Blood smear demonstrations of Anaplasma-infected

Comp Clin Pathol DOI 10.1007/s00580-015-2186-9

ORIGINAL ARTICLE

Blood smear demonstrations of Anaplasma-infected sheep in a flock Maryam Rassouli 1,2 & Khatereh Kafshdouzan 1,2 & Mohammad Saberi Zow 3 & Sajjad Ghodrati 4

Received: 13 June 2015 / Accepted: 22 September 2015 # Springer-Verlag London 2015

A b s t r a c t O v i ne an a p l a s m o s i s is c a u s e d b y t w o intraerythrocytic Anaplasma species (Rickettsiales, Anaplasmataceae). Ticks are the most important vectors in anaplasmosis. The mortality of this disease is relatively low, but it has worldwide distribution. The disease is endemic in some areas because chronic infected animals and infected vectors can act as Anaplasma reservoirs. Despite the low mortality, the economic loss which is caused by the disease is relatively high. In this research, one sheep flock which had been involved with anaplasmosis was described. Peripheral blood samples were taken and stained by Giemsa. Cell changes in the stained blood films that had been caused by anaplasmosis and management measures to prevent the disease were discussed in details. This study is important because it was done in a naturally Anaplasma-infected flock, emphasized on valuable changes in the infected blood films, and discussed the importance of preventive measures in an area.

Keywords Anaplasma . Anaplasmosis . Iran . Sheep

* Maryam Rassouli [email protected]; [email protected] 1

Pathobiology Department, Shahmirzad School of Veterinary Medicine, Semnan University, Vali e Asr area, Shahmirzad, Semnan, IranP.O. Box: 35718-95589

2

Pathobiology Department, Faculty of Veterinary Medicine, Semnan University, Semnan, Iran

3

Shahmirzad School of Veterinary Medicine, Semnan University, Semnan, Iran

4

Faculty of Veterinary Medicine, Semnan University, Semnan, Iran

Introduction Ovine anaplasmosis is caused by two intraerythrocytic Anaplasma species, Anaplasma ovis and less prevalent Anaplasma marginale. Anaplasma organisms are obligatory intracellular Gram-negative bacteria which belong to the order Rickettsiales and family Anaplasmataceae (Quinn et al. 2011). This disease is distributed worldwide (Kocan et al. 2004; Hornok et al. 2007; Quinn et al. 2011). Ticks are the most important vectors in anaplasmosis. Dermacentor spp. are biological vectors of A. ovis in the USA. In the old world, A. ovis is transmitted by Rhipicephalus bursa and most likely other ticks (Kocan and Stiller 1992; De La Fuente et al. 2007). Other hematophagous (blood feeding) arthropods and instruments contaminated with infected blood can also be transmitting intraerythrocytic Anaplasma organisms (Quinn et al. 2011). The most dominant symptoms are fever, depression, weight loss, anemia, abortion, reduced milk yield, and sometimes death (Kocan et al. 2004; Quinn et al. 2011). Sheep or ticks with persistent infection serve as reservoirs of Anaplasma in the area and cause Anaplasmaendemic areas (Kocan and Stiller 1992; De La Fuente et al. 2007). Diagnosis can be made by observation of the organisms in the stained blood films and serological and molecular tests (Quinn et al. 2011). The aim of this study is to understand the changes which are caused by intraerythrocytic Anaplasma spp. in blood films and the importance of preventive and diagnostic measures for anaplasmosis control in an area.

Comp Clin Pathol

Materials and methods Clinical history and sample collection There was a small sheep flock in Kalat region, Khorasan Razavi province, Northeast of Iran. The flock consisted of 55 ewes, 2 rams, and 25 lambs in late spring 2014. Its most dominant problem was sheep emaciation. The lambs failed to gain weight. There were two sporadic deaths of ewes in early spring 2014; after that, the owner referred to the Kalat veterinary practitioner for the flock visit. Different samples such as peripheral blood smears were taken from five ewes which showed the most severe symptoms. The samples were transferred to the Shahmirzad School of Veterinary Medicine. In the parasitology laboratory, some abnormalities were observed in the taken blood smears since the flock was visited once more 2 weeks later, in this time just peripheral blood smears were taken from 35 sheeps (26 ewes, 1 ram, 8 lambs); therefore, the total numbers of blood smears were 40 at the first and second visits. Some questions were also asked from the owner about the flock management and arthropod infestations. All samples were transferred to the parasitology laboratory, fixed by methanol, and stained by Giemsa.

Fig. 1 a Anaplasma organisms closed to RBC membrane (thin arrows) and hyperchromatophilic RBC with basophilic stipplings (thick arrow), note that the RBC is twice bigger than normal, ×200. b Four hyperchromatophilic RBCs with basophilic stipplings (black arrow), ×400. c Howell-Jolly body in a RBC (black arrow), ×500. d Anisocytosis, ×200. e Hypochromasia, ×100. Stained by Giemsa.

the previous summer when the sheep were grazing in the pasture. After diagnosing, the flock was treated by tetracycline 20 % (long acting). Some supplements were also added to the food.

