Pathological and molecular studies on elephant endotheliotropic herpesvirus haemorrhagic disease among captive and free-range Asian elephants in India.

In the present research pathology and molecular diagnosis of elephant endotheliotropic herpes virus-haemorrhagic disease (EEHV-HD) among Asian elephants was studied. Out of 76 cases, 20 were positive for EEHV infection in PANPOL and POL1 based semi-nested PCR. Out of 20 samples, 10 samples were fatal cases of EEHV-HD while 10 were of either subclinical or latent infection. Acute onset haemorrhagic disease with EEHV-HD had anorexia, facial and neck swelling, cyanotic buccal mucosa and tongue, nasal and ocular discharge, and colic. The hallmark of gross finding in all cases were severe haemorrhagic lesions in the internal organs viz. cyanosis of tongue with multifocal petechial haemorrhages, diffuse epicardial and endocardial haemorrhages, swollen liver (rounded edges) with parenchymal haemorrhages, serosal and mucosal haemorrhages in gastrointestinal tract, congested kidneys with corticomedullary haemorrhages, highly congested meninges, and brain capillaries with haemorrhages. Microscopic findings in all the cases had severe vascular changes in the visceral organs. Microthrombi was present in the vasculature of tongue, heart, lung, liver, kidney, and brain. The endothelial lining of most of the blood vessels were swollen with apoptotic changes. Amphophilic to basophilic intranuclear inclusion bodies were observed in the endothelial cells. Immunostaining using anti-EEHV DNAPOL hyperimmune sera revealed intense positive signals in the endothelium of blood vessels and their walls. Quantification of viral load in necropsy tissue samples revealed highest in the heart (7.4 × 106/µg of sample) and least in the brain (9 × 103/µg of sample). The PCR amplicons from EEHV1 specific genes (POL1(U38) and TER were subjected to partial genome sequencing which had 99.9% similarity with the EEHV1A subtype. It was concluded that Asian elephants in India are latently infected for EEHV1 and in all the fatal EEHV-HD cases, EEHV1A subtype was the causative agent with characteristic pathomorphological changes in visceral organs.

Comparative evaluation of diagnostic methods for detection of Theileria spp. in cows.

Theileriosis is a hemoprotozoan illness of cattle in tropical regions that poses a severe economic loss to dairy farmers in the form of production loss and mortality. We designed and optimized a multiplex real-time PCR by using Taq-Man® probe for detection and quantification of Theileria orientalis and Theileria annulata simultaneously by targeting 18 s rRna and MPSP (surface merozoite protein) genes, respectively. Fifty-five EDTA blood samples from clinically Theileria-suspected cows of three Theileria-endemic districts of Odisha were processed using acridine dye based fluorescent microscopy, Giemsa staining, and PCR. PCR revealed T. annulata and T. orientalis in 11/42 (26.11%) and 24/42 (57.14%) cases, respectively. Mixed infection due to both the Theileria spp. was recorded in 7/42 (16.66%). On comparison with gold standard test (PCR), the accuracy, sensitivity, and specificity were 92.72, 95.12, and 85.71% for Giemsa staining and 96.36, 97.56, and 92.85% for acridine orange dye. Multiplex real time PCR using Taq-Man probe detected two species of T. annulata and T. orientalis simultaneously. Acridine dye based fluorescent microscopy is comparatively easy and rapid method in detection of Thelieria spp.

Recombination drives the emergence of orf virus diversity: evidence from the first complete genome sequence of an Indian orf virus isolate and comparative genomic analysis.

Contagious pustular dermatitis is a disease that primarily infects small ruminants and possesses zoonotic potential. It is caused by orf virus (ORFV), a member of the genus Parapoxvirus. In this study, we evaluated an ORFV outbreak in goats in Madhya Pradesh, a state in central India, during 2017. The transboundary potential of this virus was evaluated by constructing phylogenetic trees. The complete genome sequence of an ORFV isolate named Ind/MP/17 was found to be 139,807 bp in length with 63.7% GC content and 132 open reading frames (ORFs) flanked by 3,910-bp inverted terminal repeats (ITRs). An investigation into evolutionary parameters such as selection pressure (θ = dN/dS) and nucleotide diversity (π) demonstrated that ORFV has undergone purifying selection. A total of 40 recombination events were identified, 21 of which were evident in the Ind/MP/17 genome, indicating its ability to generate new variants.

