-conversion of EHV1 and EHV4 during obtaining acute and convalescent serum samples (Hartley et al., 2005).

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Figure 2 Principle of Direct Fluorescent antibody test (FAT).

Figure 3 Complement fixation test. _________________________________________________________



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Figure 4 Principle of lateral flow test 2.5 Lateral Flow Test (LFT) This test is also known as a lateral flow immunochromatographic strip assay. LFT is used as diagnostic techniques in medical and veterinary applications. It is an easy and fast assay used for identification of interest target in sample without using any special equipment (Figure 4). This technique can be used for qualitative or specific semiquantitative identification of many interest targets such as antibodies, antigens and nucleic acid products. The assay indicates a procedural control line which shows that the assay was performed properly and validates the test result. Therefore, presence of two lines gives positive result, while indication of only control line shows negative test in experiment. However, the appearance of no lines or only test line shows invalid result and test must be repeated. This test has been successfully used in diagnosis and detection of various disease associated with equines from biological samples. The recombinant viral capsid protein p26 conjugated to colloidal gold based simple immunochromatographic lateral flow (ICLF) test was validated for specific detection of Equine infectious anemia virus (EIAV) antibodies in equine sera (Alvarez et al., 2010). Similarly, LFT was also used for detection of vesicular stomatitis virus in cattle and horse clinical samples (Ferris et al., 2012). 2.6 Virus neutralization test In virus neutralization tests, serial dilutions of heat inactivated test serum are prepared and poured in a 96 well plate and are incubated with a defined amount (generally 100 TCID 50) of infectious virus (antigen). After incubation time period, susceptible virus cells are added to the virus-serum mixture and the final serum, virus and cell combination is kept for period of 2-3 days. Depending on the virus, this may be done by microscopic examination of the plate for the indication of viral cytopathic effect (CPE). Serum containing antibodies specific to the virus in target are capable to neutralize the aliquot of virus used in the test line, hence preventing the _________________________________________________________


infection of cells when added to the plate. At the time of very high concentrations of antibody to the virus in target are present in the test sample, virus neutralization will be occur at high serum dilutions. Whenever, where some or no antibody to the virus is present in the serum sample, it will be able to neutralize the infectious virus at the first dilution used in the test. The result of the test is the target at which the serum sample has been diluted such that it no further neutralizes the entire virus in the test. This dilution indicates the titre of the serum tested. The sero-prevalence of EHV-1 and EHV-9 infections was reported by serum neutralization test (Borchers et al., 2005; Taniguchi et al., 2000). 2.7 Agar Gel Immunodiffusion (AGID) AGID technique is used for the detection, identification and quantification of antibodies and antigens present in biological samples. In this technique, a gel plate is cut to form a series of wells in agar gel. A sample aliquot of interest target is placed in one well, and antibodies are placed in nearby well and the plate was incubated for 48 hours. During incubation time the antigens in the target sample and the antibodies each diffuse out from their corresponding wells. At the point where the two diffusion lines intersect, if any of the antibodies is specific to any of the antigens then they will bind to each other and form a complex. This antigen-antibody complex precipitated gives a thin white line in the gel, helps in the visual identification of antigen recognition. AGID can be used to diagnose Equine Infectious Anemia (EIA) (Beltrao et al., 2015). It detects antibodies against the main capsid viral protein (p26) in horse serum samples and this test is simple, inexpensive and specific to identify EIAV-infected animals (Alvarez et al., 2010). 2.8 Peptide based-ELISA Petide based-ELISA, a plate based techniques for detecting and quantifying peptides, proteins, hormones and antibodies. In this technique, a synthetic peptide is to be mobilized onto a solid support and complexes with an antibody, linked to an

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enzyme. Detection is done by assessing the conjugated enzyme activity after incubation with a substrate to produce a measurable end product (Figure 5). The very important step for the detection strategy is a specific antigen-antibody interaction. Peptide-ELISAs are performed in a 96-well microtiter plate with synthetic peptide in carbonate buffer followed by incubating the plate overnight at 4°C. Now, block the plate with blocking buffer for 1 hour at 37°C followed by addition of freshly prepared diluted primary antibody into each wells and incubate the plate at 37°C for 1 hour. Subsequently, antimouse IgG, diluted in 100 μl/well antibody dilution buffer is added with the incubation at 37°C for 30 minutes. In last enhancement solution is added in the plate and incubated at 37°C for 15 minutes. The plate was washed in between each step at least five times with 1X PBST. Read the absorbance at appropriate wavelength with an appropriate time resolved plate reader.

Soutullo et al. (2001) evaluated the performance of an equine infectious an Aemia-ELISA designed with synthetic peptides. This assay could be important to prove for large throughput screening and early detection of equine infectious anaemia (EIA), when the results of the traditional Coggins test are still negative. Recently, a sensitive and specific peptide-based ELISA was developed to determine the sero-prevalance of EHV-1 and EHV-9 (Abdelgawad et al., 2015). For discrimination between serological responses to EHV 1 and EHV4 immunoglobulins-IgG based type specific ELISA was developed (Ma et al., 2013). This technique was also used to discriminate between EHV-1 and EHV-4 glycoprotein E peptides for EHV-1 and glycoprotein G (gG) for EHV-4 (Lang et al., 2013; Yasunaga et al., 1998). Recently, a glycoprotein G based peptide ELISA was developed for detection of equine herpesvirus type 4 (Bannai et al., 2016).

Figure 5 Principle of peptide- ELISA _________________________________________________________



2.9 Nucleic acid diagnostics Nucleic acid-based detection methods including polymerase chain reaction, reverse transcriptase-PCR, nested-PCR, restriction fragment length polymorphism, amplified fragment length polymorphism, random amplified polymorphic DNA, loop-mediated isothermal amplification, microarray, real-time PCR are used for identification of several equine diseases (Pusterla et al., 2007; Monego et al., 2009; Yeh et al., 2012; Quereda et al., 2000; Larrasa et al., 2002; Eischeid, 2011). 2.10 Polymerase Chain Reaction (PCR) PCR uses the enzyme DNA polymerase that amplifies a small length of targeted DNA using primers which are specific to the target. It will amplify the selected target sequence from a mixture of genome. PCR acts as an important tool for the identification of parasites due to the insufficient amount of availability of antigen and antigen products by using

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conventional assays (Gasser, 2006). PCR can utilize almost all kind of biological samples such as meat, blood, urine, skin scrapings and faeces for parasitic infection study. When compared to the conventional method the detection limit of PCR is higher. Therefore, it is useful for detecting low amount of antigen in suspected samples (Varrasso et al., 2001; Nugent et al., 2006). It can also be used for detection of many equine diseases (Oldfield et al., 2004; Ocampo-Sosa et al., 2007; Pusterla et al., 2007; Letek et al., 2008; Monego et al., 2009). Polymerase chain reaction (PCR)–based diagnostic tests can allow rapid and sensitive detection of equine infectious pathogen (Paxson, 2008). Yeh et al. (2010) developed a duplex reverse transcriptse PCR which is sensitive, specific and very rapid and is useful in both humans and as well as in horses for the simultaneous and differential diagnosis of West Nile and Japanese encephalitis viruses.

Table 2 PCR testing results for a variety of infectious equine pathogens. Pathogen West Nile Virus Strongylus edentatus, Strongylus equinus and Strongylus vulgaris Herpes viruses 4 and 1 Equine herpesvirus 1 and 4 Distinguish between EHV-1 and EHV-4 EHV1, EHV4, EHV2 and EHV5 Rhodococcus equi

PCR test RT-nPCR PCR-RLB

References Johnson et al. (2001) Traversa et al. (2007)

PCR Differential multiplex PCR PCR Multiplex PCR PCR

Rhodococcus equi Equine arteritis virus

Multiplex PCR RT-PCR

Theileria equi Babesia caballi Babesia equi Streptococcus equi Equine influenza virus Leptospira spp. Salmonella spp. Alternaria spp. Emmonsia crescens

Nested PCR and Nested PCR with hybridisation Nested PCR Nested PCR PCR RT-PCR PCR PCR PCR Single step PCR

Brendan & Michael (1993) Carvalho et al. (2000) Wagner et al. (1992) Wang et al. (2007) Khurana et al. (2015); Pal & Rahman, (2015) Chhabra et al. (2015) St-Laurent et al. (1994); Zhang et al. (2008) Wise et al. (2013)

Lawsonia intracellularis Corynebacterium pseudotuberculosis Anaplasma phagocytophilum

Faecal PCR Real-time PCR/ RAPD-PCR nPCR

Streptococcus equi Equine encephalitis virus Differentiation of B. mallei and B. pseudomallei Streptococcus equi

Species-specific PCR RT-PCR Multiplex qPCR Triplex quantitative PCR

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Battsetseg et al. (2001) Battsetseg et al. (2001) Ijaz et al. (2012) OIE, (2016) Faber et al. (2000) Amavisit et al. (2001) Dicken et al. (2010) Pusterla et al. (2002) Lavoie et al. (2000) Foley et al. (2004) Lee et al. (2015); M'ghirbi et al. (2012) Javed et al. (2016) Linssen et al. (2000) Janse et al. (2013) Webb et al. (2013)

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Table 3 Examples of Real-time Polymerase chain reaction for the diagnosis of equine infectious pathogens. Pathogen Streptococcus equi Salmonella Salmonella spp, Anaplasma phagocytophilum Streptococcus Streptococcus equi Streptococcus equi Leptospira Corynebacterium pseudotuberculosis Equine arteritis virus Equine herpes virus 1 Equine herpes virus 4 Equine herpes virus 1 Strongylus vulgaris Equine Influenza Virus Pseudomonas syringae pv. aesculi Lawsonia intracellularis Theileria equi Equine infectious anemia virus African horse sickness Babesia equi Corynebacterium pseudotuberculosis Burkholderia mallei

Target gene eqbE and SEQ2190 ompC gene of S. Heidelberg invA gene 16S rRNA 16S rRNA SeM gene sodA gene ligA and B PLD exotoxin gene EAV ORF7 gene Glycoprotein B Glycoprotein B gD and IR6 gene rDNA Matrix and hemagglutinin gene Gyrase B Aspartate ammonia lyase gene 18S rRNA gag gene NS1 ema-1 gene Phospholipase D gene ISBma2

Further, Yeh et al. (2012) developed a diagnostic algorithm which serologically differentiates West Nile virus from Japanese encephalitis virus infection and its validation in field surveillance of horses. Rakhshandehroo et al. (2014) had done an intra-specific variation study for Habronema muscae in horses using cytochrome c oxidase subunit 1 gene based identification by PCR technique. Helicobacter bacterium infection was reported by Contreras et al. (2007) using the16S rRNA gene specific PCR in equines. PCR can also be combined with other molecular methods such as reverse transcriptase or nested PCR to genotype the organisms. RT-PCR has also been developed for Western Equine Encephalitis Virus (WEEV) diagnosis (Linssen et al., 2000; Lambert et al., 2003). RT-PCR assay was successfully used for identification of west nile and japanease encephalitis virus (Lanciotti & Kerst, 2001). A multiplex PCR was designed to amplify herpes simplex virus types 1 and 2, cytomegalovirus, and varicella-zoster virus DNA present in a diverse range of clinical material (Druce et al., 2002). Multiplex PCR assays for the simultaneous identification of varicella zoster virus (VZV), herpes simplex viruses (HSV), CMV, human herpesvirus 6, and Epstein-Barr virus in cerebrospinal fluid (Quereda et al., 2000) and assays for HSV and VZV in mucocutaneous specimens (Jain et al., 2001; Nogueira et al., 2000) and CSF (Read & Kurtz, 1999) have been reported, each with improved utility over existing methods in the diagnostic setting. In a recent study herpesvirus (EHV) type 1 was detected using PCR and neuropathogenic genotype of EHV-1 was identified by DNA sequencing _________________________________________________________


Reference Webb et al. (2013) Amavisit et al. (2001) Pusterla et al. (2010) M'ghirbi et al. (2012) Javed et al. (2016) Javed et al. (2016) Javed et al. (2016) Palaniappan et al. (2005) Spier et al. (2004) Balasuriya et al. (2002) Diallo et al. (2006) Diallo et al. (2007) Goodman et al. (2007) Nielsen et al. (2008) Quinlivan et al. (2005) Green et al. (2009) Pusterla et al. (2008) Kim et al. (2008) Cook et al. (2002) Rodriguez-Sanchez et al. (2008) Ueti et al. (2005) Sharon et al. (2004) Janse et al. (2013)

