Mandeep Singh Bal

Prevalence and haemato-biochemical profile of Anaplasma marginale infection in dairy animals of Punjab (India).

OBJECTIVE To do the systematic comparison of prevalence of anaplasmosis by PCR and Giemsa stained thin blood smear (GSTBS) based parasitological assays in dairy cattle of Punjab, which has not been reported yet. To analyse the haematobiochemical alterations in infected animals to arrive at the conclusion regarding the pathogenicity induced by Anaplasma marginale (A. marginale) in latent and patent infection. METHODS Study was conducted on 320 animals (236 cows, 62 calves and 22 buffaloes) of Punjab, India. PCR on genome of A. marginale was performed by targeting msp1 β gene using specific primers BAP-2/AL34S, amplifies products of size 407 bp. Questionnaires based data on the characteristics of the infected animals and management strategies of the farm were collected and correlated. RESULTS Higher prevalence and more significant association was observed in the PCR based molecular diagnosis (P=0.00012) as compared to that in GSTBS (P=0.028 8) based diagnosis with various regions under study. With respect to the regions, highest prevalence was recorded in Ferozepur by PCR based diagnosis, while that in Jalandhar by GSTBS examination. Similar marked significant association of the PCR based diagnosis with the age of the animals under study (P=0.00013) was observed elucidating no inverse age resistance to A. marginale in cow calves. Haematobiochemical profile of infected animals revealed marked anemia, liver dysfunction and increase globulin concentrate indicating rise in immunoglobulin level to counteract infection. CONCLUSIONS PCR is far more sensitive in detecting the disease even in latent infection which may act as nidus for spread of anaplasmosis to susceptible animals in endemic areas. Severity of anaemia and liver dysfunction were comparable both in patent as well as latent infection indicating pathogenicity of both.

Molecular epidemiology of Echinococcosis from food producing animals in north India

Echinococcosis is an important medical, veterinary and economic concern in India. Ten cysts were randomly selected from each intermediate host species (cattle, buffalo, sheep, goat and pigs). Either the germinal layer (sterile cysts) or protoscoleces (fertile cysts) were collected for molecular characterization. A 434 base pair fragment of the mitochondrial cytochrome oxidase-1 gene was amplified using PCR from each isolate. Ten representative samples (2 from each intermediate host species) were sequenced in both the directions from which readable sequences were obtained from nine for phylogenetic analysis (NCBI, Blast). Phylogenetic analysis of cytochrome oxidase I gene revealed that seven (77.7%) isolates, from cattle (2), pigs (2), buffaloes (1) and goat (2) were clustered with the Indian Buffalo (G3) strain of Echinococcus granulosus, while two (22.2%) isolates from sheep were clustered with the sheep strain (G1) of E. granulosus. Phylogenetic analysis of the cytochrome oxidase-1 gene revealed that the buffalo strain (G3) and common sheep strain (G1) are cycling among livestock in north India and that these strains are highly adapted to cattle, buffalo, sheep, goats and pigs.

Multiplex PCR for detection of Trypanosoma evansi and Theileria equi in equids of Punjab, India

Multiplex PCR for simultaneous detection of Trypanosoma evansi and Theileria equi in single-step reaction was optimized and employed on 108 equids (99 horses and 9 donkeys/mules) blood samples collected from two agro-climatic zones (Sub-mountain undulating zone and Undulating plain zone) of Punjab to evaluate the status of concurrent infection and associated risk factors. The amplification products of 257 and 709 bp targeting repetitive nucleotide sequence of variable surface glycoproteins of T. evansi and 18S rRNA gene of T. equi, respectively expressed high fidelity of the primer pairs with sequence homology to neighboring geographic isolates. The overall prevalence of T. evansi and T. equi was 3.7 and 1.85%, with Undulating plain zone at higher infection risk for T. equi (OR=3.24, 95% CI=0.28-83.65); and Sub-mountain undulating zone (OR=∞, 95% CI=0.25-∞) for T. evansi. Multiplex PCR revealed higher risk of infection of both T. equi (OR=6.75, 95% CI=0.58-175.38) and T. evansi (OR=2.11, 95% CI=0.05-80.36) in the farms with inappropriate management system. The risk factor associated with the type of host species had an odds ratio of 12.35 (95% CI=0.29-508.37) for donkeys/mules versus horses for T. evansi infection. This group was also at higher risk of infection with Odds ratio (OR) of 4 (95% CI=0.14-53.99) for T. equi. The current investigation brings out various commodities at risk of infection pertaining to equid trypanosomosis and theileriosis evaluated by a rapid and sensitive multiplex PCR assay.