Determining Theileria annulata parasitemia through qPCR in lactating cows of Odisha, - a theileriosis-endemic region of India.

Study was undertaken in a theileriosis-endemic region of India during May 2018 to April 2019 among milch cows. Blood samples collected from apparently healthy (n = 65) and Theileria-suspect cows (n = 65) were screened against T. annulata and T. orientalis infection by SYBR Green‒based real time PCR using primers designed from the isolates of study area. Cows having single infection with T. annulata with/without clinical signs of inappetence, low milk yield, pale mucous membranes, fever, enlarged prescapular lymph node, soil licking, panting, coughing, salivation and lachrymation were subjected to further investigation where parasitaemia and piroplasms per 1000 erythrocytes ranged from 1.6 × 107 to 1.2 × 108 parasites/mL of blood and 3-24 piroplasms in moderate group (16/65), 4.4 × 108 to 6.9 × 109 parasites/mL of blood and >88 piroplasms in severe group (30/65) and 1.6 × 104 to 5.5 × 106 parasites/mL of blood and 0-1 piroplasms in asymptomatic or carriers (17/65), respectively. Study unfolded significant difference in T. annulata parasitaemia among apparently healthy and ill cows. Phylogenetic analysis of our T. annulata isolates (NCBI accession numbers MN098316, MN098317 and MN098318) exhibited maximum similarity with the isolates detected in other parts of India.

Characterization and Whole Genome Sequencing of Chromobacterium violaceum OUAT_2017: A Zoonotic Pathogen Found Fatal to a Wild Asiatic Elephant.

This study reports a rare fatal case of Chromobacterium violeceum OUAT_2017 strain infection in an Asiatic elephant calf in India. Necropsy revealed pus-filled nodules in liver, spleen, and lungs. Nutrient broth cultures of nodule content showed sediment of violet pigment whereas smooth, non-diffusible, violet-pigmented, homogeneous colonies appeared on nutrient agar. The organism was found to be non-haemolytic and resistant to 8 of the 24 antibiotics tested in vitro. Partial 16S rRNA gene sequence measuring 1410 bp revealed 97% homology with C. violeceum. The bacterial genome composed of 64.87% of G + C content with total size of 4,681,202 bp. The genome annotation has 42 genes responsible for multidrug antibiotic resistance with the presence of Aminoglycoside-modifying enzymes (AAC (6')) that targets streptomycin and spectinomycin. Our findings corroborated the lethal effect of C. violeceum in a new host (elephant) that enriched scientific information on epidemiological picture and whole genome sequencing as well. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01047-4.

ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS HEMORRHAGIC DISEASE OUTBREAK IN AN INDIAN ZOO.

Elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV HD) is an acute viral infection of growing Asian elephants (Elephas maximus). Four apparently healthy subadult Asian elephants aged between 6 and 10 yr at Nandankanan Zoological Park (NKZP), India, died of EEHV HD during August-September 2019. All four elephants were rescued from different reserved forests of Odisha state at less than 1 yr of age and hand reared in the NKZP. Elephants exhibited the clinical signs of lethargy, head swelling, fever, loss of appetite, abdominal distension, scant urination and defecation, signs of colic, lameness, trunk discharge, cyanosis/ulceration of tongue, erratic behavior, and recumbence before death. Period of illness varied between 28 and 42 h. Thrombocytopenia was the common significant hematological observation. No significant biochemical alterations were recorded except for higher creatinine concentrations. Analysis of blood samples in RT-PCR assay using two different sets of primers and probes that targeted terminase gene and major DNA-binding protein gene followed by cPCR and sequencing was positive for EEHV-1A in all four animals. Postmortem examination of all four carcasses showed hemorrhages in internal organs, including the hard palate, heart, lungs, stomach, mesenteric lymph nodes, mesentery, colon serosa, spleen, liver, kidney, and meninges. Histopathology showed congestion and/or hemorrhages in heart, lung, brain, kidney, and liver. There was presence of intranuclear inclusion bodies in the sinusoidal epithelial cells. The outbreak of EEHV HD that resulted in the acute death of four juvenile captive Asian elephants within <30 d, the first of its kind documented in India, is increasing the fear of similar outbreaks in the future.