(McFadden et al., 2016). Various types of PCR are used for testing a Variety of Infectious Equine Pathogens (Table 2). 2.11 Random Amplified Polymorphic DNA (RAPD) It is a type of PCR reaction which amplifies segments of DNA randomly. It uses short primers of nucleotide length varies from 8-12 nucleotides and template DNA for PCR amplification. By resolving the resulted amplified product on agarose gel electrophoresis a semi unique profile pattern can be visualized from a RAPD reaction. Although RAPD is comparatively easy to perform, but it is also a PCR dependent assay so it needs specific PCR protocol to give reproducible result. Any kind of mismatch in template and primer results in complete absence of PCR product and makes it difficult to interpret the results of RAPD. This assay has the potential to play a useful role in genetic analyses of livestock species such as horses (Cushwa & Medrano, 1996). Larrasa et al. (2002) described the development of quick and relevant DNA extraction and RAPD methods that can be used for genotyping Dermatophilus congolensis field isolates. Larrasa et al. (2004) reported the molecular typing of D. congolensis from horse skin sample by RAPD and pulsed field gel electrophoresis (PFGE) techniques. 2.12 Real time PCR The real-time PCR assay gives us the quantification of several types of biological samples using verities of fluorescent materials such as TaqMan probes, SYBR Green dye and


Scorpion primers (Nado, 2009). The pathogenic nucleic acids from various biological and environmental samples can be quantified to give the information about the extent of infection. The SYBR Green dye based Real-time PCR assays have been validated for many equine diseases from several decades. Table 3 presents an overview of real-time PCR routinely used for the detection of equine pathogens such as bacterial, viral and parasitic pathogens. Although Real-time PCR is excellent in showing sensitive and specific results but it is still uncommon in routine laboratory diagnosis especially in rural endemic areas due to its sophistication. In the Real-time amplification protocols, other procedures such as DNA extraction, choice of primers may cause heterogeneity in results and causes difficulty in standardization of assay (Bretagne & Costa, 2006). A SYBR Green based assay was developed that could detect 100% of the different WNV target region variants in their study, whereas a TaqMan assay failed to detect 47% of possible single nucleotide variations in the probe-binding site (Papin et al., 2004). Johnson et al. (2010) designed a panflavivirus RT-PCR using degenerate primers for the NS5 gene to allow the detection of a range of flaviviruses including WNV. This SYBR Green based RT-PCR was able to detect the WNV however the sensitivity was much lower compared to WNV-specific TaqMan RT-PCR assays (Johnson et al., 2010). SYBR Green has been shown to inhibit the PCR reaction to some extent and melt curve analysis is troublesome by dye

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redistribution during melting. Eischeid analyzed and reported about the behaviour of other DNA dyes in Real-time PCR and showed that EvaGreen and SYTO dyes out performed SYBR Green in real-time PCR (Eischeid, 2011). 2.13 Probe Hybridization Fragments of DNA or RNA usually around 100-1000 bases length used to detect the presence of nucleotide sequences that are complementary to the probe sequence called hybridization probe and this probe hybridizes to single-stranded nucleic acid sequence (Wetmur, 1991). Due to the nucleotide base complementarily between the target and probe, the nucleotide sequence of probe allows pairing of probe and the target (Figure 6). The labelled probe is then hybridized to the target RNA (Northern blotting) or ssDNA (Southern blotting) immobilized on a membrane or in situ. The probe is tagged with a molecular marker of either radioactive (P32, I125 etc.) molecules or non-radioactive fluorescent molecules to detect the hybridization (Digoxigenin).The probe hybridization based assays have been used for diagnosis of equine infections such as equine arteritis virus (Balasuriya et al., 2002; Westcott et al., 2003). The probe hybridization assay is relatively easy to perform. EHV-1virus strain was reported by means of Southern blot and dot-blot hybridization (Morris & Field, 1988). The probe hybridization assay was confirmed and the sensitivity was inferior to classical techniques such as virus isolation (Morris & Field, 1988).

Figure 6 Principle of probe hybridization

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2.14 Microarray The nucleic acid microarray technique is a collection of microscopic nucleic acid spots attached to a solid glass surface. Picomoles of specific nucleic acid sequence are present on each spot called probe (Bumgarner, 2013). Probes are allowed to hybridize labelled target nucleic acid (cDNA or cRNA/antisense RNA). This probe-target hybridization can be detected and also can be quantified by silver, fluorophore or chemiluminescence-labeled targets (Figure 7). This technique is also used to measure the expression levels of many expressed genes of same or different species simultaneously. Microarray has also been used for diagnosis of equine disease detection. Equine-specific microarray has been used to estimate gene expression in laminitis (Noschka et al., 2009) and articular cartilage repair (Mienaltowski et al., 2009). A recent study using microarray technology on placental tissues identified a >900-fold upregulation of mRNA encoding the cytokine interleukin-22 in chorionic girdle, which is the first time IL-22 has been reported in any cells other than immune cells (Brosnahan et al., 2012). On the basis of whole genome single nucleotide polymorphism (SNP) analysis of all available Venezuelan equine encephalitis viruses (VEE) antigenic complex genomes, verifies that a SNP-based phylogeny accurately captured the features of the phylogenetic tree based on multiple sequence alignment, and reported a high resolution genome wide SNP (Gardener et al., 2016). 2.15 Loop-mediated isothermal amplification (LAMP) LAMP is a nucleic acid amplification procedure that works under a unique amplification principle; involves two steps: these are cyclic or non-cyclic phase (Ushikubo, 2004; Parida et al., 2008).The non-cyclical step precedes the cyclical phase of amplification (Parida et al., 2008). It involves the four primers as well as the Bst DNA polymerase with strand displacement activity, play a role in this first stage of LAMP reaction. The

cyclical step builds upon the product of the non-cyclical step which involves two outer primers along with the Bst DNA polymerase. The loop primers might be involved in the cyclic step when six primers are used (Nagamine et al., 2002). LAMP assay due to its unique properties has provided a powerful diagnosis of various pathogens (Notomi et al., 2000). LAMP technique amplifies nucleic acid at a very faster rate along with maintaining high specificity, sensitivity and efficiency (Parida et al., 2008). The most inventive feature of this technique is the simplicity of its protocol (Figure 8), and the low cost of overall amplification. Alhassan et al. (2007) developed a LAMP method for diagnosis of equine piroplasmosis. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed and it should be applicable to detect the equine rotavirus infection in molecular laboratories (Nemoto et al., 2010). Equine influenza virus was also reported using LAMP assay (Nemoto et al., 2011; Nemoto et al., 2012). Novel LAMP methods was developed specific to the pathogenic bacteria found in equine secondary pneumonia, namely, the Bacteroides–Prevotella group, Klebsiella pneumoniae, Stenotrophomonas maltophilia and Staphylococcus aureus (Kinoshita et al., 2015). Two different LAMP assays targeting Escherichia coli (Hill et al., 2008) or Pseudomonas aeruginosa (Goto et al., 2010) were used by Kinoshita et al. (2015), on clinical respiratory specimens and a high accordance was found between the results of the LAMP assays and bacterial culture. Use of these LAMP assays could enable rapid detection of pathogenic bacteria and swift administration of the appropriate antimicrobials. In this way, it is possible to concurrently perform LAMP assays to detect both the primary and secondary causative pathogens of lower respiratory bacterial infections in horses in only 60 min with the naked eye; this will make it possible to institute appropriate antimicrobial therapies more quickly in horses with secondary bacterial pneumonia (Kinoshita et al., 2015).

Figure 7 Principle of microarray assay. _________________________________________________________



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Figure 8 Principle of LAMP. 2.16 Sequence analysis study Whole genome sequencing is a process that gives the complete DNA sequence of an organism's genome at a single time. High-throughput genome sequencing technologies have largely been used as a research tool and are currently being introduced in the clinics (Van et al., 2013; Gilissen, 2014; Nones et al., 2014). Genome sequencing of the domestic horse and subsequent advancements in the field of equine genomics have led to an explosion in the development of tools for mapping traits and diseases and evaluating gene expression (Finno & Bannasch, 2014). In 2011, whole-genome sequencing of an individual American quarter horse mare was performed using massively parallel paired-end sequencing (Doan et al., 2012). Several single-gene disorders in quarter horses, such as polysaccharide storage myopathy (McCue et al., 2008; Tryon et al., 2009), hyperkalemic periodic paralysis, glycogen branching enzyme deficiency (Rudolph et al.,1992), and hereditary equine regional dermal asthenia (Ward et al., 2004; Finno et al., 2009) has been reported due to wholegenome sequencing of an individual American quarter horse mare. A high-quality draft assembly was constructed and additional sequence were provided by the inclusion of bacterial artificial chromosome end sequences from a related male thorough bred horse (Leeb et al., 2006). Kinoshita et al., (2014) reported the genera Bacteroides and Prevotella _________________________________________________________


especially B. fragilis and P. heparinolytica are dominant anaerobes in lower respiratory tract infection in horses. 3 Biosensors Biosensor is an advanced technique for the detection of either the antigen or antibodies. This assay involves the use of a receptor (mostly an antibody), a disease specific antibody and a transducer that converts a biological interaction into a measurable signal (Cruz et al., 2002). These biosensors are frequently coupled to sophisticated instrumentation to produce highly-specific analytical tools, most of which are still in use only for the research and development purpose due to the high cost of instrumentation, high cost of individual sample analysis, and the need for highly trained persons to oversee the testing. Fibre optic biosensors have the potential to do multianalyte analyses in an automated format. Portable fibre optic biosensors, has been reported to detect four different analytes in one coupon (King et al., 2000). Biosenors can be used as self-contained field devices for the detection of foreign animal disease agents. West nile virus was detected using biosensors and microfluidic systems, a linear, 15 amino acid fragment of domain III of WNV was successfully used as an antigen on an amperometric immunosensor (Ionescu et al., 2007). Neng et al. (2010) reported that, a surface enhanced Raman scattering immunoassay was shown to be highly sensitive for the

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detection of anti-WNV immunoglobulin. Hu et al. (2004) developed a genetically biotinylated single chain fragment variable antibody (scFv) against Venezuelan equine encephalitis virus (VEE). Patrick et al. (2014) studied the evolution of equine influenza and the origin of canine influenza with the help of biosensor. 4 Nanotechnology The systems or devices which are related to the features of nanometre scale are broadly defined as nanotechnology. This scale of technology as it applies to diagnostics would include the detection of molecular interactions. The tiny dimensions of this technology led a basement to the use of nanoarrays and nanochips as test platforms (Jain, 2003). The potential use of this technology is to analyse a sample for an array of infectious agents on a single chip. Many research groups are considering the use of chip assays that detect several agroerrorism agents in each sample. Small, portable platforms are being designed to allow pen-side testing of animals for diseases of concern. The use of nanoparticles to label antibodies is another facet of nanotechnology. These labelled antibodies can be used in various assays to identify specific pathogens, structures or molecules. The use of gold nanoparticles, nanobarcodes, quantum dots and nanoparticle probes are the examples of nanotechnology (Yguerabide & Yguerabide, 2001). Nanopores, nanosensors, resonance light scattering and cantilever arraysare some of the additional nanotechnologies and it is anticipated that many of the specific nanotechnologies will eventually be applied to the diagnosis of endemic veterinary diseases in the future. Klier et al. (2012) reported about an aerosol formulation of biodegradable, biocompatible and nontoxic gelatine nanoparticle-bound CpG-ODN2216, to treat equine recurrent airway obstruction in a clinical study. 5 Proteomics Proteomics is the new emerging field to isolate and characterize the protein produced by various etiological agents. Different bacterial, viral as well as parasitic proteins can be targeted with the help of this technology. Hence, proteomics has potential applications in veterinary diagnostics. The usual approach of proteomic involves separation of the proteins with the help of two dimension gel electrophoresis and staining them with appropriate protein marker. The protein ‘pattern’ is different in different species; hence it can be recognized as a fingerprint. It is then analyzed by performing image analysis (Krah & Jungblut, 2004). Proteins that are up- or downregulated due to disease are compared and find by using proteome maps. A protein of interest can be cut and taken out from the gel and purified. This purified protein can be further fully characterized using peptide-mass fingerprinting and/ or mass spectrometry methods. Veterinary diagnostics may make use of proteomics to identify or look for known disease markers or patterns with biochip technology and instrumentation that combines mass spectrometry with other separation chromatography or molecular techniques in the _________________________________________________________


