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Fowl Typhoid (Salmonella Gallinarum) Outbreak in Japanese Quail (Coturnix coturnix japonica). Author(s): Renata A. Casagrande , Angelica T. Barth Wouters ...
Fowl Typhoid (Salmonella Gallinarum) Outbreak in Japanese Quail (Coturnix coturnix japonica) Author(s): Renata A. Casagrande  , Angelica T. Barth Wouters , Flademir Wouters , Caroline Pissetti , Marisa R. de Itapema Cardoso , and David Driemeier   Source: Avian Diseases, 58(3):491-494. 2014. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10796-021114-Case.1 URL: http://www.bioone.org/doi/full/10.1637/10796-021114-Case.1

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AVIAN DISEASES 58:491–494, 2014

Case Report— Fowl Typhoid (Salmonella Gallinarum) Outbreak in Japanese Quail (Coturnix coturnix japonica) Renata A. Casagrande,AB Angelica T. Barth Wouters,A Flademir Wouters,A Caroline Pissetti,C Marisa R. de Itapema Cardoso,C and David DriemeierAD A

Department of Clinical Pathology, School of Veterinary Medicine, Federal University of Rio Grande do Sul (UFRGS), Avenida Bento Gonc¸alves 9090, Agronomia, Porto Alegre-RS, 91540-000, Brazil B Instituto Federal Catarinense, SC-283, Km 8, Vila Fragosos, Conco´rdia-SC, 89700-000, Brazil C Department of Preventive Veterinary Medicine, School of Veterinary Medicine, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Brazil Received 13 February 2014; Accepted 8 May 2014; Published ahead of print 8 May 2014 SUMMARY. A fowl typhoid (FT) outbreak is reported in a flock of 400 Japanese quail (Coturnix coturnix japonica) at 91 days of age. Of these, 222 died suddenly, and necropsy revealed swollen liver and spleen with off-white to yellowish granules and reddish small intestine mucosa. Histopathology showed severe multifocal necrosis of liver and spleen (5/5), pulmonary congestion with macrophage infiltration in air capillaries (5/5), discrete interstitial nephritis (2/2), superficial necrosis of the intestinal mucosa with large numbers of coccobacilli (2/2), moderate peritonitis (2/2), and discrete airsacculitis (1/1). Anti-Salmonella immunohistochemistry (IHC) stained the cytoplasm of macrophages or free in the liver (5/5), spleen (5/5), lungs (4/5), kidneys (2/2) small intestine mucosa (2/2), cecum (1/1), bone marrow (1/1), air sacs (1/1), and ovary (1/1). In the heart (5/5), brain (2/2), esophagus (2/2), pancreas (2/2), proventriculus (2/2), gizzard (1/1), bursa of Fabricius (1/1), oviduct (1/1), and skeletal muscle (1/1) staining was observed only in the lumen of blood vessels. Salmonella Gallinarum was isolated in pure cultures of liver, spleen, lung, intestine, and blood samples of two birds. RESUMEN. Reporte de Caso—Brote de tifoidea aviar (Salmonella Gallinarum) en codornices japonesas (Coturnix coturnix japonica). Se describe un brote de tifoidea aviar en un lote de 400 codornices japonesas (Coturnix coturnix japonica) de 91 dı´as de edad, de las cuales 222 murieron su´bitamente. Se practico´ la necropsia de cinco animales y se observo´ hı´gado aumentado de taman˜o con puntos blanco amarillentos, bazo difusamente pa´lido de color blanco amarillento y la mucosa del intestino delgado se observo´ de un color rojizo. A la histopatologı´a, se observo´ en el hı´gado y en el bazo marcada necrosis multifocal (5/5), congestio´n del pulmo´n asociada con infiltracio´n de macro´fagos en los capilares ae´reos (5 /5), nefritis intersticial leve (2/2), necrosis superficial de la mucosa intestinal con gran cantidad de bacterias cocobacilares (2/2), peritonitis moderada (2/2) y aerosaculitis leve (1/1). El examen inmunohistoquı´mico para Salmonella demostro´ una fuerte tincio´n en el citoplasma de los macro´fagos o de manera difusa en el hı´gado (5/5), bazo (5/5), pulmo´n (4/5), rin˜o´n (2/2) mucosa del intestino delgado (2/2) y el ciego (1/1), me´dula o´sea (1/1), sacos ae´reos (1/1) y ovario (1/1). Por otra parte, en el corazo´n (5/5), cerebro (2/2), eso´fago (2/2), pa´ncreas (2/2), proventrı´culo (2/2), molleja (1/1), bolsa de Fabricio (1/1), oviducto (1/1) y mu´sculo esquele´tico (1/1) se observo´ tincio´n positiva so´lo en el lumen de los vasos sanguı´neos. Se aislo´ Salmonella Gallinarum en cultivo puro de muestras de hı´gado, bazo, pulmo´n, intestino y de la sangre de dos aves. Key words: salmonellosis, bacterial disease, sudden death, pathology, fowl, egg-laying Abbreviations: FT 5 fowl typhoid; H&E 5 hematoxylin and eosin; IHC 5 immunohistochemistry

