Molecular Detection of Haemoprotozoan Diseases in Anaemic Goats of Southern India: A Pilot Study

India’s livestock sector is one of the largest in the world with a holding of 11.6% of world livestock population. Among different domestic animals, goats being multipurpose animal provide meat, milk and fibre (Bhardwaj et al., 2018). Anaemia is an important clinical presentation in goats manifested by pale or white mucous membranes, exercise intolerance, tachypnea, tachycardia, possible systolic murmurs, weakness, jaundice, haemoglobinuria, melena, submandibular oedema, ascites, weight loss, prolonged capillary refill time and even collapse of the animal in extreme cases (Balcomb and Foster, 2014).

According to Smith and Sherman (2011), anaemia is classified as haemolytic anemia, blood loss anemia and due to impaired erythropoiesis. Causes of haemolytic anaemia in goats include the haemoparasitic diseases such as anaplasmosis, babesiosis and theileriosis. Anaplasmosis is caused by intra-erythrocytic rickettsial pathogen Anaplasma ovis, which is mostly found in sheep, goats, and wild ruminants. According to Torina et al. (2012), Anaplasma ovis infection in sheep and goats often has no symptoms. Co-infection with other pathogens and stress brought on by numerous conditions, such as a hot environment and transportation, frequently predispose to the disease (Aravind et al., 2020). Srivastava et al., (2022) reported 11.7% of pre-weaning mortality in goat kids was due to debility and anemia.

Babesiosis is one of the most common tick borne infections in ruminants. In acute condition it produces haemolytic anaemia, fever, inappetence, jaundice, and hemoglobinuria, all of which can be deadly in extreme instances (Hurtado et al., 2019). Babesia motasi is more prevalent in goats but may be less virulent in sheep. Haemaphysalis and Rhipicephalus ticks can spread  B.motasi (Smith and Sherman 2011). In India clinical disease in goats with mixed infection of B. ovis and B. motasi has been reported by Tufani et al. (2018).

Theileriosis encompasses a group of tick-borne protozoal diseases of ruminants affecting primarily the hemic-lymphatic system. It leads to anemia and lymphopenia in affected goats. Koch’s Blue Bodies may be identified in Giemsa-stained smears of lymph nodes or characteristic piroplasms seen in circulating RBCs (Smith and Sherman (2011). According to Yin et al. (2008) T. lestoquardi, T. uilenbergi and T. luwenshuni were characterized as highly pathogenic. Nagaraj et al. (2019) stated that goats affected with theileriosis appear to be apparently healthy. This study investigated the incidence of haemoprotozoan diseases in anaemic goats.

Goats presented to Large Animal Clinics of Madras Veterinary College Teaching Hospital, Chennai India, between March and December, 2023 was screened for the signs of anaemia. Twenty five anaemic goats were included in this study.

Screening of Anaplasma spp., Theileria spp. and Babesia spp. in blood smear

Blood smears were collected and fixed in methanol and stained with Leishman Giemsa (L-G) cocktail stain for 30 minutes and examined under oil immersion objective of the microscope (Gajendra et al., 2015). About 10 microscopic fields per smear of RBCs were screened for Theileria spp., Anaplasma spp. and Babesia spp.The sample was considered to be positive even if single piroplasm was seen

Molecular detection of Anaplasma spp. Theileria spp. and Babesia spp

Blood samples were collected from the jugular vein into a tube containing anticoagulant (EDTA) from 25 goats affected of anaemia. Genomic DNA was extracted successfully from 150 µl of blood using method adopted by Surzycki (2000) (Addition of 0.25% Sarcosyl was the modification done which aids in the lysis of cells) and DNA was stored at -20°C until further use.

Polymerase chain reaction (PCR)

The DNA samples of goats were subjected to PCR amplification with the genus specific primers and later confirmed by species specific primers of Theileria, Anaplasma and Babesia. The primers details and cycling conditions are listed in Table.1 and 2.