Discussion Results Surprisingly, after blood film observation under light microscopy, 27 samples (23 ewes, 1 ram, and 3 lambs) were positive for Anaplasma spp. All of the 5 samples that were taken from ewes at the first visit were positive, and the other 18 belonged to the ewes that had been sampled at the second visit. There were some amazing changes in the Anaplasma-infected blood smears. First of all, the intraerythrocytic Anaplasma organisms were observed as dot-shaped basophilic objects in the infected erythrocytes. Most of them were close to the reticulocyte membrane. Other changes were categorized into two groups: reticulocyte (RBC) and leukocyte (WBC) changes. Reticulocyte changes consisted of hypochromasia (low to moderate), anisocytosis, presence of Howell-Jolly bodies, hyperchromatic RBCs, and basophilic stipplings (Fig. 1). Leukocyte changes were neutrophilia, lymphopenia, and reactive lymphocytosis (low to severe) (Fig. 2). Basophilic stipplings were observed in 18 samples. Neutrophilia and lymphopenia were in all positive samples. The reactivity of lymphocytes was recorded as less than 20 %, 20–50 %, and more than 50 % of counted lymphocytes and were observed in 15, 10, and 2 samples, respectively. There were some management problems in the flock; the farmer sometimes had used the same needle for injection, and there had been no use of poisons against blood feeding arthropods since last year. The flock was attacked heavily by ticks

Ovine anaplasmosis was reported worldwide (Quinn et al. 2011), but there is no complete information about this disease, its importance, endemicity, and potential vectors in different parts of Iran. Blood smear demonstrations were limited to an experimental infection and focused on hematological panel and reticulocyte changes in the blood films (Yasini et al. 2012). This study is important because it was done in a naturally infected flock with intraerythrocytic Anaplasma spp., emphasized on valuable changes in the infected blood films, and discussed the importance of preventive measures in an area.

Fig. 2 a Monocyte-like lymphocyte with basophilic cytoplasm, ×150. b Reactive lymphocyte with amoeboid pseudopods formation in its cytoplasm, ×150. c Reactive lymphocyte with two-side projection, ×200. Stained by Giemsa.

Comp Clin Pathol

Both A. ovis and A. marginale were recorded in Anaplasma-infected sheep in Iran (Noaman 2013). Therefore, the accurate diagnosing of infective species should be done by molecular examination. Anemia in anaplasmosis is caused by extravascular opsonization and phagocytosis of parasitized erythrocytes by reticuloendothelial cells (Fraser et al. 1991). When absolute reticulocytosis is present, the animal is said to have regenerative anemia (Harvey 2001). Polychromasia is absent in stained blood films of healthy ruminants. Diffuse basophilic stippling in polychromatophilic erythrocytes commonly occurs in regenerative anemia (Harvey 2001). Anisocytosis and HowellJolly bodies are usually present in regenerative anemia. They may also be increased in animals receiving glucocorticoid therapy (Wanduragala and Ristic 1993; Harvey 2001). In this case, Howell-Jolly bodies must be differentiated from Anaplasma inclusions. Howell-Jolly bodies are bigger and more hyperchromatic than Anaplasma inclusions. In all Anaplasma positive samples, one or more of these changes due to regenerative anemia were observed. Hypochromasia was also observed in some samples (Fig. 1). Neutrophils are essential in the defense against invading pathogens such as bacteria. Neutrophilia without significant left shift may be present in different abnormal conditions such as mild and chronic inflammation (Stevens et al. 2012). In these inflammatory conditions, increase of production and release of mature neutrophils from the bone marrow occurs. In ruminants, neutrophilia in acute bacterial infections is less common and neutropenia is observed more commonly (Taylor 2000). According to the results, neutrophilia was recorded in all Anaplasma-positive samples, thus; this change may occur more commonly in chronic bacterial infections. Lymphopenia and neutrophilia can occur in a response to endogenous or exogenous glucocorticoids in animals (Collins and Suarez-Guemes 1985; Shimizu et al. 2000; Kaname et al. 2002; Yamada et al. 2002; Abraham et al. 2009). Glucocorticoids increase lymphocyte sequestration and apoptosis; some secreted cytokines in bacterial and viral infections also cause lymphopenia (Bloemena et al. 1990; Toft et al. 1993; Stevens et al. 2012). In this flock, none of glucocorticoid drugs had been prescribed and it could be concluded that the endogenous glucocorticoid secretion was increased in order to suppress the inflammatory mechanisms which had been upregulated by Anaplasma infection. Reactive lymphocytosis is caused by antigenic stimulation. These reactive lymphocytes exhibit increase in size and cytoplasmic basophilia. Most of these antigenically stimulated cells remain in peripheral lymphoid tissues, and very few of them may enter the circulation (Stevens et al. 2012). These cells were studied more in abnormal conditions of dogs and cats. In this research, reactive lymphocytosis was observed from low to high in Anaplasma-positive samples and can be

identified as big and monocyte-like lymphocytes with basophilic cytoplasms, lymphocytes with amoeboid pseudopod formation in their cytoplasms (Fig. 2). It can be proposed that endogenous glucocorticoids is caused by downregulation of lymphocytes, and reactive lymphocytes is increased in response to bacterial antigens because the immune system wants to defend against the infection by these low number of lymphocytes. On the other hand, the immune system increases the activity of the remaining lymphocytes to cover lymphopenia. The disease outbreak in the flock was 67.5 % (27 out of 40; 53–82 %; 95 % confidence interval) and more prevalent among ewes than lambs. It showed that the disease had been chronic in the flock. Economic losses had been caused by low production of meat, milk, and wool. Lamb losses by abortion and some sporadic deaths were also recorded. Anaplasmosis can be easily diagnosed and treated by suitable antibiotic therapy, and it can be prevented by farmer education. Economizing poisons not only is cost beneficial but also can be the reason of increase in blood feeding arthropods and animals’ susceptibility to some diseases such as anaplasmosis. Lack of preventive, diagnostic, and therapeutic measures lead to make endemic Anaplasma-infected areas, and the disease can be easily spread through moving the infected animals and infected blood-feeding arthropods especially ticks from one area to the other. Compliance with ethical standardsThis study was funded by 2014 Semnan University grant to the corresponding author. Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Conflict of interest Maryam Rassouli and Khatereh Kafshdouzan have received research grants from Semnan University. Mohammad Saberi Zow and Sajjad Ghodrati declare that they have no competing interests.

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