Goats have been connected with man since the birth of agriculture and animal domestication. They can adapt to a wide range of environmental circumstances which makes them a very significant socio-economic animal giving goods (meat, milk, fibre, hair) and service to humans all over the world, particularly in developing nations like India¹.Vector-borne protozoal and rickettsial infections such as anaplasmosis, babesiosis and theileriosis are the serious impediment to the health of tropical livestock and cause significant economic losses due to reduced milk and meat output, mortality, abortions, expenses on treatment and control².

Anaplasmosis in goats is one of the most serious concerns in the fast-growing small ruminant sector, as it reduces the animal's production and weight gain³. Anaplasmosis causes significant losses in livestock production of countries in tropical and subtropical regions where livestock consists primarily of sheep and goats. Among various Anaplasma species, A. marginale, A. centrale, A. bovis, A. ovis and A. phagocytophilium are obligate intracellular bacteria parasitizing erythrocytes and monocytes or granulocytes of higher vertebrates, mostly ruminants⁴.

In routine clinical practice, anaplasmosis is frequently diagnosed by using blood smear examination. But it is difficult to prove repeatedly the presence of inclusion bodies of A. phagocytophilium in leucocytes, therefore microscopic tests of blood smears should be supported by indicating the genetic material of the microbes using the PCR method⁵, ⁶.

Although, the bovine anaplasmosis has been extensively studied in the Indian subcontinent, there is a paucity of literature on anaplasmosis occurring in Indian small ruminants, especially in caprines^3,7,8. However, to the best of our knowledge, A. phagocyto-philium has never been detected by molecular techniques in Indian goat population. Hence, the present study was designed to study the clinical profile, haemato-biochemical changes and molecular detection of anaplasmosis in goats.

The results revealed anaplasmosis in all the goat flocks under study. Out of 50 suspected samples that were screened for anaplasmosis, forty-four (n=44, 88%) and forty-six (n=46, 92%) goats were found positive for anaplasmosis by blood smear examination and PCR, respectively. The blood smear examination revealed 10, 6 and 72 percent positivity for A. ovis, A. phagocytophilium and both pathogens (A. ovis + A. phagocytophilium) respectively. The data regarding the species-wise prevalence of anaplasmosis by PCR recorded an equal infection among tested animals for A. ovis and A. phagocytophilium (14%). While the mixed infection (A. ovis + A. phagocytophilium) was observed in 64 percent goats (Table 1).

The age-wise prevalence of anaplasmosis in goats was found higher in the age group above 12 months (n=32, 69.56%) followed by the age group 3 to 12 months (n=12, 26.09%) and the lowest prevalence was recorded in the age group below 3 months (n=2, 4.35%). The analysis of sex-wise data revealed higher occurrence of anaplasmosis in females (n=42, 91.30%) as compared to males (n=4, 8.70%).

The anaplasmosis positive goats showed clinical signs such as fever (103.69 ± 0.11 vs 100.92 ± 0.12°F), anorexia, presence of tick infestation, congested, pale or papery white mucous membranes (Fig. 1), swollen lymph nodes and respiratory distress. In few cases, clinical signs such as lameness, nasal discharge and recumbency were also recorded.

The blood smear examination of anaplasmosis suspected goats which were positive for A. ovis revealed presence of intra-erythrocytic one or more small round dark basophilic inclusion bodies located centrally, sub-marginally or marginally (Fig. 2), while the intra-cytoplasmic morulae made up of numerous delicate punctiform initial bodies (rod, round or oval-shaped) within neutrophils or mononuclear cells of colours ranging from dark blue to purple-grey placed in vacuole surrounded by a membrane were evident in blood smears of suspected goats found positive for A. phagocytophilium (Fig. 3).

The haematological analysis of blood samples of anaplasmosis positive goats (n=46) revealed a highly significant increase (p<0.01) in total leucocyte count and absolute granulocyte count as compared to healthy control goats (n=12). Whereas, a highly significant (p<0.01) decrease in total erythrocyte count, haemoglobin concentration and packed cell volume as well as in absolute counts of monocytes were evident in anaplasmosis affected goats. The absolute lymphocyte count from anaplasmosis affected and healthy control goats not revealed any significant difference (Table 2).

Biochemical analysis of serum samples from anaplasmosis positive goats (n=46) revealed a highly significant increase (p<0.01) in serum levels of AST, ALT, creatinine and blood urea nitrogen whereas a significant increase (p<0.05) in the levels of total bilirubin were observed in anaplasmosis infected goats as compared to healthy control (Table 3).