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future. These instrumentations are designed to specifically select, separate by molecular mass, and identify the complex mixture of proteins in a sample, which can be compared to known samples for diagnostic purposes. In equine medicine, proteomics is been used in the diagnosis of different metabolic as well as orthopedic diseases which show some of the alteration in the expression levels of marker proteins (Amaya, 2014). In the proteomic marker analysis conducted in biopsy samples of horse muscles, it was found that three significantly increased proteins: alpha actin, tropomyosin alpha chain and creatine kinase M chain (CKM). CKM was represented by multiple spots probably due to posttranslational modification, one of which appeared to be unique for tying-up suggesting that altered energy distribution within muscle cells is part of the disease etiology (Freek et al., 2010a). In another study they have identified, 20 differential spots representing 16 different proteins. Evaluation of those proteins complies with adaptation of the skeletal muscle after normal training involving structural changes towards a higher oxidative capacity, an increased capacity to take up long-chain fatty acids, and to store energy in the form of glycogen. Intensified training leads to additional changed spots. Alpha-1antitrypsin was found increased after intensified training but not after normal training. This protein may thus be considered as a marker for overtraining in horses and also linked to overtraining in human athletes (Freek et al., 2010b). In an another study, which was conducted on the proteomics, study of cerebrospinal fluid, a total of 320 proteins were confidently identified across six healthy horses, and these proteins were further characterized by gene ontology terms mapped in UniProt, and normalized spectral abundance factors were calculated as a measure of relative abundance and these results provide an optimized protocol for analysis of equine CSF and laid the basement for future studies involving the CSF study of equines in the context of pathogenic disease states (Carolyn et al., 2014). The analysis of osteoarthritis and osteochondrosis conducted by Elisabetta et al. (2012) has identified some putative protein markers which can be further tried for the definitive early diagnosis of osteoarthritis in the horses. A highly sensitive proteomic comparison together with insightful data mining enabled us to identify proteins and pathways involved in early OA which could aid the development of early OA diagnostic markers and therapeutics (Peffers et al., 2012). In case of a very unpredictable disease of equines, laminitis identification and measurement of novel protein biomarkers present in blood that predict the onset and resolution of laminitis would both aid clinical management of at-risk equine patients and shed light on underlying mechanisms with the intent of developing novel preventive strategies and therapeutic approaches (Joseph et al., 2008). Conclusion A profound change has been occurred in recent years in veterinary diagnostics with the introduction of new biotechnological assays which completely changed the


scenario of time-tested, traditional diagnostic techniques of veterinary disease diagnosis. These new biotechnological methods, includes the production of more specific antigens by the use of recombination, expression vectors and synthetic peptides. When coupled with the use of monoclonal antibodies, the sensitivity and specificity of a number of traditional diagnostic assays have been significantly improved. Various forms of PCR have become a routine diagnostic tool in veterinary laboratories for rapid screening of large number of samples during disease outbreaks to develop prevention and control measures and also to make specific typing determinations for research purpose. Other technologies are likely to be widely adopted in the future as they demonstrate the ability to improve the diagnostic capabilities while reducing the time and, perhaps cost associated with more conventional technologies. Proteomics has the potential to look at the broader picture of protein expression for a pathogen of interest or by infected animals and it may lead to a special niche of veterinary diagnostics. Nanotechnologies hold the promise of screening numerous pathogens in a single assay, while not yet implemented in veterinary laboratories. Nanotechnology has become the choice for mobile and pen-side testing of animal diseases due to its small size and easy handling. Biotechnology and its applications hold the great promise for improving the speed and accuracy of diagnostic tests for veterinary pathogens. Much developmental work will be required to realise the potential with well-characterised, validated assay systems that provide improved diagnostic capabilities to safeguard animal health. Traditionally, pathogens were detected by microscopic and other conventional methods of various biological samples. Later on several molecular and serological assays have been employed for diagnostic purpose. These assays are shown highly effective and sensitive results for the detection of parasites regardless of the type of infection and sample. Among the various available techniques, some are used for treatment monitoring along with the diagnosis of parasites. Thus they became a useful tool in the clinical decision making process. The molecular and serological methods are also useful in vast epidemiological studies, because they are also involved in the geographical distribution study of parasites, genetic diversity of populations, susceptibility of infections and mutations in parasites. Detailed knowledge about the genetic characteristics, morphology and behaviour of parasitic disease in the affected population is provided by the molecular tools. Although, the cost of molecular diagnosis is higher than the conventional methods, they are highly used in veterinary clinical diagnosis, epidemiological studies and treatment monitoring of animals. The suitable molecular tests showing rapid, sensitive, accurate and reliable result and which can detect all or most targeted pathogens in a multiplex amplification system should be developed. Moreover, for faster surveillance strategies and monitoring of parasitic epidemiology automated technology should be developed to process the large number of serum samples for antibody _________________________________________________________


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ISSN No. 2320 – 8694

EVOLVING VIEWS ON ENTERIC VIRAL INFECTIONS OF EQUINES: AN APPRAISAL OF KEY PATHOGENS Shubhankar Sircar1, Sharad Saurabh1, Jobin J. Kattoor1, Pallavi Deol1, Kuldeep Dhama1, Sandip K Khurana2 and Yashpal S. Malik1,* 1

ICAR-Indian Veterinary Research Institute, Izatnagar 243 122, Uttar Pradesh, India ICAR-National Research Centre on Equines, Hisar - 125 001, Haryana, India

2


KEYWORDS ABSTRACT Equines Enteric Virus Epidemiology Diagnosis Control

Equines, the earliest known mammalian species, have been found highly susceptible to several enteric pathogens including viruses, fungi, parasites and bacteria. This review conserves with the key viral pathogens that affects foals and horses such as rotavirus, adenovirus, coronavirus, parvovirus, picobirnavirus etc. With the advent of next generation sequencing approaches the list of new enteric viruses has expanded. Viruses like Cyclovirus, Kirkovirus and Anellovirus are the new members identified in equines recently. Close proximity of horses to human settlements and/or other domestic animals pretense the threat of infectious diseases spread to humans/animals and vice-versa. Therefore, horse diseases are not only of veterinary importance but also are among important factors for public health. Herein, we intend to appraise current status of key enteric viruses that cause diarrheic disorders in foals and horses.

* Corresponding author E-mail: [email protected] (Yashpal S. Malik) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.



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1 Introduction Equines are among the earliest known mammalian species that have been incessantly domesticated by the human race since 4000 BC. Apart from being companion animals to humans, they have also been used as amusing or sporting animal. During late 19th century, equine microbiology came under existence with the discovery of Burkholderia mallei as the feared pathogen of glanders and farcy of horse. Later on, a filterable agent was identified capable of causing African horse sickness (Slater, 2013). With the help of classic virological methods, numerous equine viruses have been added to this list such as equine influenza virus, West Nile virus, equid herpesviruses, equine encephalitis viruses, equine arteritis virus, equine infectious anemia virus, equine coronavirus, Hendra virus and vesicular stomatitis virus. Though, the study of equine infectious diseases has been an important part of veterinary sciences since ages, a large number of the equine diseases still remains unfamiliar. Close proximity of horses to human settlements and to other domestic animals pose the threat of infectious diseases spread to humans/animals and vice-versa. Zoonotic pathogens such as Alphaviruses, Hendra virus, Influenza A virus, Rabies virus and West Nile virus are reported to infect horses (Johnson, 2011; Cullinane & Newton, 2013; Onmaz et al., 2013; Slater, 2013). It is therefore noteworthy that horse diseases are not only of veterinary importance but also pose potential threat of zoonosis/reverse zoonosis (Khurana et al., 2015; Mukarim et al., 2015) and are among important factors from human public health perspective. Several enteric pathogens leading to multifactorial diseases have been detected and isolated from diseased foals and horses. The major enteric pathogens associated with neonatal foal diarrhea are rotavirus, coronavirus, Clostridium perfringens type A, Salmonella spp., Rhodococcusequiand Clostridium difficile (Fielding et al., 2015; Franco Ayala & Oliver Espinosa, 2015; Barr, 2016). Foals co-infecting with number of infectious agents has also been documented in diarrheic foals (Slovis et al., 2014). One of the prime causes of equine enteritis is the viral infections. A recent metagenomics study highlighted several new viruses like Cyclovirus, Kirkovirus and Anellovirus along with known enteric viruses such as parvovirus, adenoviruses, coronaviruses, rotaviruses and picobirnaviruses in equines (Li et al., 2015). In last few years, several reviews appeared on various aspects of equine health excluding enteric viral pathogens of equines, except a few that highlighted coronavirus and rotavirus infections in equines (Bailey et al., 2013; Papp et al., 2013; Dhama et al., 2014; Pusterla et al., 2015a). Viral etiological agents which are able to cause diarrheic outbreaks are comprehended in Table 1. In the event of emergence of several new viral infections worldwide including Ebola and Zika (Dhama et al., 2015a; Singh et al., 2016) there is urgent need to understand the burden of viral infections in different animal hosts. Here, we intend to review current status of major enteric viruses of

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economic importance that cause diarrheic disorders in foals and horses. Furthermore, current updates in the rotavirus and coronavirus infection in equines are covered. 2 Adenovirus Adenoviruses belong to the Adenoviridae family, are unenveloped and have icosahedral nucleocapsid, with 90–100 nm size, encapsulating a dsDNA genome of 26–45 Kbp (Davison et al., 2003). Adenoviruses are known to affect a wide vertebrate’s host range and are genetically/antigenically heterogeneous (Harrach et al., 2011). Presently, this family comprises of 5 genuses as Mastadenovirus, Atadenovirus, Aviadenovirus, Ichtadenovirus and Siadenovirus (Harrach et al., 2011). Horses and foals are affected by adenoviruses of genera Mastadenovirus, have been primarily connected with respiratory and gastrointestinal tract infections (Reubel & Studdert, 1997; Cavanagh et al., 2012). Equine adenoviruses are separated into 2 serotypes and consequent molecular and phylogenetic readings established them as distinct species which are nowadays called as Equine adenovirus 1 (EAdV-1) and Equine Adenovirus 2 (EAdV-2) (Reubel & Studdert, 1997; Cavanagh et al., 2012). Out of these two, EAdV-1 is primarily associated with infections in the respiratory tract of young foals and horses whereas EAdV-2 has been reported mainly from horses having diarrheal illness (Studdert & Blackney, 1982). The first equine adenovirus was reported and isolated in USA (Todd, 1969) it was followed by isolation reports from Germany and Australia whereas physical characterization of the virus was published in 1973 of an adenovirus isolated from the pneumonic lung tissue of an Arabian foal (Ardans et al., 1973). Antibodies pertaining to both the Equine Adenovirus types were reported in New South Wales, Australia (Giles et al., 2010). In a study during 1982 on the diarrheal outbreaks in young foals, researchers isolated and identified an Equine adenovirus strain which did not contain the hemagglutinationinhibiting antibody to EAdV-1 (Studdert & Blackney, 1982). Similar research findings were published in New Zealand revealing two serologically different EAdV strains isolated from thoroughbred foals suffering from diarrhea and respiratory disease (Horner & Hunter, 1982).These research findings supported the assumption that adenoviruses found in the fecal specimens should be considered as the prototype for EAdV-2. Compared to EAdV-1 adenovirus there has been less studies done with respect to EAdV-2 adenovirus with the very first sequence data appeared on the GenBank database for EAdV-2 in the year 1997 (Reubel & Studdert, 1997) (GenBank L80007.1). The study by Ruebel and Studdert also established the first molecular evidence that EAdV-2 is distinct to EAdV1, not only in antigenic aspect but is quite different on molecular level too.

Evolving Views on Enteric Viral Infections of Equines: An Appraisal of Key Pathogens.

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Table 1 Enteric viruses reported in equines having relevance in causing enteritis highlighting their main features. Sl No. 1.

Viruses

Genome

Genus

Species/serotypes

Adenoviruses

dsDNA

Mastadenovirus

Equine Adenovirus 1

Relevance in enteric diseases No

2.

Coronaviruses

ssRNA

Betacoronavirus

Equine Adenovirus 2 Betacoronavirus-1

Yes Yes

3.

Rotaviruses

dsRNA

Rotavirus A

Yes

4. 5. 6. 7.

Picobirnavirus Parvovirus Anellovirus Cyclovirus

dsRNA ssDNA ssDNA ssDNA

Picobirnaviruses Copiparvovirus Mutorquevirus Cyclovirus

G3P[12], G14P[12], G3P[3], G5P[7], G6P[1], G8P[1], G10P[1], G10P[11] and G13P[18] Equine picobirnavirus Ungulate Copiparvovirus 3 Unclassified Unclassified

A whole genome characterization of an EAdV-2 strain was also reported recently in the year 2015 in Australia (Giles et al., 2015) (GenBank Acc. No. KT160425). Interestingly, till this review in September 2016 there were only 2 sequence report available for EAdV-2 in the GenBank database out of which one is complete genome and another is 5.5 kbp in length comprising the hexon and endopeptidase genes. Unlike EAdV-1, which can be easily isolated and grows fastidiously in primary tissue culture derived from equine fetal kidney (EFK) cells (Studdert, 1978), EAdV-2 responds very poorly to cell culture and has been infrequently isolated (Horner & Hunter, 1982; Studdert & Blackney, 1982). The major technique used for the diagnosis and characterization of adenovirus is PCR which is based on the hexon gene of adenovirus genome which contains highly conserved regions (Reubel & Studdert, 1997). With the available literature regarding the infection of adenovirus in equines EAdV-1 has been studied widely in comparison to EAdV-2. Despite the significant improvement in the development of diagnostic techniques there has been scarcity of information regarding EAdV-2 which requires the attention of veterinarians and scientist working in the field of equine infectious diseases.