Fowl typhoid (FT) is a severe systemic disease caused by Salmonella Gallinarum which affects poultry and fowl. This disease is a cause of high mortality and morbidity rates and low egg production and, therefore, has great economic importance (3,11,13,14,15,18,20,21). Salmonella Gallinarum is the etiologic agent of septicemic disease, mainly in chickens and turkeys (20), although outbreaks have been described in other poultry species (2,21). Salmonelloses are important avian diseases and are constantly surveyed in commercial poultry production (3,20,21). In Brazil, companies that breed pure lineages and house second- and thirdgeneration female breeders have to be free of Salmonella Gallinarum, Salmonella Pullorum, Salmonella Enteritidis, and Salmonella Typhimurium. In turn, stocks have to be free of Salmonella Gallinarum and Salmonella Pullorum and free of or surveyed for Salmonella Enteritidis and Salmonella Typhimurium. However, in Brazil only D

Corresponding author. Avenida Bento Gonc¸alves 9090, Agronomia 91540-000, Porto Alegre-RS, Brazil. E-mail: [email protected]

occasional surveillance is required in commercial egg and broiler companies as well as in properties where backyard chicken are raised or other poultry species are grown, including quail (7). The lack of constant surveillance in production systems of backyard chicken or other poultry species may pose a hazard to the health of industrial flocks as a whole because these animals may be reservoirs of pathogens and maintain them in the environment (3,17). In Brazil, salmonellosis is still an economic concern, with approximately 100 outbreaks reported between 2005 and 2012 (16). This study describes clinical, pathologic, and immunohistochemical findings in an outbreak of FT in a Japanese quail (Coturnix coturnix japonica) flock at the beginning of their egg-laying stage. CASE REPORT

A farm located in the state of Rio Grande do Sul, Brazil had been producing and selling quail eggs for human consumption for over 5 yr. The flock was formed by approximately 2000 birds of different

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ages and lots, housed in battery cages arranged as rows and columns, and separated by fecal collection trays. This production system is quite common in Brazil. A 400-quail lot at the beginning of the egg-laying phase (42 days of age) was purchased by the farm in April 2012. Upon arrival it became clear that mortality was above, and egg-laying capacity below, those of other flocks of the same age. After 7 wk, when quail were 91 days old, a steep increase in mortality was recorded that started in one cage with 20 quail on the top floor of the battery and then spread to another cage on the same floor. Soon these findings were recorded in the whole battery. Mortality lasted for 2 wk. In total, 222 quail died (55.5%). Some were found dead while others were prostrated, presented ruffled feathers, and soon died. The other lots of quail from different sources and ages did not present any clinical change or mortality. MATERIALS AND METHODS Five quail that died during the visit of the authors to the farm were necropsied. Liver, spleen, heart, and lung (5/5); brain, bone marrow, kidneys, esophagus, proventriculum, small intestine, and pancreas (2/5); bursa of Fabricius, air sac, ovary, cecum, and skeletal muscle (1/5); and oviduct, gizzard, and trachea (1/5) samples were collected, fixed in 10% buffered formalin, processed for routine histopathologic analysis, and stained by hematoxylin and eosin (H&E). Liver, spleen, lung, intestine, and blood samples of two birds were collected separately and aseptically and submitted to aerobic bacterial growth in blood agar supplemented with 5% goat blood and MacConkey agar. Samples were incubated at 37 C for 24–72 hr. The presence of Salmonella was also investigated in these samples. Selective enrichment was carried out using tetrathionate broth and RappaportVassiliadis broth, both of which were incubated at 42 C for 24 hr. After that, samples were plated on XLD agar and incubated at 37 C for 48 hr. Isolates were identified based on their biochemical profile. All colonies for which all biochemical assays were positive for Salmonella spp. were sent to a reference laboratory and serotyped. Additionally, an antibiogram of the isolated colonies was carried out. Immunohistochemical (IHC) studies were performed on the all tissues for detection of Salmonella using the polyclonal antibody antiSalmonella (dilution 1:1000, BiogenesisH Ltd., Poole, UK) (19) with 0.05% protease XIV (Sigma, St. Louis, MO) for 15 min for antigen retrieval as well as for a modified avidin-biotin peroxidase complex method (kit LSAB-HRP, K0690, DakoCytomationH, Glostrup, Denmark) using 3-amino-9-etilcarbazol (AEC, K3469, DakoCytomationH) as the chromogen.