Complete mitogenome sequence analysis of an Indian Black Bengal goat, Capra hircus, and its phylogenetic consequences.

Capra hircus (Black Bengal goat) is a prized goat breed reared across the Southeast Asian region. Known for its excellent chevon quality and adaptability to hot and humid climates, it supports the livelihood of millions of farmers. The present study reveals the complete mitogenome of a Black Bengal goat of Indian origin utilizing a next-generation sequencing approach (GenBank acc. no.-MZ073671). This newly assembled whole mitochondrial genome (16,637 bp) is composed of a non-coding control region (D-loop region), two ribosomal RNA (rRNA genes), 13 protein-coding genes, and 22 transfer RNA (tRNA). Overall, the current mitogenome shares 99.77% sequence homology with previously reported mitogenomes of Capra hircus. The phylogenetic analysis inferred that the current mitogenome shares a close relationship with the Teressa goat breed of the Andaman and Nicobar Islands.

One Health intervention for elimination of anthrax in an endemic district of Odisha: A baseline and endline study.

Background: This study was to compare a baseline and endline survey which were conducted to assess the changes in knowledge, attitude and practices about anthrax disease among the communities after One Health intervention for the elimination of human anthrax in an endemic district of Odisha. Methods: A total of 2670 respondents were interviewed during the baseline and 2511 for the endline survey using a structured questionnaire by multi-stage sampling method. Descriptive statistics were used and logistic regression was performed to estimate the relationship between the variables and knowledge of anthrax. Results: Out of the total participants in the study, males were about 76.25% in baseline and 72.08% in endline and about half of the total respondents were illiterate. Majority of the respondents had reported agriculture as their main occupation during both surveys. More than 50% of the respondents had livestock in their houses and farming was the main purpose for keeping them in both surveys. Around 20.26% of respondents knew about anthrax in baseline which raised to 53.64% after One Health intervention. Almost 21.29% of livestock owners had vaccinated their animals against anthrax disease throughout baseline, which increased to 66.5% during the endline survey. Conclusion: This study highlights a significant surge in both knowledge and practices related to anthrax within the community after the implementation of intervention packages based on the One Health approach. The outcome of our study signified the importance of One Health interventions to address the health challenges related to zoonotic diseases in tribal communities. The data could be useful for local Governments to incorporate such an approach in their health policy to eliminate human anthrax.

Investigation of human anthrax outbreak in Koraput district of Odisha, India.

BACKGROUND: Anthrax is a zoonotic infection resulting from the bacteria Bacillus anthracis. Humans contract cutaneous anthrax by coming into contact, and gastrointestinal (GI) anthrax by consumption of infected animals or animal products. An outbreak investigation was conducted to confirm the occurrence of the anthrax outbreak, comprehend its extent, understand the epidemiological characteristics, identify the outbreak's cause, and propose control measures. METHODS: A descriptive epidemiology was carried out for this outbreak investigation. We defined a suspected human cutaneous anthrax case as appearance of skin lesions and symptoms (itching/redness/swelling) and a suspected case of GI anthrax as appearance of diarrhoea/abdominal pain/vomiting in a resident of Koraput district after being associated with slaughtering and/or consumption of carcass during 5th April to 15th May 2023. The etiological hypothesis was formulated using descriptive epidemiological methods. Laboratory confirmation was performed by real-time polymerase chain reaction (RT-PCR). Statistical analyses were conducted using SPSS 25. RESULTS: A total of 47 clinically suspected anthrax cases were identified during the outbreak in five villages of Koraput district in Odisha. The epidemic curve indicated multiple point-source exposures starting from 13th April 2023. About 10 cases were identified by RT-PCR testing as confirmed cases of anthrax. No death was recorded in this outbreak investigation. CONCLUSIONS: Based on a thorough examination of epidemiological survey results and laboratory findings, we conclude that the outbreak was of human cutaneous and GI anthrax. Exposures from handling dead animals were associated with cutaneous anthrax, whereas eating uncooked meat of dead sheep was associated with gastrointestinal anthrax.

Prevalence of Newcastle Disease Virus in Wild and Migratory Birds in Haryana, India.