PCR assay was carried out in thermal cycler. Each PCR reaction was added with 10 ρmole of genus specific primers and species specific primers encoding the 18S rRNA of Theileria spp., 16S RNA of Anaplasma spp. and 16S RNA of Babesia spp. and made up to the final volume of 20 μl with nuclease free water. Red dye master mix (2x, Ampliqon III) was used to prepare PCR reaction mix. Each 20 μl reaction mixture comprised 5 μl of template DNA, 10 μl Red dye PCR Master Mix (2X, Ampliqon), 1 μl of each primers at 10 pmol/μl (forward and reverse primer) and 3 μl nuclease free water. In addition to this test DNA samples, a negative control (DNA extracted from known negative healthy goat blood sample) were included in each of the PCR amplification. 10 μl of amplified PCR product was loaded for electrophoresis in 1.5% Agarose gel along with 100 bp DNA ladder (Lee et al., 2012). The images were captured and documented using gel documentation system (Bio Rad., USA).

Among 25 blood samples and blood smears 12 samples (48 per cent), were found positive for haemoprotozoan disease.

Microscopic examination

The Leishman Giemsa (L-G) stained blood smears were screened for Anaplasma spp., Theileria spp. and Babesia spp. Among 25 blood smears from goats, 6 cases (24 per cent) were found to be positive for Anaplasma spp. and1case (4 per cent) was positive for Theileria spp. showing characteristic piroplasms stages in the RBCs (Fig 1a,1b).



Molecular detection

The amplicon of genus specific Anaplasma ovis, Babesia ovis, Babesia motasi and Theileria luwenshuni was observed as a single band of 347 bp, 636 bp, 642 bp and 389 bp, respectively (Fig 2-5).






Out of 25 blood samples, 12 (48 per cent) cases were found positive for haemoprotozoan. In which 8 cases were found positive for Anaplasma ovis and one case found positive for Babesia motasi, Babesia ovis each and two cases were found positive for mixed infection (Anaplasma ovis with Babesia ovis and Theileria luwenshuni; Anaplasma ovis with Babesia ovis) by PCR.

In the present study, anaemic goats had highest incidence of Anaplasma ovis (8 cases; 32 per cent) which is in agreement with findings of Aravind et al. (2020). Due to co-infection with other pathogens and stress brought on by numerous conditions, such as a hot environment and transportation, frequently predispose goats to the diseases (Aravind et al., 2020).

In this study, one animal was found positive for Theileria luwenshuni, Anaplasma ovis and Babesia ovis mixed infection. Similarly a study conducted by Nagaraj et al., (2019), observed that the prevalence of T.luwenshuni were 32.58 percent in apparently healthy goats.

Theileriosis was confirmed by detection of Koch’s Blue Bodies (KBB) using FNAC from swollen pre-scapular lymph nodes (Banka et al., 2020). According to Yin et al. (2008), blood smear examination is reliable for detection of acute cases; it has a limited value for detection of infection in chronic cases where only a few number of piroplasms exist. A study conducted by Jayalakshmi and Premalatha (2020), blood smear examination revealed 18.54% goats were positive for haemoparasites like Anaplasma spp and Theileria spp. of which 15.32% was Anaplasma spp and 3.22% animals showed mixed infection of Theileria spp and Anaplasma spp.

In this study, one case (4 per cent) was found positive for Babesia motasi and Babesia ovis each. Remaining two cases (8 per cent) were found positive for mixed infection (Babesia ovis, Anaplasma ovis and Theileria luwenshuni; Babesia ovis and Anaplasma ovis). Babesia ovis and Babesia motasi also reported by others in goats (Uilenberg, 2001; Smith and Sherman, 2009; Haq et al., 2017) and Theileria luwenshuni in sheep (Dhaygude, 2021). According to Rather et al. (2016), among ruminants the goats had resistance against babesiosis so prevalence of the babesiosis in goats was reported to be 0.75% per cent compared to other ruminants. But climatic changes are expected to increase the risks of vector borne diseases due to rise of environmental temperature and change in rainfall pattern, it leads to an increase in the vector population (Sharma, 2011). Ingle (2019) reported an overall prevalence of 29 per cent for Babesia species using PCR where 11% were positive for B. ovis. According to Ingle (2019) the use of PCR in the surveillance of babesiosis will enable the detection of asymptomatic carrier animals that could not be detected using conventional methods.

The present study revealed that anemia is common in goats and almost 50% of anemia is due to haemoprotozoan diseases. Anaplasma ovis is the common infection followed by mixed infection of Babesia and Theileria. Polymerase chain reaction was found useful molecular technique for diagnosis and surveillance of haemoprotozoan diseases in goats.
 

The authors thank Dean, Madras Veterinary College and Director of Clinics, TANUVAS, Chennai for providing necessary facility and support for this work.

on behalf of all authors I declare that there is no conflict of interest.