Yes No No No

References Cavanagh et al.(2012) Giles et al. (2015) Nemoto et al. (2015b) Ghosh & Kobayashi (2014)

Li et al. (2015) Li et al. (2015) Li et al. (2015) Li et al. (2015)

(Holmes, 2001). The different animals reported to have Coronavirus mediated infections are swine, cattle, horse, dog, cat and avian species like chicken and turkey (Saif et al., 1991; White & Fenner, 1994; Studdert, 1996; Jamieson et al., 1998; Guy et al., 2000; Lai & Holmes, 2001; McIntosh, 2002; Strauss & Strauss, 2002; Ksiazek et al., 2003; Van der Hoek et al., 2004; Brian & Baric, 2005; Weiss & Navas-Martin, 2005; Decaro & Buonavoglia, 2008; Boileau & Kapil, 2010; Woo et al., 2012). Further, based on the antigenic properties coronaviruses are divided into 3 major antigenic groups which infect several animal hosts and humans (group 1 and 2) and also avian species (group 2). The group 1 comprise of human coronavirus (strain 229E), canine coronavirus, porcine transmissible gastroenteritis virus and feline infectious peritonitis virus. The group 2 is represented by human coronavirus strains (OC43 and HKU1), murine hepatitis virus, bovine coronavirus, porcine hemagglutinating encephalomyelitis virus, canine respiratory coronavirus (Erles et al., 2007), and bubaline coronavirus (Decaro et al., 2010). Viruses such as turkey coronavirus and infectious bronchitis virus are included in group 3 (Resta et al., 1985; Studdert, 1996; Davis et al., 2000; Guy et al., 2000; Lai & Holmes, 2001; McIntosh, 2002; Strauss & Strauss, 2002; Van der Hoek et al., 2004; Smith & Denison, 2012; Woo et al., 2012; Smith et al., 2013).

3 Coronavirus Coronavirus is an enveloped, ssRNA, positive sense virus having a helical symmetry with a genome length of 26 to 32 Kb, which is largest among the RNA viruses. The Coronaviridae family is divided into 2 subfamilies (Torovirinae and Coronavirinae). The later subfamily classified into four different genera viz. Alphacoronavirus, Betacoronavirus, Deltacoronavirus and Gammacoronavirus based on the serological and molecular differences (Woo et al., 2012). These are mainly associated with respiratory, neurologic, hepatic or gastrointestinal disorders in animals _________________________________________________________


The earliest known Equine Coronavirus (ECoV) recognized by electron microscopy (EM) in the foals diarrheic fecal samples and adult horses (Bass & Sharpee, 1975; Huang et al., 1983). Later, in 2000 Davis and colleagues developed an antigen capture ELISA for the detection ECoV in the feces of diarrheic foal along with immunohistochemistry of affected foal’s intestine suffering from neonatal enterocolitis (Davis et al., 2000). In the same year, an ECoV strain NC99 was isolated and its N protein gene was characterized using the previously described BCoV primers (Guy et al., 2000). The ECoVs have been found highly related with bovine coronaviruses (Imagawa

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et al., 1990; Guy et al., 2000). Subsequently, the complete genomic constellation of ECoV (strain NC99) was achieved which consists of 2 replicase polyproteins, 4 accessory proteins (ns2, p4.7, p12.7, and I) and 5 structural proteins viz. spike, hemagglutinin esterase, membrane, envelope and nucleocapsid (Zhang et al., 2007). Diagnosis of coronaviruses has been routinely done through EM in the earlier times (Reed et al., 1983; Biermann et al., 1991; Guy et al., 2000). Using BCV antibodies serum neutralization tests has also been employed to detect ECoV infections in horses (Bass & Sharpee, 1975; Imagawa et al., 1990; Anzai et al., 2001). Molecular techniques like nested PCR and quantitative PCR have been actively used in the diagnosis of ECoV (Guy et al., 2000; Slovis et al., 2010). During early stages of infection highly sensitive technique like real-time RT-PCR has been used (Pusterla et al., 2013; Miszczak et al., 2014). In 2015, a RT-LAMP was also developed for the isothermal detection of ECoV which can be economical in comparison to other diagnostic tests (Nemoto et al., 2015a). Recently a one-step real time RT-PCR was also developed and demonstrated for the sensitive detection of ECoV in respiratory and fecal samples (Miszczak et al., 2016). Improving the specificity of the detection one ELISA was recently developed for the specific detection of ECoV in naturally infected horses and record the seroprevalence in them (Kooijman et al., 2016). In earlier studies, ECoV was suspected to cause enteritis in foals but their pathogenicity in young foals remained unproven (Davis et al., 2000; Van der Hoek et al., 2004; Arguedas, 2007). Mainly the tissue tropism for ECoV infections have been found to be inside the gastrointestinal tract of young foals and horses (Miszczak et al., 2014; Fielding et al. 2015; Pusterla et al., 2015b). Multiple ECoV outbreaks have occurred in the ÜSA and Japan (Oue et al., 2011; Oue et al., 2013; Pusterla et al., 2013). Besides the possible respiratory and mechanical transmission of coronaviruses in equines, infections spreads through fecal-oral route (Studdert, 1996; Anzai et al., 2001). Although, signs of upper respiratory tract infections are predominant with ECoV infected foals but their infrequent detection in nasal secretions shows their lack of tropism to the upper respiratory tract of young horses. In an experimental study on Japanese draught horses, the horses were experimentally inoculated with ECoV strain NC99 and Obihiri 12-2 to confirm and investigate the clinical signs and virus shedding pattern of ECoV in horses. Though, nasal secretions came positive in PCR assay, experiments could not define whether this was due to nasal replication of the virus and subsequent shedding or due to environmental factors or both (Nemoto et al., 2014). Subsequently, in 2015 a study reported the whole genome sequencing of three earlier isolated ECoV strains from Japan Obihiri 12-1, Obihiri 12-2 and Tokachi09. The study found the three strains genetically similar to NC99 strains of USA with minor exceptions in NS2, NSP3 and p4.7 genes (Nemoto et al., 2015b). _________________________________________________________


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It has been usually observed that ECoV infection in horses spread when they are sheltered together, which has been confirmed by the presence of BCoV and other related coronavirus antibodies in the feces of horses (Imagawa et al., 1990; Anzai et al., 2001). Better management practices regarding the sheltering and transit of young foals and adult horses can arrest the infection from spreading to other places. 4 Rotaviruses Rotaviruses are the foremost cause of diarrhea in neonates of humans, animals and avian (Estes & Kapikian, 2007; Dhama et al., 2015b). Till date, nine different species have been identified in rotaviruses from (A-I) as per the antigenic differences in the major inner capsid protein and nucleotide sequence identities gene encoding the VP6 protein (Matthijnssens et al., 2012a; Mihalov-Kovacs et al., 2015), although recently reported species I needs further endorsement by the ICTV. Rotaviral genome is made up of eleven dsRNA segments that encode 6 structural proteins viz; VP1, VP2, VP3, VP4, VP6 & VP7 and 5/6 non-structural proteins i.e. NSP1– NSP5/6). The major coat proteins (VP4 & VP7) induce the neutralizing antibodies production and are considered important due to the genetic classification which is based on these genes. As per the latest update of Rotavirus Classification Work Group (RCWG) till 27 th June, 2016 there are 32 G-genotypes (VP7) and 47 P-genotypes (VP4) reported in various host species (https://rega.kuleuven.be/cev/viralmetagenomics/virusclassification/ rcwg). Among all the species of rotavirus, only group A rotaviruses (RVA) has been detected in the equines so far (Matthijnssens et al., 2012b). The first detection of equine RV has been reported in the year 1975 from England (Flewett et al., 1975). Since from this, equine RVs have been known to be the major causes of diarrhea in foals (Imagawa et al., 1991; Collins et al., 2008; Frederick et al., 2009). Based on the serological reports equine RVAs have been shown to be ubiquitous in the equine populations (Pearson et al., 1982). Numbers of diagnostic techniques have been employed for detecting RVs in diarrheic foals and adult horses. Electron microscopy was the first technique to be applied for the identification of RVs in foals but unfortunately it requires costly instruments and expertise. Moreover the diagnostic sensitivity of EM turns out to be very low as it can’t detect virus particles lower than 107per ml of stool samples (McIntosh, 1996). Culturing the RV in cell line is again a daunting task. Equine RV has been isolated in MA-104 cell line in the year 1981 when field sample from a diarrheic foal has been successfully adapted in UK (Imagawa et al., 1981). RNA-PAGE has been widely applied for the detection of equine RVs due to the peculiarity of different RV groups to migrate differently on the RNA-PAGE. Certain studies has reported the typical migration pattern being observed with equine RVA isolates with the segment 3 & 4 migrating together close to each other and unlike the other RVA isolates


here the segment 7, 8 and 9 migrate as a triplet (Hardy et al., 1991). ELISA and other immunodiagnostic have been now used for rapid detection of RV which are usually based on the group specific VP6 gene product (outer coat protein). Many test kits available for human RV detection has also been employed in the detection of equine rotaviruses, though the sensitivity of ELISA has been found more in respect to other immunodiagnostic test (Nemoto et al., 2010a). The indication of widespread RVA infections in horses was evident by the finding of RVA specific antibodies in adult horses (Pearson et al., 1982; Eichhorn & Huan-Chun, 1987). It was also evident by the detection of RVA in equine population from different countries which includes the United Kingdom (Strickland et al., 1982), the United States of America (Kanitz, 1977), Australia (Studdert et al., 1978; Tzipori & Walker, 1978) Germany (Elschner et al., 2005), New Zealand (Durham et al., 1979;Schroeder et al., 1983), France (Puyalto-Moussu & Taouji, 2002), Greece (Ntafis et al., 2010), Italy (Monini et al., 2011), the Netherlands (Van der Heide et al., 2005), Argentina (Barrandeguy et al., 1998), Venezuela (Ciarlet et al., 1994) and India (Gulati et al., 2009). RT-PCR assays are being used as test of choice nowadays for the detection as well as for genotyping of equine RVA strains (Gouvea et al., 1994; Tsunemitsu et al., 2001; Garaicoechea et al., 2011). Hybridizing probe based diagnostics and genotyping has also been attempted in bovine rotavirus which can be opted for equine strains too (Minakshi et al., 2005). More recently, one isothermal technique RT-LAMP has been developed targeting the VP4 with P[12] specificity (Nemoto et al., 2010b). Moreover, in USA RT-PCR kit is commercially available for early and quick detection of RVs (Slovis et al., 2010). As submissions of molecular sequence of rotavirus have been increased in public database, sequence based typing of rotavirus groups got interest. Till now, there have been 6 Ggenotypes (Imagawa et al., 1994; Isa et al., 1994; Isa et al., 1996) and 6 P-genotypes (Garaicoechea et al., 2011) are reported in equines (Table 2). Table 2 G and P genotypes of equines rotaviruses reported till date. G-Type G3 G5 G8 G10 G13 G14

P-Type P[1] P[3] P[7] P[11] P[12] P[18]

Among the widely reported and commonly found genotype combinations of equine RVA, G3P[12] and G14P[12] have been described several times worldwide (Tsunemitsu et al., 2001; Elschner et al., 2005; Collins et al., 2008; Ntafis et al., 2010; Garaicoechea et al., 2011; Nemoto et al., 2011 Papp et _________________________________________________________


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al., 2013). The G3 RVA strains have been further differentiated into 2 subtypes (G3A and G3B) as per reactivity with monoclonal antibodies (Browning et al., 1992). In the year 1991, a highly unfamiliar equine RVA stain (L338) carrying G13P[18] genotype combination was reported from UK (Browning et al., 1991a). Using the RNA-RNA hybridization technique, it was shown that the strain L338 possess a distinctive genotype of G13 and P[18] and unusual NSP1 genetic makeup which found distinct to other human and animal RVAs (Wu et al.,1993; Taniguchi et al., 1994; Kojima et al., 1996). Certain bovine-like equine RVAs have also been reported which are highly similar to bovine RV strains employing labeled probes and RNA-RNA hybridization techniques from UK and Japan, respectively (Imagawa et al., 1991; Imagawa et al., 1993; Isa et al., 1996), Similarly, an unusual G6 and G10P6[1] rotavirus was detected in equines between 2003 and 2005 in addition to G1 strains from India, but later this report was taken back following the wrong classification of G6 RVA strains as G16 (Matthijnssens et al., 2012b). In Argentina also an uncommon feline-like RVA was also reported in diarrheic foal (Garaicoechea et al., 2011). Certain unusual equine RVA genotype combinations has also been discovered such as G3P[3], G5P[7], G6P[1], G8P[1], G10P[1], G10P[11] and G13P[18], (Gulati et al., 2007; Garaicoechea et al., 2011). Till date only three inactivated equine RVA vaccine have developed by Argentina, Japan and USA and are being used in several countries contains the common genotype of G3P[12] strain (Imagawa et al., 2005). During three decades (1981-2010) there have been several studies in Japan leading to the genetic analysis and characterization of equine RVAs (Takagi et al., 1994; Tsunemitsu et al., 2001; Fukai et al., 2006; Nemoto et al., 2012;). Till 2013 only few reports for the whole genome characterization and analysis of equine RVA strains have been documented (Ghosh et al., 2012; Matthijnssens et al., 2012b; Mino et al., 2013). The strains characterized for their complete genomic constellations were three from Europe (Strain: L338; 03V04954 and 04V2024), three from Argentina (Strain: E30; E403 and E4040), one from South Africa (Strain: EqRV-SA1) and four from Japan (Stain: B1; HH-22; CH-3 and OH-4) (Ghosh et al., 2012; Matthijnssens et al., 2012b; Ghosh et al., 2013; Mino et al., 2013). In February 2015, a report appeared in which 23 equine RVA strains from late 1990s and 2009-2010 along with the vaccine strain HO-5 were characterized through the next generation sequencing (Nemoto et al., 2015c) taking the total count of whole genome reports for equine RVA to 37 isolates till dates (Table 2). In the whole genome and further phylogenetic analysis of 37 isolates revealed that the G3 strains carried a uniform genetic constellation for their 11 gene segments and were closely related to the HO-5 vaccine strain irrespective of time they were confirmed (Nemoto et al., 2015c). In disparity, the G14 strains showed some divergence in respect to VP7 and NSP4 gene.