RESULTS

Necropsy of the five quail revealed good body condition, ecchymosis in the subcutaneous tissue in the abdominal region and thigh muscles, and celomic cavities with a mild accumulation of turbid fluid. Livers were moderately swollen and dark red with offwhite to yellowish foci randomly distributed (Fig. 1A). In addition, spleens were swollen and diffusely pale, with off-white to yellowish foci and occasional petechial hemorrhage (Fig. 1B) alongside small intestines with reddish mucosa. Histopathology showed severe multifocal necrosis of liver and spleen with a fibrinoid aspect in some foci (Fig. 1C, D; 5/5). Also observed were mild pulmonary congestion with moderate macrophage infiltration in air capillaries (5/5), kidneys presenting discrete, multifocal interstitial infiltrates of macrophages and lymphocytes (2/ 2), small intestine with superficial necrosis of the intestinal mucosa associated with hemorrhage and large numbers of coccobacilli adhered to the mucosa and lumen (2/2), and air sacs with discrete,

multifocal interstitial infiltrates of macrophages and lymphocytes (1/1). The serous membrane of intestines, ovaries, and oviduct was diffusely reactive and presented discrete macrophage and lymphocyte infiltration (2/2). Anti-Salmonella immunoreaction was observed in the cytoplasm of macrophages or free in the necrotic or sinusoidal areas of the liver (5/5), in the red pulp of the spleen (Fig. 1E) (5/5), in pulmonary air capillaries (4/4), in the lamina propria, lumen, and surface of enterocytes of the small intestines (2/2) (Fig. 1F), and in the cecum (1/1) as well as in interstitial inflammatory foci of kidneys and vascular sinusoidal areas of the bone marrow (1/1) along with inflammation foci in air sacs (1/1) and in the cortical stroma of ovaries (1/1). In all these organs, positive staining was also observed in the cytoplasm of monocytes or free in the lumen of blood vessels. Staining was observed only in the lumen of vessels in the heart (5/5), brain (2/2), esophagus (2/2), pancreas (2/2), proventriculus (2/2), gizzard (1/1), bursa of Fabricius (1/1), oviduct (1/1), and skeletal muscle (1/1). In all organ specimens or blood samples analyzed to isolate bacteria and serotyped, pure cultures revealed the presence of Salmonella Gallinarum. The antibiogram revealed that these strains were susceptible to sulfamethoxazole-trimethroprim, sulfonamide, trimethroprim, gentamicin, enrofloxacin, doxycycline, and ceftiofur and were resistance to erythromycin. The quail in this lot were treated with sulfamethoxazoletrimethroprim for 3 days in the drinking water (120 g/1000 liters). This treatment was repeated after 1 wk with no decrease in mortality. Therefore, the remaining animals in this lot were euthanatized 2 wk later and the cages they had occupied were washed, disinfected with sodium hypochlorite, and left out in the sun for 30 days. Afterwards the cages received a new flock of quail, and none of the birds died. Contact was made with the person who had sold the first lot of quail to find out whether the other birds also presented the same health issues; however, he refused to provide this information. DISCUSSION

Although there are few reports of FT in quail, in the present study we observed that this disease may lead to considerable economic losses associated with high mortality and reduced egg-laying. The Salmonella Gallinarum culture of blood and different tissues, considered together with the presence of lesions and positive antiSalmonella immunoreaction in several organs, suggests acute septicemic disease. As observed in this outbreak, FT may be characterized by acute septicemic disease, often without clinical manifestations but with continuous mortality within days or weeks (12,13,14,17,18). In the present study, as in other reports, treatment with antibiotics did not reduce mortality because there is no drug that is efficacious in eliminating infection in fowl (14,18,21). In many European and North American countries, treatment is not considered because flocks that test positive for Salmonella Gallinarum have to be euthanatized to eradicate the disease (21). The macro- and microscopic lesions observed in the present study are typical of FT (5,6,10,12,14,18,20,21) and were characterized by necrotic lesions in the liver and spleen and by nephritis, airsacculitis, and peritonitis. Pericarditis and myocarditis, which are also typical lesions of this disease, were not observed in the lot (10,12,20,21). Bacterial isolation and serotyping proved efficacious to identify Salmonella Gallinarum. Anti-Salmonella IHC may be used as a method to diagnose salmonellosis in poultry, mainly when there are only formalin-fixed tissues and no new biologic material for bacterial

