Prevalence of Anaplasma marginale in Water Buffaloes (Bubalus bubalis) in the State of Uttar Pradesh, North India

Anaplasma marginale, a tick-borne obligate intraerythrocytic rickettsia is one of the most common causative agent of bovine anaplasmosis worldwide and is endemic in several regions of Asia, Africa, Australia, North and South America (Kocan et al., 2010). The effects of bovine anaplasmosis on the health and productivity of cattle have been known since long but buffaloes, bison, African antelopes, mule deer can become persistently infected with A. marginale (Kuttler, 1984). The clinical symptoms of anaplasmosis include haemolytic anaemia, weight loss, high fever, tachycardia, jaundice, decreased milk yield, gastrointestinal signs, abortion and sudden death (Kocan et al., 2003, 2004).
 
Transmission of A. marginale in cattle and buffaloes occurs biologically through ticks and mechanically by blood-feeding flies and blood contaminated fomites (Scoles et al., 2005). Transplacental transmission of A. marginale occurs in cattle, resulting in healthy but persistently infected calves (Kocan et al., 2015). Microscopic examination of stained blood smears is the conventional method for detecting A. marginale infection. However, molecular techniques and serological assays have proven to be more sensitive and specific for the detection of subclinical infection and carrier status of the animal for anaplasmosis (Bisen et al., 2021). Major surface proteins of A. marginale play a vital role in the interaction and invasion of the pathogen into the host cells. Six major surface proteins (MSPs) MSP1a, MSP1b, MSP2, MSP3, MSP4 and MSP5 have been identified and studied in A. marginale as promising molecules in the diagnosis and vaccine development (Palmer et al., 1985; Kocan et al., 2003, 2004; de la Fuente et al., 2010). Major surface protein 5 is a highly conserved surface protein that has been proven effective as a diagnostic antigen (de Echaide​ et al., 1998). India is a developing agricultural country and is the largest buffalo milk producer in the world. Buffaloes make an important contribution to the agricultural economy. According to 20th livestock census (Department of Animal Husbandry and Dairying, Government of India, 2019); water buffalo population is 109.85 million which contributes to about 20.5% of the total livestock population of our country. Uttar Pradesh state has the largest water buffalo population, nearing 33 million (Department of Animal Husbandry and Dairying, Government of India, 2019). Like cattle, buffaloes are also susceptible to many tick-borne parasitic infections causing huge production and economic losses to the dairy farmers (Vidotto et al., 1998). Bovine anaplasmosis caused by A. marginale in cattle has been reported from several parts of India but there are meagre reports on its occurrence in water buffaloes in India. Therefore, present investigation on the prevalence of A. marginale infection in water buffaloes in U.P, North India by microscopy and by PCR was undertaken for determining the status of this infection in this part of the country.

Blood samples of two hundred twenty eight water buffaloes were collected from Bareilly, Moradabad and Pilibhit districts of U.P from July 2017 to June 2019. Blood samples were collected from asymptomatic buffaloes and those with signs of fever, anaemia, tick infestation and reduced milk production for screening of Anaplasma marginale infection. The blood samples were collected from the ear vein and jugular vein of the animals. Thin blood smears were prepared by puncturing the ear vein using 18G needle, air dried and fixed in methanol for 2 min. The thin blood smears were stained with a working dilution of 10% Giemsa stain for 40 min. The smears were washed with tap water, air dried and examined under oil immersion lens (X 100) of microscope for demonstration of A. marginale. Whole blood (~5 ml) was drawn from the jugular vein into EDTA coated vacutainers (BD, USA) and stored at -20°C for isolation of genomic DNA. The present investigation was performed at the Division of Parasitology, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly (U.P) from July 2017 to June 2019. The percentage rickettsia was determined by counting the infected RBCs in proportion to the total RBCs in at least 15-20 microscopic fields. Percentage of rickettsia infected RBCs was determined using the following formula.
                