An outbreak of acute Trypanosoma evansi infection in crossbred cattle in Punjab, India

An outbreak of acute trypanosomosis caused by Trypanosoma evansi in Punjab, a northern state of India was investigated in a cattle farm comprising 78 adults (lactating and dry) and 27 calves. The outbreak in the affected animals exhibiting high fever (105°F), circling, tremors, convulsions and death within 24–48 hours was confirmed based on examination of Wright-Giemsa stained blood smears, morpho-metric measurements and by polymerase chain reaction by using T. evansi specific primers yielding species specific 227 bp PCR product. Pretreatment parasitaemia estimated in three clinical cases revealed 12.5×104, 11.2×104 and 16.7×104 tryps/µl of blood and cow that was treated with isometamedium chloride revealed parasitemia of 1.4×104 tryps/µl of blood 24 hours after treatment while no parasitemia was found after 24 hrs of treatment in other two cows, treated with quinapyramine sulphate and chloride combination (Triquin). Post-mortem revealed moderate enlargement of spleen. Biochemical parameters revealed increase in cholesterol, bilirubin and total proteins.

Molecular epidemiology, risk factors and hematochemical alterations induced by Theileria annulata in bovines of Punjab (India).

Abstract Bovine tropical theileriosis, caused by Theileria annulata, is one of the economically important fatal tick borne haemoprotozoan diseases of dairy animals. The aim of present investigation was to map the distribution of T. annulata in bovines of Punjab state of India in relation to various risk factors including age, sex of animals, location and management of farms. In a cross sectional study, a total of 1278 blood samples were randomly collected from twenty districts falling in five major agro-climatic zones of Punjab. All the samples were screened by blood smear examination followed by polymerase chain reaction targeting SSU rRNA gene for Theileria spp. PCR positive samples (n = 386) for Theileria spp. were then analyzed for T. annulata by amplification of Tams1 gene. Overall prevalence of T. annulata was found to be 29.26% in Punjab, with highest in western Zone (40.49%, 95% CI = 35.57-45.41) and lowest in submountain zone (18.90%, 95% CI = 13.73-24.06). The propensity of incidence of T. annulata was found to be highest in cross bred cattle (32.40%, 95% CI = 29.87-34.94), followed by indigenous cattle (19.64%, 95% CI = 10.67-28.61) and buffaloes (19.2%, 95% CI = 14.99-23.41). Between the two sexes, incidence of T. annulata was higher in female animals. Calves less than 6 months of age were found to be more prone to theileriosis.

Detection of anthelmintic resistance in sheep and goat against fenbendazole by faecal egg count reduction test

Anthelmintic resistance against commonly used anthelmintic fenbendazole was evaluated by employing faecal egg count reduction test (FECRT) in naturally occurring gastrointestinal (GI) nematodes in the semi organized sheep and goat farms of Ludhiana and Amritsar districts. A total of 80 animals (20 each for sheep and goat in both districts) were randomly selected and their faecal samples were examined by qualitative and quantitative parasitological techniques. Results indicate presence of high level of resistance against fenbendazole in both sheep and goat population of Ludhiana and Amritsar districts. More resistance was observed in the GI nematodes from animals reared in Amritsar district as compared to Ludhiana district. The level of anthelmintic resistance observed was apparently more in sheep than goats.

Pathological observations on clinical Anaplasma marginale infection in cattle

Gross and histopathological changes were recorded in a pregnant cattle died of clinical anaplasmosis, a tick transmitted economically important disease caused by Anaplasma marginale. Grossly emaciated carcass along with pale visible mucous membranes and pale serosal surface, splenomegaly and hepatomegaly was observed. Microscopically, in lungs variable extend of interstitial pneumonia, emphysema along with infiltration of mononuclear cells was seen. Spleen showed extensive increase in red pulp area with massive proliferation of lymphocytes. In liver marked thickening of capsule with fatty changes along with retention of bile was seen. Gall bladder showed congestion, glandular hyperplasia and thickening wall. Myocardium showed degeneration and necrosis.