Newcastle disease virus (NDV) can infect approximately 250 avian species and causes highly contagious Newcastle disease (ND) in domestic poultry, leading to huge economic losses. There are three different pathotypes of NDV, i.e., lentogenic, mesogenic, and velogenic. Wild resident (wild) and migratory birds are natural reservoirs of NDV and are believed to play a key role in transmitting the virus to domestic poultry. The present study was conducted to determine the prevalence of NDV in wild and migratory birds in the state of Haryana, India, during two migratory seasons (2018-19 and 2019-20). In total 1379 samples (1368 choanal swabs and 11 tissue samples) were collected from live (n = 1368) or dead birds (n = 4) belonging to 53 different avian species. These samples belonged to apparently healthy (n = 1338), sick (n = 30), and dead (n = 4) birds. All samples were tested for NDV by real-time reverse transcription-PCR using M gene specific primers and probe. Of the 1379 samples, 23 samples from wild birds [Columba livia domestica (n = 12, 52.17%), Pavo cristatus (n = 9, 39.13%), and Psittaciformes (n = 2, 8.69%)] were found positive for NDV. Only one of the 23 samples (from P. cristatus) was positive for F gene, indicating it to be a mesogenic/velogenic strain. These results indicate that both lentogenic and velogenic strains of NDV are circulating in wild birds in Haryana and that further studies are needed to characterize NDV strains from wild/migratory birds and domestic poultry to determine the extent of virus transmission among these populations. This study considers the disease transmission risk from domestic pigeons and parrots to commercial poultry and vice versa, and the results emphasize the need for strict biosecurity strategies to protect commercial poultry in the region.

Prevalencia del virus de la enfermedad de Newcastle en aves silvestres y migratorias en Haryana, India. El virus de la enfermedad de Newcastle (NDV) puede infectar aproximadamente a 250 especies de aves y causa la enfermedad de Newcastle (ND) altamente contagiosa en la avicultura comercial, lo que genera enormes pérdidas económicas. Hay tres patotipos diferentes del virus de Newcastle, que incluyen, lentogénico, mesogénico y velogénico. Las aves silvestres residentes (silvestres) y migratorias son reservorios naturales del virus de Newcastle y se cree que desempeñan un papel clave en la transmisión del virus a las aves domésticas comerciales. El presente estudio se realizó para determinar la prevalencia del virus de Newcastle en aves silvestres y migratorias en el estado de Haryana, India, durante dos temporadas migratorias (2018-19 y 2019-20). En total, se recolectaron 1379 muestras (1368 hisopos coanales y 11 muestras de tejido) de aves vivas (n = 1368) o muertas (n = 4) pertenecientes a 53 especies de aves diferentes. Estas muestras pertenecían a aves aparentemente sanas (n = 1338), enfermas (n = 30) y muertas (n = 4). Todas las muestras se analizaron para detectar al virus de Newcastle mediante transcripción reversa y PCR en tiempo real utilizando iniciadores y una sonda específicos del gene M. De las 1379 muestras, 23 muestras de aves silvestres [Columba livia domestica (n = 12, 52.17 %), Pavo cristatus (n = 9, 39.13 %) y Psittaciformes (n = 2, 8.69 %)] resultaron positivas para el virus de Newcastle. Solo una de las 23 muestras (de P. cristatus) fue positiva para el gene F, lo que indica que se trata de una cepa mesogénica/velogénica. Estos resultados indican que tanto las cepas lentogénicas como las velogénicas del virus de Newcastle están circulando en las aves silvestres de Haryana y que se necesitan más estudios para caracterizar las cepas del virus de Newcastle de las aves silvestres/migratorias y de las aves domésticas para determinar el alcance de la transmisión del virus entre estas poblaciones. Este estudio considera el riesgo de transmisión de la enfermedad de las palomasdomésticas y loros a las aves comerciales y viceversa, y los resultados enfatizan la necesidad de estrategias estrictas de bioseguridad para proteger las aves comerciales en la región.

Detection of Newcastle disease virus and assessment of associated relative risk in backyard and commercial poultry in Kerala, India.