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Figure 1 Whole genome backbone consisting of 11 segments of equine group A rotavirus (RVA) strains with known genomic constellations. SL. No

Year of Isolation

VP7

VP4

VP6

VP1

VP2

VP3

NSP1

NSP2

NSP3

NSP4

NSP5

1

Strain Name RVA/Horse-wt/ARG/E403/2006/G14P[12]

2006

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E12

H7

Matthijnssens et al. (2012)

2

RVA/Horse-wt/ARG/E4040/2008/G14P[12]

2008

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E12

H7


3

RVA/Horse-wt/ARG/E30/1993/G3P[12]

1993

G3

P[12]

I6

R2

C2

M3

A10

N2

T3

E12

H7


4

RVA/Horse-wt/IRL/03V04954/2003/G3P[12]

2003

G3

P[12]

I6

R2

C2

M3

A10

N2

T3

E2

H7


5

RVA/Horse-wt/IRL/04V2024/2004/G14P[12]

2004

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


6

RVA/Horse-wt/ZAF/EqRV-

2006

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


7

1991

G13

P[18]

I6

R9

C9

M6

A6

N9

T12

E14

H11


8

RVA/Horse-tc/GBR/L338/1991/G13P[18] SA1/2006/G14P[12] RVA/Horse-wt/ARG/E3198/2008/G3P[3]

2008

G3

P[3]

I3

R3

C3

M3

A9

N3

T3

E3

H6

Mino et al. (2013)

9

RVA/Horse-tc/GBR/H-1/1975/G5P9[7]

1975

G5

P[7]

I5

R1

C1

M1

A8

N1

T1

E1

H1

Ghosh et al. (2013)

10

RVA/Horse -tc/JPN/OH -4/1982/G6P[5]

1982

G6

P[5]

I2

R2

C2

M2

A13

N2

T6

E2

H3

Ghosh et al. (2013)

11

RVA/Horse -tc/JPN/BI/1981/G3P[12]

1981

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7

Ghosh et al. (2013)

12

RVA/Horse -tc/JPN/HH -22/1989/G 3P[12]

1989

G3

P[12]

I6

R2

C2

M3

A10

N2

T3

E2

H7

Ghosh et al. (2013)

13

RVA/Horse -tc/JPN/CH -3/1987/G14P[12]

1987

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7

Ghosh et al. (2013)

14

RVA/Horse-tc/JPN/HO-5/1982/G3P[12]

1982

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7

Nemoto et al. (2015c)

15

RVA/Horse-tc/JPN/JE29/1997/G3P[12]

1997

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


16


1997

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


17

RVA/Horse-tc/JPN/JE76/1996-1997/G3P[12]

1996

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


18


1996

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


19


1997

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


20

RVA/Horse-tc/JPN/No.1/2010/G3P[12]

2010

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


21


2010

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


22


2010

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


23


2010

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


24


2010

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


25


2010

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


26


2010

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


27


2009

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


28


2009

G3

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


29


1997

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


30


1997

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


31


1996

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


32

RVA/Horse-tc/JPN/JE87/1996-1997/G14P[12]

1996

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


33


1997

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


34


2010

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


35


2010

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7

Nemoto et al. (2015)

36


2010

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


37


2010

G14

P[12]

I2

R2

C2

M3

A10

N2

T3

E2

H7


_________________________________________________________


Reference


The NSP4 gene of G14 strains and also G3 strains clustered along with bovine RVAs and bovine-like equine strain OH-4 and HH-22, whereas rest all other genes were identical to HO5 vaccine strain (Nemoto et al., 2015c). The common backbone of the equine RVAs has been shown in Table 2 comprising all the whole genome isolates. Almost all the common whole genome equine RVA strains characterized previously showed conserved genotypic constellation among them and were divergent in nature to the other RVA isolates (Matthijnssens et al., 2012b). Whereas few equine RVA strains characterized showed unique genetic makeup and elaborated the possibility of interspecies jumping of RVAs from one species to the other (Figure 1). The strains showing the possibility of interspecies transmission were equine G3P[18] RVA strain L338 (Matthijnssens et al., 2012b), H-1 strain believed to be transmitted from pigs to equines (Ghosh et al., 2012), whereas strain E3198 seemed to be resultant of canine/feline RVA strain to equines (Mino et al., 2013). Further, strengthening the need to analyze more common and uncommon equine RVA strains around the world the analysis done by two research groups were remarkable (Matthijnssens et al., 2012b; Ghosh et al., 2013). These reports established the fact that more or less out of the 11 segments of RVAs the interspecies transmission event are constantly changing the nature of equine RVA being detected in the horse population worldwide. While a report from UK reported that the strain L338 has no closeness with any other rotavirus and thus confirms that few strains remains solely adaptable to equine with no sign of interspecies transmission (Ciarlet et al., 2001). Although out of the eleven segments of RVA, eight showed the conserved nature among the equine RVAs, whereas only VP6, VP7 and NSP4 genes showed existence of 2 diverse genotypes: I2/I6, G3/G14 and E2/E12, respectively. These reports also hypothesized the presence of distinguished lineages present among the common equine genotype constellations. At least three lineages of equine genomic constellation are circulating among equine rotaviruses since 1990s including G3-P[12]-I6-E2 lineage I, G14-P[12]-I2-E2 lineage I, and G14-P[12]-I2-E2 lineage II (Nemoto et al., 2015c). Though these lineages are interim therefore more complete information will be needed to decipher the relationship of these lineages. Unusually the three complete genome reports of equine RVA from Argentina showed the presence of E12 NSP4 genotype which were recently recovered from guanacos and the unusual bovine RVA strain (G15P[11]) from Argentina (Matthijnssens et al., 2009). Further, this E12 genotype was also detected in all analyzed isolates from 1998 to 2008 in Argentinian foals (Garaicoechea et al., 2011). Certain unpublished data also shown the incidence of E12 NSP4 genotype in Argentinean goat and cattle population. But it was surprising to find that NSP4 E12 genotype is only confined to equine RVA strains to much of extent which may be the success indicator of quarantine procedures involved in the transportation of horses which _________________________________________________________


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restricted its transmission to other continents. But no such geographical barrier is present between the African and European equine RVA strains as two strains (04V2024 and EqRV-SA1) shared nearly identical genotype G14P[12] identified in South Africa and Ireland, respectively. Presence of bovine RVA like NSP4 gene in conserved equine backbone from Japan points towards the probability of equine rotavirus as an acceptor of gene segments from different host species and vice-versa (Nemoto et al., 2015b). Detection of equine origin G3 genotype of VP7 in humans from Asia, Australia and Europe confirms the possible host species jump event of equine rotavirus to human hosts (Malasao et al., 2015; Cowley et al., 2016). Lately, similar reports emerged from Spain and Hungary concerning the emergence of unusual G3P[8] type rotaviruses in humans wherein the VP7 gene represents an equine like G3 (Arana et al., 2016; Dóró et al., 2016). These evolutionary phenomenon i.e. reassortment and species jumping leads to rotavirus genetic diversity which eventually pose a threat to human and other animal host species. Therefore, a more rigorous surveillance programs are prerequisite to establish geographical relationship among the equine rotavirus strains. 5 Picobirnavirus Picobirnaviruses (PBVs) are small, un-enveloped viruses containing bi-segmented dsRNA genome. They contains ~2.5 kbp segments 1 and ~ 1.7 kbp for the segment 2 (Malik et al., 2014). The segment I encodes for the single structural capsid protein gene and segment 2 encodes the viral RNA polymerase (RdRp). It is placed in a new viral family namely Picobirnaviridae and is assigned a new taxonomic order which contains a single genus Picobirnavirus. The genus include only two species i.e. Human Picobirnavirus and Rabbit Picobirnavirus (Malik et al., 2014). The human PBV serves as the type species whereas the rabbit PBV serves as the designated species according to the ICTV. Based on the RdRp gene segment 2, PBVs are grouped into 2 genogroups (genogroup-I and II). Recent studies also proposed possible new genogroup namely III, IV and V based on the diversity of RdRp sequences (Smits et al., 2014; Li et al., 2015). The proposed new groups include strains from human (group III and V), dromedary (group V) and equine (group IV and V). Equine PBV strains have been identified in horse blood plasma (Li et al., 2015). With the advancement of molecular diagnostic techniques such as RT-PCR (Ganesh et al., 2012; Malik et al., 2013; Takiuchi et al., 2016) and qRT-PCR (Malik, unpublished data) for picobirnaviruses, they have been detected in faecal and respiratory samples from over 20 animal species including rodents, aves and large animals like rats, hamsters, guinea pigs, giant ant eater, dogs, pigs, bovine calves, buffalo calves, foals camels, snakes worldwide and exhibit vast genetic diversity (Ganesh et al., 2011; Malik et al., 2011; Smits et al., 2011; Gillman et al., 2013; Malik et al., 2013; Malik et al., 2014; Ng et al., 2014; Ribeiro et al., 2014; Woo et al., 2014, Verma et

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al., 2015; Takiuchi et al., 2016). Despite the broad host range, pathogenicity of PBVs alone or as co-infecting agents remains unclear. In equines, PBVs have been reported for the first time in the year 1991 in foals in Ireland and Britain (Browning et al., 1991b), followed by the RT-PCR detection in a female foal from Kolkata, India (Ganesh et al., 2011). Recently, equine PBVs co-infections were detected in plasma of a horse showing depression, loss of appetite (Li et al., 2015). Sequence analysis of viral RdRp and capsid genes showed four highly diverse picobirnaviruses including a novel fused picobirnavirus genome. These few reports of equine PBVs deserves attention regarding their epidemiological study and towards the development of diagnostic tools. 6 Miscellaneous Enteric Viral Infections Several other viruses have also been shown to induce diarrhea like symptoms in neonatal or young foals but their relevance to enteritis less studied. Parvovirus in animals is usually known for its diarrheal symptoms but in equines it is reported in aborted equine fetus (Wong et al., 1985). Of late, parvovirus was recorded in cerebrospinal fluid (CSF) of a horse exhibiting neurological signs (Li et al., 2015). The study also enlist some other enteric viruses with no relevance to enteritis in equine namely anelloviruses, cyclovirus, kirkovirus etc. The scanty data on these viruses especially in equines is still lacking. Conclusion Many diseases have their multifactorial causes with different symptoms and etiologies. A peculiar type of immune response is prompted by a particular pathogen whereas, other coinfecting pathogens make it complex. In veterinary medicine, the documentation of GIT infections with co-infecting pathogens has not been well studied. Especially in equines, the knowledge of co-infections is very scarce. Different enteric viruses have different symptoms and their spread can be controlled by better diagnosis and intensive care of the foals. Prognosis for a young foal with diarrhea varies according to the causative virus and hence the severity of clinical signs also changes. Apart from vaccine development other options such as herbal preparations can be also tried as potential preventive alternative. Since enteric infections are a big challenge worldwide we need to explore better preventive options available as anything might prove efficient and miraculous in preventing these infection in days to come. For inactivation of the environmental contamination through virus shedding, proper disinfectants must be opted as well as selection of better hand sanitizers must be taken care. Therefore, better management practices, nutritional management, diagnosis and treatment strategy according to the causative virus is important for the equine health.

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ISSN No. 2320 – 8694

LYME BORRELIOSIS IN THE HORSE: A MINI-REVIEW J.H. van der Kolk* Division of Clinical Veterinary Medicine, Swiss Institute for Equine Medicine (ISME), Vetsuisse Faculty, ALP Haras, University of Bern, Länggassstrabe 124, 3012 Bern, Switzerland Received – November 06, 2016; Revision – November 20, 2016; Accepted – December 05, 2016 Available Online – December 17, 2016 DOI: http://dx.doi.org/10.18006/2016.4(Spl-4-EHIDZ).S196.S202

KEYWORDS Lyme borreliosis Horse

ABSTRACT Lyme borreliosis is a multisystemic tick borne disease (also called Lyme disease in humans) which is caused by the Borrelia burgdorferi sensu lato species complex. It is a thin, elongated Gram-negative bacterium and exhibiting motility with flagellar projections. Affected animals mainly show cranial or peripheral neuropathies and uveitis as the more commonly seen extraneural manifestation. Serological evidences confirm its higher occurrence in elderly horses than young ones. Although, incidence of equine Lyme borreliosis is low, its diagnosis is a real challenge. As no indisputable test exists for detecting antibodies to B. burgdorferi, histopathology remains the gold standard and might reveal vascular sclerosis and pleocellular inflammatory infiltrates in neural tissue. Despite antibiotic treatment clinical signs might progress or recur. This review converses with the disease etiology, pathobiology in brief and its mangment from publich health point of view.