Prevalence of gastrointestinal parasitism in small ruminants in western zone of Punjab, India

Aim: The aim of this study was to explore the prevalence of gastrointestinal parasitism in small ruminants in relation to various risk factors in the western zone of Punjab. Materials and Methods: During the study, 603 fecal samples (391 of sheep and 212 of goats) were examined qualitatively by floatation and sedimentation techniques, and quantitatively by McMaster technique. Results: Out of the 603 fecal (391 sheep and 212 goats) samples examined, 501 were found positive for endoparasitic infection with an overall prevalence of 83.08%, consisting of 85.16% and 79.24% in sheep and goats, respectively. Egg per gram in sheep was apparently more 1441.88±77.72 than goats 1168.57±78.31. The associated risk factors with the prevalence of gastrointestinal tract (GIT) parasites showed that females (85.97%) were significantly more susceptible than males (69.23%). Age wise the adults (>6 months) were significantly more prone to parasitic infection as compared to young ones (<6 months). Seasonal variation was recorded throughout the year and was significantly highest during monsoon (90.10%), followed by winter (83.84%) and summer (78.35%). Conclusion: The study revealed an overall prevalence of 83.08% of GIT parasitic infections in small ruminants constituting 85.16% in sheep and 79.24% in goats in the western zone of Punjab. The most relevant risk factors for the prevalence of gastrointestinal parasitism in ruminants were sex, age, and season.

Outbreaks of Anaplasmosis in Dairy Cattle in Punjab, India

Two severe outbreaks of bovine anapalsmosis caused by Anaplasma marginale were recorded in two districts (Ferozepur and Patiala) of Punjab state in the year 2016. Mortality of animals was recorded in two dairy cattle herd comprising of a total of 260 animals in Ferozepur (n=218) and Patiala (n=42) districts. There was history of purchase of animals at one farm. Out of 260 cattle at risk, 40 were critically ill and 9 died of disease with morbidity, mortality and case fatality rate of 15.38 %, 3.46 % and 22.50 % respectively. Fifteen blood samples were collected from diseased (n=11) and healthy animals (n=4) for haematological analysis, parasitological and polymerase chain reaction (PCR) based diagnosis. Clinical signs in infected animals included high temperature, icterus, anemia, anorexia and decreased milk production. Necropsy findings revealed splenomegaly and severe jaundice. Mild tick infestation was observed at both the farms. Ticks collected from both the outbreaks were identified as Rhipicephaus (Boophilus) microplus. Thin blood smears from infected animals were found positive for Anaplama marginale organisms & disease was further confirmed by molecular approach (PCR). Affected animals were successfully treated with tetracycline, haematinics and antipyretics. PCR was found to be more sensitive in detecting the disease especially in latent infections. Animal owners were advised to follow quarantine measures before mixing new animals in a herd and strategic acaricidal treatment for effective tick control.

Diagnosis and management of bovine babesiosis outbreaks in cattle in Punjab state.

Aim: The aim of the present study was to diagnose severe outbreaks of bovine babesiosis in Punjab state, in the year 2015 and to suggest control and preventive measures to animal owners. Materials and Methods: Mortality of animals was recorded in two cattle herd comprising a total of 465 cattle in Sangrur (n=125) and Faridkot (n=340) districts. There was a history of purchase of animals at one farm. 23 blood samples were collected from diseased (n=15) and healthy animals (n=8) for hematological analysis, parasitological, and polymerase chain reaction (PCR)-based diagnosis. Ticks were also collected from animals for identification. Results: Out of 465 cattle at risk, 28 were critically ill and 14 died of disease with morbidity, mortality, and case fatality rate of 6.02%, 3.01%, and 50.00%, respectively. Clinical signs and necropsy findings were suggestive of babesiosis. Ticks collected from both the outbreaks were identified as Rhipicephalus (Boophilus) microplus. Thin blood smears from infected animals (especially with clinical sign of hemoglobinuria) were found positive for Babesia bigemina organisms; however, molecular diagnosis (PCR) further confirmed the disease. Animals were successfully treated with diminazene aceturate, hematinics, and antipyretics. Conclusions: Two fatal outbreaks of babesiosis in cattle were diagnosed with application of conventional parasitological, hematological, and molecular diagnostic techniques. PCR was found to be far more sensitive in detecting the disease, especially in latent infections. Animal owners were advised to follow quarantine measures before mixing new animals in the herd and strategic acaricidal treatments for effective tick control.