Molecular detection of A. marginale infection in water buffaloes
 
Genomic DNA was isolated from 100 µl of whole blood from each sample using DNAeasy® blood and tissue kit (Qiagen, Germany) in accordance with the manufacturer’s protocol. Extracted DNA was eluted in 50 µl of DNA elution buffer and stored at -20°C till further use. The concentration of genomic DNA was quantified by Nanodrop spectrophotometer. Genomic DNA of A. marginale isolated from blood samples showing high parasitaemia by light microscopy was used as positive control in the PCR. Genomic DNA isolated from the whole blood of water buffaloes that were maintained in tick free conditions of the experimental sheds of ICAR-Indian Veterinary Research Institute, Izatnagar and were negative in the microscopic examination of several blood smears and in two rounds of nested-PCR were used as negative controls in PCR assays.

Blood samples from 228 water buffaloes belonging to the three districts of U.P were screened for A. marginale infection. The msp5 gene of A. marginale was used for the PCR amplification and for determining the prevalence of A. marginale infection in these animals. Primers were designed to amplify full length msp5 gene (633 bp) of A. marginale as described by Visser et al., (1992) (Table 1) and custom synthesized based on GenBank accession number M93392. Each PCR reaction was carried out in a standard 25 µl reaction volume using thermal cycler (S1000-BIO-RAD). The master mix used for the primary PCR reaction consisted of 2.5 µl of 10X PCR Green buffer (Thermo Scientific, USA), 0.5 µl of 10 mM dNTP mix (Thermo Scientific, USA), 0.25 µl of Dream Taq DNA polymerase (Thermo Scientific, USA), 1.0 µl each (10 pmol) of the forward (msp5- FOR) and reverse (msp5-REV) primers (Eurofins, India) and 2.0 µl of template DNA. The final volume was made up to 25 µl using nuclease free water. Initial denaturation of the genomic DNA at 94°C for 4 min followed by 35 cycles of denaturation at 94°C for 45 sec, annealing at 52°C for 45 sec, primer elongation at 72°C for 1 min and final elongation at 72°C for 10 min was the protocol used for PCR amplification of the msp5 gene.



For enhanced sensitivity of the PCR assay, nested-PCR was also performed. Forward  (msp5-N FOR-1) and  reverse (msp5-N REV) primers were custom synthesized (Table 1) to amplify 457 bp fragment of msp5 gene. Subsequently, second round of nested-PCR was performed on this primary PCR product using internal forward (msp5-N FOR-2) and reverse (msp5-N REV) primers to amplify a product of 343 bp (de Echaide et al., 1998). The PCR reaction conditions were identical for carrying out conventional and nested-PCR. Primary PCR product (1:2 to 1:10 dilution) was used as template in nested-PCR. The PCR products were checked for amplification by electrophoresis on 1.5% agarose gel and visualized in gel documentation system (Syngene, UK). A statistically significant number of PCR amplicons (n=20) (both primary and nested-PCR products) were sequenced for confirmation of the Anaplasma species. Sensitivity of the nested-PCR was determined by tenfold dilution of the genomic DNA retrieved from the blood samples positive for A. marginale infection by microscopic examination. The specificity of the nested-PCR primers was also checked with genomic DNAs of Babesia bigemina, Theileria annulata and Trypanosoma evansi.

The microscopic investigation of Giemsa stained peripheral blood smears of 228 water buffaloes detected 18 (7.89%) animals positive for the blue/purple coloured, rounded inclusion bodies of A. marginale located at the margins of the erythrocytes. These results are in concurrence with the findings in South-West Gujarat where a prevalence of 7.07% of A. marginale infection was observed in water buffaloes by routine microscopy of Giemsa stained blood smears (Maharana et al., 2015). In the present investigation, 6.58% (15/228) of animals were found to be clinically infected and showed signs of pyrexia, progressive weakness, paleness of mucous membrane, tachycardia and tick infestation. The average level of parasitaemia in clinically positive animals was found as 1570.5± 876.8 ´ 106 infected RBCs per ml of blood which coincided with the observations of Gale et al., (1996). Rickettsiaemia level exceeding 109 infected erythrocytes per ml of blood is indicative of acute infection, generally characterized by clinical manifestations. Recovery from acute anaplasmosis results in persistent infection and in such cases the level of rickettsiaemia is generally below the threshold levels of microscopic detection (106 infected erythrocytes per ml of blood). Moreover, A. marginale organisms remain undetected during conventional microscopic investigation and microscopy is, therefore, not a reliable tool for detecting presymptomatic or carrier animals (Carelli et al., 2007; Aubry and Geale, 2011).