* Corresponding author E-mail: [email protected] (J.H. van der Kolk) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.



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1 Introduction Lyme borreliosis caused by the Borrelia burgdorferi sensu lato species complex, is a multisystemic tickborne disease (also called Lyme disease in humans). About a century back, Afzelius was the first to notice expanding skin lesion in a human patient, and named it as erythema migrans, which has now become clear to be as the initial skin manifestation of Lyme disease. Subsequently, in 1976 Burgdorfer and his associates first time reported the etiologic agent of this disease and also performed an epidemiological evaluation on a cluster of children suffering with arthritis in the city Old Lyme, Connecticut, USA. This became the first report on Lyme disease (Mast & Burrows 1976; Steere et al., 1977; Burgdorfer et al., 1982; Butler et al., 2005; Steere 2006; Staneck et al., 2012). Hitherto studies have revealed geographical restiction of this bacteria, as in Europian countries, five genospecies of B. burgdorferi s.l. have been found to affect humans, namely B. afzelii, B. burgdorferi s.s., B. garinii, B. spielmanii and B. Bavariensis. Among these, the first three are the predominant species. While in North America, B. burgdorferi s.s. is the only species that is pathogenic for humans. In Asian region, B. garinii is more commonly reported (Staneck et al., 2012). In the equine species, diagnosis of Lyme borreliosis remains a challenge like in other species. As a consequence, the question if Lyme borreliosis in horses is overdiagnosed remains valid even to date (Bartol, 2013). This review converses with the recent information available on Lyme borreliosisin the horse. 2 Etiology Borrelia sps. are gram-negative, flagellated, thin and elongated motile bacteria. This bacterium possess 21 plasmids (nine circular and 12 linear), which is the largest number of plasmids to be found in any known bacterium. Moreover, it also shows genetic complexity with at least 132 functioning genes, intracellular localisation, immune evasion, and auto-regulation, which makes this spirochaete a formidable infectious pathogen (Qiu et al., 2004; Stricker et al., 2005).These bacteria belong to the phylum Spirochaetes and are grouped in the B. burgdorferi s.l. genospecies complex that contains at least 20 proposed genospecies such as B. afzelii, B. garinii, B. burgdorferi sensu stricto, B. andersoni, B. japonica, B. lusitaniae, B. sinica, B. tanuki, B. turdii, B. valaisiana, and B. bissettii (Stanek et al., 2004; Wang et al., 1999; Becker et al., 2016). Phylogenetically all these genospecies are classifed into two major clades: one that represents Europian and Asian species and another consists of species found in North America and Europe. Furthermore, the two groups in the “American” cluster (B. burgdorferi s.s. and B. Bissettiae) occur in Europe as well as in North America. This raises concern over the common ancestor of this cluster whether it originated in North America or Europe (Becker et al., 2016).

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In Austria, B. afzelii is the predominant genospecies in clinically normal horses (Muller et al., 2002). Lately, B. lusitaniae infection has been noticed in horses in Italy (Veronesi et al., 2012). 3 Epidemiology In Europe, Ixodes ricinus is the main vector of B. burgdorferi s.l. in comparison to black-legged ticks (Ixodes scapularis) which is more common in the USA. The small rodents also act as reservoirs (Gern et al., 1998, Humair et al., 1998). Additionally, birds have been found to play a major role in the ecology of Lyme borreliosis, where ticks have been transported over large distances and across geographical barriers by avian hosts (Humair, 2002). To note, particular vector adaptations determine the geographic distribution range of genospecies (Becker et al., 2016). It has been noticed that B. burgdorferi s.s. may persist in clinically healthy horses (Chang et al., 2000). The seroprevalence of Lyme borreliosis in horses varies with geographical areas. In some areas of the north-eastern USA it is about 33-50% (Funk et al., 2016; Magnarelli et al., 2000), 9.8-42.8% in Brazil (Basile et al., 2016), 31-48% in France (Maurizi et al., 2010), 29% in Denmark (Hansen et al., 2010), 26% in Poland (Stefanciková et al., 2008), 24% in Italy (Ebani et al., 2012), 6% in Turkey (Bhide et al., 2008), and 5.5% in Korea (Lee et al., 2016). Older horses are more prone for positive test response than younger ones (Ebani et al., 2012; Funk et al., 2016). Of note, no significant differences in the mean seroprevalence were observed in the respective years in Italy (Ebani et al., 2012). It has been indicated that the majority of horses that were positive on initial testing did not have a different test result 5-17 months later (Funk et al., 2016). Recently, Lee et al. (2016) showed statistically significant differences according to breed and region where variances might be attributed to the ecology of vector ticks and climate conditions. The presence of viable B. burgdorferi spirochetes observed in clinically healthy horse’s urine in an endemic region (Manion et al., 1998) has raised concern whether non-tick transmission of this bacterium may occur by direct urine/mucosal contact (Butler et al., 2005). 4 Pathophysiology The attachment of an outer-coat protein (OspA) displayed on the lumenal side of the gut of ticks (like black-legged ticks Ixodes scapularis or Ixodes ricinus) to a receptor (TROSPA) favours B. burgdorferi to persist in the gut and avoid elimination (Pal et al., 2004). The infection may be acquired through larvae or nymphs feeding on small to medium sized wild animals harbouring the B. burgdorferi as reservoir. Adult ticks have been found to only engorge on larger animals (deer, sheep, cows and horses). Of the note, B. burgdorferi s.l. DNA has been more commonly detected in female ticks, followed by

Lyme borreliosis in the horse: A mini-review

nymphs and larvae, and least in adult male ticks (Wodecka, 2003). After the attachment of ticks to a host, the spirochetes present in the midgut of ticks move through the midgut wall and haemocoel, reach the salivary glands and get inoculated with the saliva of ticks into the host 2-3 days after attachment (Piesman et al., 1987). On few occasions, inoculation may occur earlier if spirochetes are already present in the salivary glands of the infected tick (Alekseev et al., 1995). Though, for proper B. burgdorferi transmission to occur an infected tick must attach for at least 24 hours on the animal (Thanassi & Schoen 2000), its transmission to the host has been seen to occur as early as 18 hours after attachment (Alekseev et al., 1995). Concurrent infection with other tick-borne pathogens like Anaplasma phagocytophilum (Persing 1997) and Theileria equi (Basile et al., 2015) can occur. The predominant migration of B. burgdorferi within connective tissues may provide protection to this bacterium from humoral antibodies (Divers et al., 2001). 5 Clinical Presentation Of the 16 equine cases with histologically confirmed Lyme borreliosis recently reviewed, 12 were geldings, while the remainder was mares. Breeds included 6 Thoroughbreds, 2 Paints, 2 Ponies, 2 Quarter Horses, and one each of Haflinger, Arabian, and Morgan. The breed was not known in one of the cases studied. The horses were not vaccinated against Borrelia (Johnstone et al., 2016). Incubation period of this bacterium in the equine species has not been established yet. Clinical signs in horses attributed to B. burgdorferi include low grade fever and lethargy (Burgess & Mattison 1987b; Magnarelli et al., 1988; Johnstone et al., 2016), weight loss (Johnstone et al., 2016), changes in behavior (Johnstone et al., 2016), dysphagia (Johnstone et al., 2016), lameness (Browning et al., 1993), arthritis (Burgess et al., 1986; Hahn et al., 1996; Passamonti et al., 2015; Johnstone et al., 2016), neck stiffness (Johnstone et al., 2016), episodic respiratory distress (Johnstone et al., 2016), muscle tenderness (Divers et al., 2003) and fasciculations (Johnstone et al., 2016), anterior uveitis (Burgess et al., 1986; Hahn et al., 1996; Johnstone et al., 2016), cranial nerve deficits (Johnstone et al., 2016), ataxia (Johnstone et al., 2016), meningo-encephalitis (Burgess & Mattison 1987b; James et al., 2010; Imai et al., 2011), abortion (Sorensen et al., 1990), cardiac arrhythmias (Johnstone et al., 2016) and foal mortality (Burgess et al., 1987a). Ataxia was characterized by general proprioceptive deficits and was frequently reported in conjunction with limb paresis. Signs of generalized lower motor neuron weakness, facial nerve deficits with paresis or muscle fasciculations have been observed less frequently. Dysphagia, tongue paresis and fasciculations are also clinically evident. The variation reported in presenting complaints reflects the multisystemic nature of Lyme borreliosis. Uveitis has been reported to be the most frequent extraneural manifestation of Borrelia infection (Johnstone et al., 2016).

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The variation of the clinical presentation in B. burgdorferi infected horses might be unapparent due to co-infection with pathogens like A. phagocytophylum. Such variation might also occur due to infection with different genospecies of B. burgdorferi as has been seen in case of human beings (Butler et al., 2005). 6 Differential Diagnosis The differential diagnosis is large not only due to the great variety in clinical signs but also associated with different B. burgdorferi genospecies and possible co-infection. 7 Diagnosis The diagnosis of borreliosis in horses as well as in other species remains a challenge as persistent B. burgdorferi infections without any clinical symptoms have been documented in horses too (Chang et al., 2000). Antibodies can be detected at 5-6 weeks in Ponies exposed to ticks infected with B. burgdorferi, with the highest antibody levels induced at 3 months after exposure (Chang et al., 2000). Preference might be given to culture of B. burgdorferi from equine skin biopsies (Chang et al., 2000) combined with a two-step serology protocol (ELISA or IFAT supplemented by protein immunoblotting like Western blot or reverse line blot) (Trevejo et al., 1999; Magnarelli et al., 2000; Butler et al., 2005). The development and validation of a new fluorescent beadbased multiplex assay for the detection of antibodies to outer surface protein A (OspA), OspC and OspF antigens of B. burgdorferi in horse serum has been reported. This assay has improved analytical and diagnostic sensitivities compared to Western blot analysis. Multiplex analysis is a valuable quantitative tool that simultaneously detects antibodies indicative for natural infection with and/or vaccination against the Lyme pathogen (Wagner et al., 2011). Commercial C6 testing identified most infected horses but also resulted in false positive and false negative interpretations (Johnson et al., 2008; Wagner et al., 2013; Schvartz et al., 2015b). A recent study indicated that the available serologic tests (a point-of-care C6 enzyme-linked immunosorbent assay (ELISA), an whole-cell IFAT, an ELISA confirmed with Western blot, and the Lyme multiplex assay for antibodies against B. burgdorferi) all lacked agreement when used to assess the exposure to B. burgdorferi of horses from a lowprevalence population. Samples found positive by whole-cell IFAT and Lyme multiplex assay, detecting antibodies against Osp C during early antibody responses, could yield negative results by ELISA. The differences between the diagnostic tests owes to varying sensitivities for Osp C antibodies detection, supported by the low anti-Osp C titers in the Lyme multiplex assay. Caution against the use of serologic “screening” in the absence of clinical suspicion has been advocated accordingly (Schvartz et al., 2015a).

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Overall, 94% of the suspected horse samples were seropositive by luciferase immunoprecipitation systems (LIPS) test, and heat map analysis revealed that seropositive samples often were immunoreactive with at least two of the three antigens (against the synthetic VOVO antigen, comprising repeated immunodominant C6 epitopes as well as OspC immunodominant epitopes). These results suggest that LIPS tests employing multiple recombinant antigens offer a promising approach for the evaluation of antibody responses in Lyme borreliosis (Burbelo et al., 2011).

5/10 horses with histologically confirmed Lyme borreliosis. Of the 5 cases that had a negative PCR, 4 were positive with Warthin–Starry stain and one showed an indication of intrathecal antibody production (Johnstone et al., 2016). The distribution and histologic features including vascular sclerosis (indicating chronicity) and pleocellular inflammatory infiltrates present in horses with histologically confirmed Lyme borreliosis are rather characteristic (Johnstone et al., 2016).

Due to limitations of false negative results of serum tests during B. burgdorferi associated uveitis and their failure to identify an active infection, a combination of cytologic assessment, antibody, and/or PCR testing of ocular fluids has been suggested to be worthwhile when the clinical suspicion is high for Lyme uveitis.

In comparison to oral administration of doxycycline or parenteral sodium ceftiofur, tetracycline @ 6.6 mg/kg BW IV bid for 3 weeks has been found to be superior for treatment of B. burgdorferi infected ponies (Divers et al 2003). Besides, avoiding tick-infested areas as well as careful grooming of the horse for early removal of ticks are the best preventive measures. For prevention of tick-infestation, various kind of insecticidal sprays can be used but most of these have not been approved for horses and also their efficacy is unproven yet (Butler et al., 2005). However, the use of canine tick sprays on horses till so far has not revealed any adverse effects (Divers et al., 2001).