BACKGROUND: Newcastle disease (ND) is an economically important viral disease affecting the poultry industry. In Kerala, a state in South India, incidences of ND in commercial and backyard poultry have been reported. But a systematic statewide study on the prevalence of the disease has not been carried out. OBJECTIVES: A cross-sectional survey was performed to detect the presence of Newcastle disease virus (NDV) in suspect cases and among apparently healthy commercial flocks and backyard poultry, in the state and to identify risk factors for NDV infection. METHODS: Real-time reverse transcription-PCR (RT-PCR) was used to detect the M gene of NDV in choanal swabs and tissue samples collected from live and dead birds, respectively and the results were statistically analysed. RESULTS: The predominant clinical signs of the examined birds included mild respiratory signs, huddling together and greenish diarrhoea. Nervous signs in the form of torticollis were noticed in birds in some of the affected flocks. On necropsy, many birds had haemorrhages in the proventriculus and caecal tonsils which were suggestive of ND. Of the 2079 samples tested, 167 (8.0%) were positive for the NDV M-gene by RT-PCR. Among 893 samples collected from diseased flocks, 129 (14.5%), were positive for M gene with pairwise relative risk (RR) of 15.6 as compared to apparently healthy flocks where 6 out of 650 (0.9%) samples were positive. All positive samples were from poultry; none of the ducks, pigeons, turkey and wild birds were positive. Commercial broilers were at higher risk of infection than commercial layers (RR: 4.5) and backyard poultry (RR: 4.9). Similarly, birds reared under intensive housing conditions were at a higher risk of being infected as compared to those reared under semi-intensive (RR: 6.7) or backyard housing (RR: 2.1). Multivariable analysis indicated that significantly higher risk of infection exists during migratory season and during ND outbreaks occurring nearby. Further, lower risk was observed with flock vaccination and backyard or semi-intensive housing when compared to intensive housing. When the M gene positive samples were tested by RT-PCR to determine whether the detected NDV were mesogenic/velogenic, 7 (4.2%) were positive. CONCLUSIONS: In Kerala, NDV is endemic in poultry with birds reared commercially under intensive rearing systems being affected the most. The outcome of this study also provides a link between epidemiologic knowledge and the development of successful disease control measures. Statistical analysis suggests that wild bird migration season and presence of migratory birds influences the prevalence of the virus in the State. Further studies are needed to genotype and sub-genotype the detected viruses and to generate baseline data on the prevalence of NDV strains, design better detection strategies, and determine patterns of NDV transmission across domestic poultry and wild bird populations in Kerala.

Prevalence of Newcastle disease and associated risk factors in domestic chickens in the Indian state of Odisha.

Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a contagious disease that affects a variety of domestic and wild avian species. Though ND is vaccine-preventable, it is a persistent threat to poultry industry across the globe. The disease represents a leading cause of morbidity and mortality in chickens. To better understand the epidemiology of NDV among commercial and backyard chickens of Odisha, where chicken farming is being prioritized to assist with poverty alleviation, a cross-sectional study was conducted in two distinct seasons during 2018. Choanal swabs (n = 1361) from live birds (commercial layers, broilers, and backyard chicken) and tracheal tissues from dead birds (n = 10) were collected and tested by real-time reverse transcription polymerase chain reaction (RT-PCR) for the presence of matrix (M) and fusion (F) genes of NDV. Risk factors at the flock and individual bird levels (health status, ND vaccination status, geographical zone, management system, and housing) were assessed using multivariable logistic regression analyses. Of the 1371 samples tested, 160 were positive for M gene amplification indicating an overall apparent prevalence of 11.7% (95% CI 10.1-13.5%). Circulation of virulent NDV strains was also evident with apparent prevalence of 8.1% (13/160; 95% CI: 4.8-13.4%). In addition, commercial birds had significantly higher odds (75%) of being infected with NDV as compared to backyard poultry (p = 0.01). This study helps fill a knowledge gap in the prevalence and distribution of NDV in apparently healthy birds in eastern India, and provides a framework for future longitudinal research of NDV risk and mitigation in targeted geographies-a step forward for effective control of ND in Odisha.

Prevalence of Newcastle Disease Virus in Commercial and Backyard Poultry in Haryana, India.

