Amrita Sharma

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 Prevalence of Babesia bigemina and Trypanosoma evansi in Dairy Animals from Punjab, India, by Duplex PCR: A Step Forward to the Detection and Management of Concurrent Latent Infections

Specific duplex polymerase chain reaction (PCR) was employed on 411 (386 cattle and 25 buffaloes) blood samples of dairy animals from 9 districts of Punjab, India, for simultaneous detection of Babesia bigemina and Trypanosoma evansi. The results were compared and correlated with conventional Giemsa stained thin blood smear (GSTBS) examination and haematological alterations to know the clinical status and pathogenicity of infections. The Bg3/Bg4 and TR3/TR4 primers were used in duplex PCR for B. bigemina and T. evansi amplified products of 689 bp and 257 bp, respectively. The overall prevalence by duplex PCR was found to be 36.49, 2.43, and 3.41% for T. evansi, B. bigemina, and dual infection, respectively. A more significant difference was observed for dual infection status (P ≤ 0.005) as compared to T. evansi (P ≤ 0.05) and B. bigemina (P ≤ 0.01) among various districts under study. A very low prevalence of T. evansi (0.73%) and B. bigemina (0.48%) was seen by GSTBS. The highly sensitive, specific, and cost-effective duplex PCR was able to detect latent T. evansi and B. bigemina infection in cattle and buffaloes. Haematological evaluation revealed marked pathology in B. bigemina infected group and in dual infected group in contrast to that infected with T. evansi alone.

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.

Spatial distribution, risk factors and haemato-biochemical alterations associated with Theileria equi infected equids of Punjab (India) diagnosed by indirect ELISA and nested PCR.

Equine piroplasmosis is a febrile, tick-borne disease of equids predominately caused by obligatory intra-erythrocytic protozoa Theileria equi in the Indian sub-continent. A cross-sectional study was carried out on 464 equids (426 horses and 38 donkeys/mules) in Punjab, India to assess the level of exposure to equine piroplasmosis by 18S rRNA gene nested polymerase chain reaction (nPCR) and equine merozoite antigen-2 (EMA2) indirect-ELISA (enzyme linked immunosorbent assay), to investigate risk factors and haemato-biochemical alterations associated with the infection. The endemicity of the disease was confirmed by positive PCR amplification in 21.77% and positive antibody titers in 49.78% equid samples. There was a fair agreement between these two diagnostic techniques (Kappa coefficient=0.326). The spatial distribution analysis revealed an increasing trend of T. equi prevalence from north-eastern to south-western region of Punjab by both the techniques correspondingly, which proffered a direct relation with temperature and inverse with humidity variables. The relatively prominent risk factor associated with sero-positivity was the presence of other domestic animals in the herd, while the propensity of finding a positive PCR amplification was higher in donkeys/mules, animal kept at unorganised farm or those used for commercial purposes as compared to their counterparts. There was a significant increase in globulins, gamma glutamyl-transferase, total bilirubin, direct bilirubin, indirect bilirubin, glucose levels and decrease in total erythrocyte count, haemoglobin, packed cell volume by animals, which were revealed positive by nPCR (may or may not positive by indirect-ELISA) and increase in creatinine, total bilirubin, direct bilirubin, glucose and decrease in total erythrocytes count by animals, which were revealed positive by indirect-ELISA (alone). To our knowledge, this study, for the first time, brings out a comprehensive report on the status on spatial distribution of T. equi in Punjab (India) state, thoroughly investigated by molecular and serological techniques, evaluating various environmental and demographic risk factors along with the haemato-biochemical alterations in the exposed animals.

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.

PCR and ELISA vis-à-vis microscopy for detection of bovine anaplasmosis: a study on associated risk of an upcoming problem in North India.

This investigation demonstrates the status of bovine anaplasmosis caused by A. marginale in bovines from Submountain and Undulating Zone of Punjab. Out of 184 suspected animals, 25 (19.51%), 47 (31.71%), and 78 (68.75%) were positive by microscopy, indirect ELISA, and PCR assay, respectively. The microscopy showed 29% sensitivity and 99% specificity, while ELISA showed 32% sensitivity and 79% specificity in concordance with PCR assay. Five false negative samples by msp1β PCR were reconfirmed for Anaplasma spp. targeting 16S rRNA gene. The sequence analysis showed the presence for A. marginale specific restriction site, indicating variation in the local strains of the organism resulting in no amplification with msp1β gene primers. Of 82 samples positive by PCR, 57 were negative by ELISA indicating lower efficacy of ELISA to detect early anaplasmosis. The assessment of risk factor with results of PCR technique indicated that cattle (Odds ratio = 2.884), particularly those of age > 1 years (Odds ratio = 2.204) of district Pathankot (Odds ratio = 3.182) of Submountain Zone (Odds ratio = 2.086), were at high risk of anaplasmosis. All three districts of Submountain Zone are at higher risk indicating the impact of biotic and abiotic factors on the incidence of disease.