The PCR of the full length msp5 gene resulted in an amplicon of 633 bp in 29.39% (67/228) of animals (Fig 1). However, nested-PCR was highly sensitive in the detection of rickettsia in buffaloes using primers designed by de Echaide et al., (1998). In the nested-PCR assay, A. marginale infection was revealed as the primary-PCR product of 457 bp in 35.5% (81/228) of buffaloes (Fig 2) while secondary-PCR generated amplicon of 343 bp in 64.0% (146/228) of these animals (Fig 3). The analytical sensitivity of the PCR assay for detection of genomic DNA of A. marginale using present set of primers was 200 picograms. A significant number of PCR amplicons (n=20) were randomly sequenced which revealed nucleotide sequence specific to A. marginale. The PCR primers used in the present assay did not generate any amplicon when the genomic DNA of T. annulata, B. bigemina and T. evansi were used as template. Based on the msp5 gene PCR assay, the prevalence of A. marginale was reported to be 57.5% in buffaloes in Sri Lanka (Zhtldyz et al., 2019). In Colombia, a prevalence of 13.1% was observed in buffaloes using semi-nested PCR targeting the msp5 gene of A. marginale (James-Duenez et al., 2018). Molecular detection of A. marginale infection in buffaloes by PCR based on msp4 gene observed a prevalence of 8.0% in North-East Thailand (Saetiew et al., 2015). A study on buffaloes in Pakistan by Ashraf et al., (2013) using 16S RNA gene reported a prevalence of A. marginale in 7.0% of animals. The nested-PCR based on msp5 gene has been reported as the most sensitive assay (de Echaide et al., 1998; Scoles et al., 2005; Singh et al., 2012; Mason et al., 2017; Bisen et al., 2021) for detecting A. marginale, A. centrale and A. ovis. Very few reports on the prevalence of A. marginale infection in water buffaloes are available from India. A three year old buffalo manifesting clinical signs of anaplasmosis and its complete recovery after treatment was reported by Vatsya et al., (2013). A prevalence of anaplasmosis in 18.18% (4/22) buffaloes was reported by msp1β gene based PCR (Sharma et al., 2013). Based on the amplification of 576 bp msp5 DNA, the detection rate of A. marginale was found to be 18.33% (11/60) in buffaloes in coastal South Gujarat (Kumar et al., 2019). A study on A. marginale using msp5 based nested-PCR observed a prevalence of 73.1% in carrier cattle of North-west India (Singh et al., 2012). Similar trends were observed in a study conducted in carrier cattle of North India which revealed a prevalence of 87.9% by nested-PCR based on msp5 gene of A. marginale (Bisen et al., 2021). In the present study, a large number of buffaloes found positive for anaplasmosis by microscopy and PCR were asymptomatic but only 6.58% of these buffaloes presented clinical signs of anaplasmosis. The clinical picture of A. marginale infection was revealed in only 1.90% of buffaloes in a study in coastal South Gujarat by Kumar et al., (2019). Buffaloes are reported to be less susceptible to A. marginale infection than cattle as they are naturally resistant to tick infestation and can reduce the intensity of infection by preventing the multiplication of the pathogen in the cells (Amira et al., 2020). Detection of persistently infected buffaloes and cattle is important to control the movement of infected animals into disease-free regions and also to formulate and implement strategic control measures against the spread of infection.

The present study carried out in water buffaloes in U.P, North India detected a large percentage of these animals as positive (64.0%) for A. marginale by nested-PCR. However, a large buffalo population needs to be screened for anaplasmosis in different geographical regions of the country for a complete epidemiological study of the infection.

The authors declare that they have no conflict of interest.

The authors are thankful to the Director ICAR-Indian Veterinary Research Institute, Izatnagar for providing necessary facilities for this research work. The authors acknowledge the grants received from CAAST/ACLH-NAHEP (Grant No.NAHEP/CAAST/2018-2019) of ICAR-World Bank funded “National Agricultural Higher Education Project” for supporting this research work.