Newcastle disease virus (NDV) causes Newcastle disease (ND) in poultry. The ND is a highly contagious disease, which is endemic in several countries despite regular vaccination with live or killed vaccines. Studies on NDV in India are mostly targeted toward its detection and characterization from disease outbreaks. A surveillance study was undertaken to determine NDV prevalence throughout the state of Haryana from March 2018 to March 2020 using a stratified sampling scheme. The state was divided into three different zones and a total of 4,001 choanal swab samples were collected from backyard poultry, commercial broilers, and layers. These samples were tested for the M gene of NDV using real-time RT-PCR. Of the 4,001 samples tested, 392 were positive (9.8% apparent prevalence; 95% CI: 8.9-10.8%) for the M gene. Of these 392 M gene positive samples, 35 (8.9%; 95% CI: 6.4-12.3%) were found to be positive based on F gene real-time RT-PCR. Circulation of NDV in commercial and backyard poultry highlights the importance of surveillance studies even in apparently healthy flocks. The information generated in this study should contribute to better understanding of NDV epidemiology in India and may help formulate appropriate disease control strategies for commercial and backyard birds.

Identification and phylogenetic characterization of bovine ephemeral fever virus (BEFV) of Middle Eastern lineage associated with 2018-2019 outbreaks in India.

Bovine ephemeral fever virus (BEFV) is an evolving arbovirus reported across tropical, subtropical and temperate climatic zones globally. This study reveals prominent BEFV outbreaks in India, emerging annually during monsoons in subtropical areas accompanied by a congenial abundance of the vector population. PCR-based detection of viral genomic RNA in the blood samples collected during outbreaks of 2018-2019 for the first time confirmed the presence of BEFV in India. Phylogenetic analysis based on the glycoprotein gene of BEFV showed the current isolates to have high sequence homology with Middle Eastern lineage with nearly 97%, identity to Turkey (BEFV Ad12/TUR) and Israel (Israel 2006) isolates.

Complete Genome Sequences of Newcastle Disease Virus Isolates from Backyard Chickens in Northern India.

The molecular characterization of three Newcastle disease viruses (NDV) isolated from backyard chickens in the state of Haryana, India, was undertaken. Two genotype II strains and one genotype XIIIc class II isolate with genome sizes of 15,186 and 15,192 nucleotides (nt), respectively, were identified.

Seroprevalence of Capripoxvirus infection in sheep and goats among different agro-climatic zones of Odisha, India.

AIM: The study was undertaken to assess the prevalence of antibodies to Capripoxviruses among small ruminants of Odisha, India. MATERIALS AND METHODS: A total of 500 random serum samples collected from 214 sheep and 286 goats across 10 agro-climatic zones of Odisha, were screened using whole virus antigen-based indirect ELISA for antibodies against Capripoxviruses. Results were analyzed by suitable statistical methods. RESULTS: Screening of 500 serum samples showed seropositivity of 8.88% and 31.47% in sheep and goats, respectively, for Capripoxviruses. The prevalence rate according to agro-climatic zone ranged from 0% (North Eastern coastal plain zone) to 48.57% (North central plateau zone) for goat pox, and 0% (Western undulating zone and North central plateau) to 22.22% (South Eastern ghat zone) for sheep pox. The difference in prevalence rates among the various agro-climatic zones was statistically significant (p<0.05) for goats, but not for sheep. Antibody prevalence rates among various districts were recorded to be the highest in Jagatsinghpur (30%) for sheep pox and Dhenkanal (80%) for goat pox. CONCLUSIONS: The study revealed serological evidence of Capripoxvirus infection in sheep and goat populations in the study area, in the absence of vaccination. Systematic investigation, monitoring, and reporting of outbreaks are necessary to devise control strategies.

Prevalence of carrier state theileriosis in lactating cows.

AIM: The objective of this study was to examine the carrier status of theileriosis among apparently healthy cross-bred jersey cattle population of Odisha using conventional blood smear examination and polymerase chain reaction (PCR). MATERIALS AND METHODS: A total of 34 apparently healthy cross-bred Jersey lactating cows were considered in this study. Blood samples were subjected to microscopic examination after staining with Giemsa stain and PCR based molecular diagnosis using two sets of primer, i.e., N516/N517 and TorF1/TorF2 specific for Theileria annulata and Theileriaorientalis, respectively. RESULTS: Examination of blood samples revealed presence of theileria parasites to a magnitude of 20.59% for T. annulata, 8.82% for T. orientalis, and 2.94% for both. CONCLUSION: Molecular diagnosis was found to be much more sensitive than conventional method for diagnosis of theileriosis. T. annulata was found to be the predominant species affecting the exotic cattle. T. orientalis was detected in apparently healthy cows.