Of note, horses treated with antibiotics revealed a decline in ELISA titres as compared to control horses (P ≤ 0.05) while the untreated horses showed increased ELISA titres (OR = 0.5; 95% C.I. = 0.3-0.9). Such decline in ELISA titres was low in comparison to the previously reported results in experimentally infected and treated ponies. Horses exposed in the field with B. burgdorferi having high ELISA values when treated with either oxytetracycline or doxycycline may show only a small decline in ELISA values (Divers et al., 2012). It should be realized that negative serology and normal CSF analysis do not exclude the diagnosis of Lyme borreliosis and it has been stated that histopathology might represent the most definitive test for borreliosis in horses. However, when presented with a horse displaying ataxia, cranial nerve deficits, and weight loss, with historic or current evidence of uveitis, collapse, or dysphagia, one should consider Lyme borreliosis regardless of CSF analysis or serological results (Johnstone et al., 2016). 8 Pathology Duration of disease before death has been observed to range from 2 to 730 days with a median of 120 days (IQR 33–180 days) (Johnstone et al., 2016). Spirochetes can be visualized in affected tissues of the horses with Lyme borreliosis by Steiner silver impregnation and immunohistochemistry, predominantly within the dense collagenous tissue of the dura mater and leptomeninges (Imai et al., 2011). Leptomeningitis, lymphohistiocytic leptomeningeal vasculitis, cranial neuritis, and peripheral radiculoneuritis with Wallerian degeneration are the lesions observed during histopathology, which are consistent with a diagnosis of neuroborreliosis (James et al., 2010; Johnstone et al., 2016). In comparison, lesions in B. burgdorferi s.s. infected ponies have been reported to be limited to the skin, observed as perivascular and perineural lymphohistiocytic aggregates in the superficial and deep dermis (Chang et al., 2000). B. burgdorferi PCR of nervous tissue obtained positive results in _________________________________________________________


9 Management/Treatment (including prognosis)

Of interest, a 12-year-old thoroughbred horse with B. burgdorferi infection responded well to doxycycline treatment (10 mg/kg BW PO q 12 h for 60 days) and returned to normal exercise. However, the horse was found to again develop a stiff neck and rapidly progressive neurologic deficits along with severe ataxia and vestibular deficits after 60 days of treatment. The condition of the horse got deteriorated rapidly despite administering IV oxytetracycline, and it was euthanatized (James et al., 2010). Eight horses out of sixteen horses with histologically confirmed Lyme borreliosis received antibiotic treatment, including doxycycline, minocycline, oxytetracycline, or ceftiofur. In these cases, clinical signs either continued to progress or, despite an initial improvement, plateaued or showed recurrence and subsequent progression (Johnstone et al., 2016). Of importance, persistently high serum titres observed after treatment of Lyme disease in horses, without the presence of clinical signs, may not be a reason to follow more prolonged treatment (Divers et al., 2012). 10 Public Health Significance Lyme borreliosis is regarded as an important tick borne zoonosis although the equine species is not considered as a main reservoir for human infection. However, the presence of viable B. burgdorferi spirochetes has been observed in urine of clinically healthy horses (Manion et al., 1998). Lyme borreliosis is the most common human tick-transmitted disease in the northern hemisphere. A complete presentation of the disease is an extremely unusual observation in which a skin lesion results from a tick bite and is followed by heart and


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ISSN No. 2320 – 8694

AN OVERVIEW OF OZONE THERAPY IN EQUINE- AN EMERGING HEALTHCARE SOLUTION Jyotsana Bhatt1,*, Abas Rashid Bhat1, Kuldeep Dhama2 and Amarpal3 1

Research Scholar, Division of Surgery, Indian Veterinary Research Institute, Izatnagar-243122 (UP), India Principal Scientist, Division of Pathology, Indian Veterinary Research Institute, Izatnagar-243122 (UP), India Head, Division of Surgery, Indian Veterinary Research Institute, Izatnagar-243122 (UP), India

2 3


KEYWORDS Ozone therapy Equine Oxidative stress Antioxidant Hydrogen peroxide

ABSTRACT The significance of ozone therapy has been increasingly realised in recent times particularly in equine medicine. The beneficial effects of ozone therapy are basically engendered by the mild oxidative stress it creates upon interacting with the extra-cellular and intracellular components. However therapeutic benefits of treatment could be obtained only when it is used within the therapeutic window. Higher doses may be counterproductive and lower doses ineffective. It is now well proved that it up regulates the antioxidant system of the patient and may provide relief from many chronic degenerative diseases upon prolonged use. Ozone therapy has shown encouraging results in the treatment of wide spectrum of diseases and disorders in equines including bacterial and viral infections. Obvious benefits of ozone therapy have been reported in Equine infectious anemia, chlamydial abortions, lymphomas and equine ehrlichiosis. This article provides an insight into the mechanism of action involved in ozone therapy and reviews various conditions which could be treated with the use of ozone therapy in equines.

* Corresponding author E-mail: [email protected] (Dr Jyotsana Bhatt) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.



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1 Introduction Ozone molecule comprises of three atoms of oxygen arranged in a dihedral shape. This allotropic form of oxygen is less stable than oxygen due to the presence of mesomeric states (Elvis & Ekta, 2011) and reacts with many other compounds like carbon and nitrous oxide. Ozone is 1.6 fold denser and 10fold more soluble in water than oxygen. It is the third most potent oxidant after fluorine and per-sulfate. Ozone is an unstable gas that cannot be stored and should be used at once because it has a half life of 40 min at 20°C and 140 min at 0°C (Cakir,2014). In nature ozone is produced by the effect of electrical discharge on atmospheric oxygen during lightening or by ultraviolet radiation. It is a powerful oxidant and has many applications owing to its oxidant property. It is a highly reactive molecule which is able to inactivate microorganisms, boost the immune system and is also able to induce analgesic effect (Duricic et al., 2015). Ozone has many health benefits if used within the therapeutic window but may cause tissue damage if used above it. Ozone oxygenates every cell of the body and increases the stability of healthy cells. Medical ozone is a mixture of oxygen and ozone having less than 5% of ozone at maximum concentration and rest is pure oxygen (Bocci, 2006). Unfortunately many clinicians and veterinary professionals are unaware of the therapeutic benefits and mechanism of action of ozone upon its interaction with biological fluids. As in most of the cases it is the dose which would decide the therapeutic or harmful effects of ozone. But it is now possible to perfectly tame the cytotoxicity of ozone by potent antioxidant system of the body. During last two decades scientists have made great efforts to understand the scientific mechanisms underlying the beneficial effects of ozone therapy at both basic research and clinical level. Ozone therapy has now been established as treatment of choice for many equine diseases like Equine infectious anaemia. Ozone therapy is also able to boost the antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase etc. in the body (Mandhare et al., 2012). 2 Ozone productions As in nature, ozone is produced by the action of lightening on oxygen, ozone generation can be achieved by passing oxygen across an electric arc having a potential difference of about 10,000 Volt in an ozone generator according to the following reaction3O2 + 68,400 cal------2O3 An ozone generator must be made up of high quality ozone resistant material like teflon, glass, 316 stainless steel, silicone etc to resist oxidation (Mandhare et al., 2012). It should also be equipped with a photometer to measure the accurate concentration of ozone produced. There must also be a _________________________________________________________


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provision for temperature and humidity control as temperature and humidity also play important role in ozone production. A variety of ozone generators are available nowadays. However, three types of ozone generators are commonly used for production of medical ozone. I. Corona discharge type- This type of ozone generators use corona discharge tube (high voltage electric field). They are cost effective and use ambient air as a source of oxygen. This type of ozone generators produce a concentration of 3-6% of ozone, however they also produce nitrous oxide as a by-product. A research showed the use of meshed–plate electrode in place of conventional plate has advantage of decreasing corona onset voltage and reduced decomposition of ozone thereby providing maximum ozone generation concentration and ozone generation efficiency (Park etal., 2006) II. Ultraviolet lamp type- This type of ozone generators utilise a light source that generates a narrow-band ultraviolet light with a wavelength of approximately 185 nm. They produce ozone with concentration of 0.5% or less but this type of ozone generators are cost effective than the corona discharge type generators as they consume less electricity. Added advantage of such type of generator is that it doesn’t produces nitrous oxide (http://www.silvermedicine.org/ozone-therapygenerators.html). III. Cold plasma type- In this type of generator oxygen gas is exposed to a plasma created by dielectric barrier discharge. These ozone generators breaks oxygen molecule in atoms of oxygen, which are very reactive and combines the available oxygen molecules to form a molecule of ozone. A maximal concentration of 5% is produced in such generators (http://www.silvermedicine.org/ozone-therapygenerators.html). Ozone molecule is very unstable so it must be prepared immediately before use. Within less than an hour after preparation only half of the mixture remained ozone while the rest half transformed into oxygen. Because of this characteristic it is impossible to store medical grade ozone for long period of time (Nogales et al., 2008). 3 Mechanism of action of ozone Ozone on coming in contact with the blood acts on different targets and initiates a cascade of reactions thereby producing several beneficial effects. Unlike oxygen, ozone is very active gas and reacts as it comes in contact with blood or any other biological fluid. In order of preference, ozone reacts with polyunsaturated fatty acids (PUFA), protiens, antioxidants such as ascorbic acid and glutathione.

An overview of ozone therapy in equine- an emerging healthcare solution

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Figure 1 Schematic representation of ozone therapy procedure in horses. On reaction with bio molecules it produces one molecule of reactive oxygen species (ROS) mainly hydrogen peroxide and two molecules of lipid oxidation products(LOP). The main reaction with bio molecules is depicted below: R-CH=CH-R + O3+H2O ----- R-CH=O + R-CH=O + H2O2(Bocci et al.,1993) The by-products of the reaction tend to act in two different ways. ROS act immediately by reacting with erythrocytes which are available in the blood stream and disappear. This may be termed as early phase reaction, which is short lived. LOP, on the other hand, are distributed in the tissues and act on receptor molecules located at different locations in body, undergoing marked dilution within the circulatory system and thus their action may be termed as late phase reaction, which lasts longer. ROS, particularly hydrogen peroxide, activates pentose phosphate pathway which is determined by the significant increase in the ATP formation (Bocci, 2005). LOPs produced in the reaction are mainly malonaldehyde and 4hydroxynonenal, which are very stable and toxic as well in vitro as compared to ROS. Fortunately, they undergo marked dilution in the circulation and gets metabolised upon blood distribution and redistribution in the body thus they reach at their target sites only in the submicromolar concentration thereby minimising their toxic effects.

with NO regulates the vasodilation by activating cGMP (Bocci,2006). The mechanism of beneficial effects of ozone therapy may be summarised in the following waysI. II.

III. IV.

It increases availability and delivery of oxygen, glucose and ATP within ischemic tissues. It enhances implantation of bone marrow stem cells at the site of lesion, which can provide angiogenesis, neovascularization and tissue regeneration. It activates a neurohumoral reaction responsible for improving quality of life. It induces up-regulation of the expression of antioxidant enzymes and heme-oxygenase I and extends preconditioning benefits (Bocci,2006).

Ozone however could be toxic to the respiratory system, if inhaled, because respiratory tract lining has minimal amount of antioxidant coverage. In contrary to that blood has adequate amount of antioxidants to completely tame up the ozone toxicity, if used within the limits of therapeutic range. It is advisable that to enhance the safety of ozone therapy, prior to initiation of the therapy, antioxidant level of the patient body should be measured and should be strengthened by administration of antioxidants like vitamin C , α-tocopherol etc (Sagai & Bocci,2011). 4 Methods of administration of ozone

Small concentration of LOPs extend their beneficial effect by up-regulation of the antioxidant enzymes like superoxide dismutase, glutathione peroxide etc (Iles & Liu,2005). They also induce oxidative stress protein like Hemoxygenase-1(HO1) or Heat shock protein. HO-1 degrade the heme molecule into CO and bilirubin (Snyder & Barañano, 2001). Bilirubin acts as a powerful antioxidant molecule whereas CO along

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4.1 Auto-hemotherapy Autohemotherapy was first described by Wherli & Steinbart in 1954. It may be major or minor autohemotherapy.