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Fig. 1. Fowl typhoid in Japanese quail. (A) Mildly swollen liver with off-white to yellowish foci randomly scattered. (B) Swollen spleen with off-white to yellowish foci (arrow). Fibrinoid necrosis in (C) liver (arrow) and (D) spleen (arrow) (H&E, 4003). Anti-Salmonella immunohistochemistry: (E) Spleen presenting a reaction in the cytoplasm of macrophages in necrotic areas (2003). (F) Small intestine with reaction in the cytoplasm of macrophages in lamina propria (2003). Avidin-biotin ligated to peroxidase.

isolation. However, through this technique it is not possible to determine the serotype involved (19). Several factors may facilitate infection by Salmonella Gallinarum in poultry. Here, it was not possible to determine how the quail

became infected, although there is the suspicion that they arrived in the farm already carrying the pathogen as the quail of other lots did not develop the disease. It is possible that these quail hosted the bacterium and manifested the disease due to the stress associated

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with transport, to the new environment, and to the beginning of the egg-laying period. The consumption of feces contaminated with Salmonella Gallinarum is the main route of FT transmission (4,6). In the present case report the bacterium was isolated in intestines and detected by IHC, which suggests that feces may indeed have been the contamination route in these quail. Therefore, because quail of different flocks were housed in distinct batteries, contact with feces was prevented, thereby blocking the spread of the disease to the other quail in the farm. However, to confirm this hypothesis a survey of Salmonella spp. should have been carried out in fecal samples of the other flocks. Salmonella Gallinarum may be vertically transmitted and cause persistent infections in poultry flocks (3,20,21); nevertheless, some experimental studies have not isolated the bacterium from eggs laid by infected egg-layers (5,6). As previously reported for chicken, Salmonella Gallinarum may be isolated in the ovary and oviduct (5,6,20). The presence of the bacterium was confirmed by IHC in the organs of the reproductive system of quail in this report. In the outbreak investigated, vertical transmission was a likely initial source of infection. The infected quail excreted the bacterium in feces, transmitting it to other individuals. To confirm this hypothesis it would have been necessary to know whether other flocks bred from the same breeders also carried the bacterium, but it was not possible to obtain this information from the seller. Another relevant factor is the variation in susceptibility of different poultry or fowl species to FT (4,21). Additionally, varying degrees of susceptibility occur within species (1,5,6,8,12). Studies on chickens have proven that free-range hens are more resistant to FT than are commercial battery flocks (1). Other studies have pointed to the genetic background as a source of varying susceptibility profiles between commercial lineages of poultry (5,6,8,12). Few studies have investigated the susceptibility of quail to Salmonella Gallinarum (9,10). The authors observed that young quail may be highly susceptible to FT and present an acute manifestation of the disease, which is then accompanied by high mortality. In conclusion, FT is a deadly disease in quail. In this sense, more attention should be given to salmonellosis surveillance efforts in flocks of these birds; such efforts may help optimize diagnosis and control outbreaks. REFERENCES 1. Alvarez, M. T., N. Ledesma, G. Te´llez, J. L. Molinari, and P. Tato. Comparison of the immune responses against Salmonella enterica serovar Gallinarum infection between naked neck chickens and a commercial chicken line. Avian Pathol. 32:193–203. 2003. 2. Awaad, M. H., H. M. Hafez, M. Z. El-Dimerdash, and H. Krauss. Some epidemiological aspects of Salmonella Gallinarum infection in Japanese quail (Coturnix coturnix japonica). Zentralbl. Veterinarmed. B. 28:704–712. 1981. 3. Barrow, P. A., and O. C. Freitas Neto. Pullorum disease and fowl typhoid—new thoughts on old diseases: a review. Avian Pathol. 40:1–13. 2011. 4. Barrow, P. A., M. A. Lovell, C. K. Murphy, and K. Page. Salmonella infection in a commercial line of ducks; experimental studies on virulence, intestinal colonization and immune protection. Epidemiol. Infect. 123: 121–132. 1999. 5. Berchieri, A. Jr., A. Murphy, K. Marston, and P. A. Barrow. Observations on the persistence and vertical transmission of Salmonella

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ACKNOWLEDGMENTS We are grateful to Dr. Ricardo Evandro Mendes for translating the summary to Spanish. This work was financially supported by the Brazilian National Council of Scientific and Technologic Development (CNPq).