For species detection, partial MSP4 gene of A. ovis and 16S rRNA gene of A. phagocytophilium were targeted by PCR. The PCR resulted coamplification of 92 bp and 172 bp amplicons specific to the primer pair positions, respectively (Fig. 4). The PCR detected equal presence of A. ovis and A. phagocytophilium infection in 14 percent samples, while the mixed infection or coinfection (A. ovis + A. phagocytophilium ) was recorded in 64 percent samples indicating overall prevalence of 92% in clinically suspected goats.

The clinical signs of anaplasmosis observed in goats in the present study were in agreement with the findings reported by earlier research workers¹¹, ¹². Endogenous pyrogens generated by the Anaplasma organisms damage the erythrocytes and activate numerous haemopoietic and thermo regulatory centres throughout the body resulting in anaemia, high fever, weakness and weight loss¹³. The pale mucous membranes found in our study could be related to anaemia which is caused by immune-mediated erythrocyte destruction in which the host produces auto-antibodies against Anaplasma species and its erythrocytes. The antibodies cover parasitized erythrocytes, which are phagocytosed by the spleen, liver and bone marrow's mononuclear phagocytic system¹⁴.

In the current study, highest age-specific prevalence of anaplasmosis was seen in female goats older than 12 months of age which is found consistent with findings of earlier workers¹⁵. Furthermore, they stated that the adult animals have more opportunities for exposure to ticks carrying the pathogen than young animals. This could be explained by the fact that the adult goats were more exposed to tick infestation carrying Anaplasma spp. because they went through more tick seasons.

The high prevalence of anaplasmosis in female goats can be attributed to extended breeding and lactation, stress of breeding, milking and repeated hormonal changes associated with the pregnancy and parturition¹⁵, ¹⁶. These findings of earlier research workers were in agreement with findings of present study.

In routine clinical practice, anaplasmosis is frequently diagnosed by using blood smear examination. But it is difficult to prove repeatedly the presence of inclusion bodies of A. phagocytophilium in leucocytes, therefore microscopic tests of blood smears should be supported by indicating the genetic material of the microbes using the PCR method⁵, ⁶. These findings recorded regarding diagnostic morphology of A. ovis and A. phagocytophilium in positive blood smears and findings observed during molecular detection of these pathogens in present study were also in consonance with previous reports in different animal species^6,9,10,17-20.

More or less similar haematological findings of anaplasmosis in goats were reported by earlier investigators^{7,8,13,16,21,22}. Furthermore, these earlier workers stated that the significant decrease in erythrocyte count, haemoglobin concentration and packed cell volume might be due to extravascular haemolysis of erythrocytes caused by parasitic damage to erythrocytes which is phagocytosed by the reticuloendothelial system¹³. A significant increase in total leucocyte count might be due to parasite and its toxins which stimulates lymphoid tissues and stem cells in the bone marrow for phagocytosis of infected erythrocytes²¹.

The biochemical changes observed in the present study were found in consonance with earlier studies of anaplasmosis in goats^21,23-25. The increased levels of total bilirubin in anaplasmosis affected goats can be correlated with haemolysis of parasitized erythrocytes in the reticuloendothelial system, hepatic dysfunction and haemolytic anaemia whereas the increase in serum creatinine and BUN levels in anaplasmosis affected goats can be attributed to kidney dysfunction and muscle catabolism. The elevated AST and ALT level in the anaplasmosis affected animals is attributed to hepatic injury due to an increased load of phagocytosed parasitized erythrocytes in the hepatic reticuloendothelial system²⁴.

This study utilized MSP4 and 16S RNA genes for speciation of Anaplasma ovis and A. phagocytophilium . These genes were also used by earlier researchers for differentiation of A. ovis and A. phagocytophilium infection in goats⁹, ¹⁰. As compared to the findings of present study, earlier researchers recorded more or less similar prevalence of A. ovis and A. phagocytophilium infection in goats by blood smear examination and PCR from China and other countries^26-29.

In conclusion, our study documented 92 percent overall prevalence of anaplasmosis in the goat population with a high incidence of mixed infection or coinfection (A. ovis + A. phagocytophilium). Although the clinical diagnosis of anaplasmosis in goats can be precisely made from blood smear examination, the polymerase chain reaction (PCR) showed more specificity and sensitivity in terms of species detection and assessment of overall prevalence. This study also reports the first molecular detection of A. phagocytophilium infection in Indian goat flock.

NS : Non-significant, *Significant (P<0.05), **Highly Significant (P<0.01)

^*Significant (P<0.05), ^**Highly Significant (P<0.01)

References