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In major autohemotherapy method around 250ml of blood is collected from the animal in heparin or 3.13% sodium citrate anticoagulant and the blood is ozonated outside the body of the animal for 5-10 minutes and then this ozonated blood is slowly infused back into the animal’s body in over 15 minutes by intravenous routes. Ozonated autohemotherapy (O3-AHT) was used in the treatment of atherosclerotic ischemia of lower limb in human patients (Tylicki et al., 2004).While in minor autohemotherapy method, around 5ml of venous blood is collected from the patient without anticoagulant and ozonated outside the body for 1 minute. The ozonated blood is then injected intramuscularly (Borrelli &Bocci, 2009). This minor autohaemotherapy approach had been used in treatment of chronic laminitis in a 10 years old mare (Coelho et al., 2015) and for mechanical lumbar pain in riding horses (Vigliani et al., 2005). 4.2 Insufflation Insufflation of ozone is done in the body spaces like rectal, vaginal and ear canal. Rectal insufflation of ozone is most commonly practiced. Humidified ozonated gas can be introduced through the rectal opening to treat conditions like diarrhoea and inflammatory bowel disease caused by infections such as Rotavirus and Ehrlichia in horses. Rectal sufflation of medical ozone was also used in the treatment of patients with type 2 diabetes and diabetic feet (Martínez-Sánchez et al., 2005). 4.3 Ozone bagging In this method an oxygen-ozone mixture is pumped into an ozone resistant bag which is then placed around the area to be

treated. In this method superficial lesions can be treated as ozone is absorbed through the skin. The method has been applied for the treatment of cutaneous infections like chronic wounds and ulcers (Bocci, 2013). 4.4 Ozonated oil In ozonated oil method the ozone is used with oil as a carrier of ozone. Ozone is bubbled in oil like olive, sesame or sunflower oil until it forms a gel like consistency, the gel can be used to treat several conditions like skin infections, insect stings, ulcers, vulvovaginitis and periodontitis (Shoukheba & Ali, 2014 ). 4.5 Ozony blanket In ozony blanket method an ozonated silicone blanket is placed around the horse body to ozonate the whole body of the animal. It could be used to treat several local and systemic conditions. This system proved to be very effective in treating various equine diseases (http://ozonyozone.weebly.com/ozonyblanket.html (accessed on 24/11/2016) 5 Medical application of ozone therapy in equine Owing to its versatile biological action, ozone therapy is able to treat a wide variety of diseases and conditions. Equine medicine is a potential field for therapeutic exploitation of this modality as it has a capacity to reverse many serious illnesses. Initial studies suggested that ozone therapy could be very effective in treating many equine diseases by controlling infection, mitigating inflammation and improving anti-oxidant status.

Table 1 Some of the important studies showing beneficial outcome of ozone therapy in clinical cases of equine. S.no.

Scientist / Investigator Coelho et al., 2015

Disease condition

2

Vigliani et al., 2005

Mechanical lumbar pain

30µg/ml

3

Shinozuka et al., 2008 Akey & Walton, 1985

Mastitis

0.8mg/l

1

4

5

Sechi et al., 2001

6

Ozbay et al., 2016 Ouf et al.,2016

7

Chronic laminitis

Inactivation of Venezuelan equine encephalomyelitis virus Cutaneous wound healing Facial nerve paralysis Antifungal effect

Ozone concentration 19mg/L

After effects

IM, at suprascapular region, Near deep digital flexor tendon IM, at interspinous and paravertebral level

Recovered to obel grade IV to grade II of lameness

Into the affected quarter

Signs of clinical mastitis reversed reduction of 99.99997% of the viral particles

0.025 mg/L

Physical exposure for 45 minutes

1·18– 9·5 mg/ ml

Topically as ozonised oil

1.1 mg/kg body weight 4 µg/ml 0.5 and 0.25 µg/ml

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Site of administration

Intraperitoneally As ozone gas spray As ozonised oil

Effective analgesic effect

Better healing with increased production of various growth factors Lower stimulation thresholds in ozone treated group Better recovery with least side effects


Several reports and clinical trials have appeared in the recent literature to prove the efficacy of ozone therapy as an adjunct therapy as well as a sole therapeutic agent. The following text describes various medical applications of ozone therapy in treating equine diseases. 5.1 Chronic laminitis Chronic laminitis is a crippling disease of equine. It does not easily respond to standard medical therapy. Ozone therapy could be a solution for such cases. A case of chronic laminitis in a 10 year mare diagnosed with Obel grade IV chronic laminitis on right forelimb was successfully treated with ozone therapy (Coelho et al., 2015). On presentation the horse exhibited signs of laminitis like shifting lameness, high temperature, pain on palpation etc. On radiological examination distal phalanx of right limb was displaced by 30 degrees. Therapeutic regimen comprised of hoof trimming followed by intramuscular, peritendinous and intrarectal administration of medical ozone. The injection of ozone was made at two suprascapular points located cranial and caudal to the scapula on both sides, two points were selected in scapular region, one point in the middle of the radial region and one near the deep digital flexor tendon. The selected points were clipped and aseptically prepared and a 10 ml mixture of ozone-oxygen having a concentration of 19 mg ozone per ml was injected at each point. No NSAIDs or any other medication was used in the treatment regimen. Intrarectal insufflation of ozone was also performed. Ozone therapy was given twice a week for 10 weeks. Six months after therapy mare was able to walk properly along with normal relationship between the dorsal hoof wall and the distal phalanx on radiographic examination. Animal recovered up to the Obel grade II lameness with the help of this therapy (Coelho et al., 2015). 5.2 Mechanical lumbar pain and spinal muscle disorders in riding horse Riding and thoroughbred horses which are commonly used for racing purpose frequently suffer from back pain. Its probable causes include bone or soft tissue injuries. In a study 30 horses suffering from back pain were treated with local infiltration of 15 ml of oxygen-ozone mixture at the ozone concentration of 30µg/ml into the affected muscle at interspinous and paravertebral level. This study proved efficacy of ozone therapy in pain management. Hence, ozone therapy could be considered as an alternative to NSAIDS treatment in cases of lumbar pain (Vigliani et al., 2005). It has been observed that during its administration, ozone produces an itchy sensation which is replaced by the analgesic effect as animal does not feel pain on palpation at the later stage. Supplementation with antioxidant in the form of vitamin C to maintain oxidantantioxidant balance could benefit the patient during ozone therapy (Ballardini, 2005).

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5.3 Clinical mastitis in mares Mastitis in mare is not quite common as in dairy cattle but can affect the health and productivity of mare adversely. Gram negative organisms accounted for mastitis in 42% of cases including Klebsiella and E.coli in 11.8% and 5.9% of cases, respectively (McCue & Wilson, 1989). These gram negative microbes tend to develop endotoxic shock upon treatment with antibiotics. A study was conducted to compare the amount of endotoxin produced upon antibiotic therapy and ozone therapy.Results showed that in comparison to antibiotic therapy, ozone therapy (0.8mg/L) resulted in less amount of endotoxin production (Shinozuka et al., 2008). It is observed that ozone therapy helps in reversing the local and systemic signs of acute clinical mastitis in animals. The therapeutic effects of ozone therapy could be attributable to increase in leukocytic function and increased respiratory burst. Ozone therapy can be preferred over antibiotic therapy in treating clinical mastitis as no milk withdrawal time is required with ozone therapy (Ogata & Nagahata, 2000). However, it is not clear whether ozone functions by killing the pathogen or by improving the host defence mechanism. 5.4 Enhancement of antioxidant capacity Oxidative stress can adversely affect the physical ability of performing horses. Ozonated autohemotherapy (OAHT) has shown to increase the antioxidant capacity of the blood. In a study 10 thoroughbred horses were examined for their biological antioxidant potential (BAP) after treating with ozone at the concentration of 20µg/kg body weight .Same horses were used as control and for treatment. Control blood and serum samples were collected one month before the OAHT and for treatment group 1, 2, 3, 7 and 14 days later. Diacronreactive oxygen metabolites (d-ROMs) and biological antioxidant potential (BAP) was measured from serum samples to calculate oxidative stress index (OSI).BAP was increased significantly on day 3 and 7 in OAHT group as compared to control group (Tsuzuki et al., 2015). 5.5 Inactivation of Venezuelan equine encephalomyelitis virus Humidified ozone gas is used as a sterilizing agent for medical instruments (Faddis, 1993). Ozone in liquid phase application at a concentration of 0.025 mg per liter was able to inactivate arbovirus within 45 min of exposure. This study showed a reduction of 99.99997% of the viral particles as compared to the control levels. Hence ozone proved to be an effective candidate as a sterilizing agent in some applications for biological safety cabinets and other equipment used in vector studies with arboviruses (Akey & Walton, 1985). 5.6 Cutaneous wound healing Ozonated olive oil proved to be effective in healing of cutaneous wounds.

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In a study using guinea pigs as animal model the ozonated olive oil showed better wound healing in terms of lesser residual wound area, increased number of collagen fibres and fibroblast along with upregulation of various growth factors like PDGF, TGF-β and VEGF in comparison to the group treated with simple olive oil and control group (Kim et al., 2009). Ozone used for wound treatment causes reduction in septic process and accelerates wound healing and also decreases the cost of antibiotic therapy (Białoszewski & Kowalewski, 2003).A research showed increased angiogenesis, an enhanced vascular endothelial growth factors and cyclin D1 expression as a result of using ozonated sesame oil in cutaneous wound healing study model in SKH1 mice (Valacchi et al., 2011). In a study, effect of Oleozon (ozonised sunflower oil) was tested on Mycobacteria, staphylococci, streptococci, enterococci, Pseudomonas and Escherichia coli, which showed encouraging antimicrobial effects of oleozon on tested organisms (MIC range 1·18–9·5 mg/ ml) (Sechi et al., 2001) . 5.7 Neurological malfunctions (facial nerve paralysis) In a case record of 450 horses with signs of neurological disease, facial nerve paralysis was the most common type of cranial nerve injury (Tyler et al., 1993). Ozone therapy provides promising results in facial nerve regeneration. In a study comprised of fourteen Wistar albino rats, all animals underwent surgery in which the left facial nerve was exposed and crushed. Treatment with saline or ozone began on the day of the nerve crush. The ozone group received an ozone dose of 1.1 mg/kg/d intraperitoneally (IP) for 30 days. Left facial nerve stimulation thresholds were measured before crush, immediately after crush, and after 30 days. Post-crushing, the ozone-treated group had lower stimulation thresholds than the saline group. In this study regeneration of the facial nerve was evaluated by assessing electrophysiological thresholds and by histopathological examination. This proves that ozonetherapy exerted beneficial effect on the regeneration of crushed facial nerves (Ozbay et al., 2016). 5.8 Antifungal potential of ozone Fungal infection possesses a serious threat to animal health eventually leading to decreased productivity and work potential. Trichophyton equinum was recognized as the most common cause of equine ringworm in the United States, Canada, South America, and Europe (Georg et al., 1957). M. canis, T. Mentagrophytes varmentagrophytes, T. verrucosum, M. Praecox and M. Gypseum are also reported to be associated with equine ringworm (De Vroey et al., 1983). According to Ouf et al. (2016) Ozone therapy proved to be a better alternative in curing fungal infection and also prevents side effects of synthetic antifungal drugs due to their long term application. Fungal wall comprised of approximately 80% carbohydrates and 20% of proteins and glycoproteins with multiple disulfide bond making it possible site for oxidative inactivation by ozone. MIC for growth and spore germination for M. Gypseum and M. canis was 4 µg/ml in the case of ozone _________________________________________________________


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applied as gas and was 0.5 and 0.25 µg/ml in the case of ozonized oil. Application of ozone in the form of ozonized oil appears to be more efficacious than gaseous ozone. 5.9 Other applications Several studies have been conducted to develop a non invasive technique of ozone therapy in which an ozone blanket is used to ozonate the whole body of the horse (http://ozonyozone.weebly.com/ozony-blanket.html accessed on 24/11/2016). This method has shown promising results in a wide range of ailments in horses. The method was developed by South African scientist Gail Pedra (http://ozonyozone.weebly.com/ozony-blanket.html accessed on 24/11/2016). These researchers treated a foal suffering from African Horse Sickness Virus in 2006 by ozonation of the whole body. This method is applied to treat illness by covering the animal inside an ozony blanket. Several conditions in which ozony blanket yields significant outcomes are sarcoma, African horse sickness, lyme disease, pro-active sport therapy etc. The ozony blankets have been used widely in South Africa and approximately1000 horses have been successfully treated for disease control, prevention or enhancement of sports performance. This technique has various benefits as it is a completely portable device, even recumbent animal is able to receive treatment and multiple horses can be handled at one time. Conclusion The benefits of ozone therapy have been documented in numerous studies. It has been proved to be effective as an alternative treatment or as an adjunct treatment for many equine diseases like lameness, mastitis, bacterial and viral diseases, neurological and musculoskeletal disorders etc. Ozone acts to induce mild oxidative stress that can induce production of several anti-oxidant enzymes to benefit the patients and gradually being accepted as therapeutic modality in orthodox medicine. Medical grade ozone generators and photometers are essential for its optimum production and potential useful effects. There could be several methods of its administration, which should be standardised for maximum therapeutic benefits. Conflict of interest Authors would hereby like to declare that there is no conflict of interests that could possibly arise. References Akey DH, Walton TE (1985) Liquid-phase study of ozone inactivation of Venezuelan equine encephalomyelitis virus. Applied and Environmental Microbiology 50: 882-886.doi 0099-2240/85/100882-05$02.00/0 Ballardini E (2005) Oxygen-Ozone Therapy for spinal muscle disorders in the horse. RivistaItaliana di Ossigeno-


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