Gursimran Filia

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 Mycobacterium tuberculosis and Mycobacterium bovis in Sahiwal cattle from an organized farm using ante-mortem techniques.

Aim: The aim of this study was to investigate the prevalence of bovine tuberculosis (TB) and detection of Mycobacterium bovis in cattle from an organized dairy farm. Materials and Methods: A total of 121 animals (93 females and 28 males) of 1 year and above were studied for the prevalence of bovine TB using single intradermal comparative cervical tuberculin (SICCT) test, bovine gamma-interferon (γ-IFN) enzyme immunoassay, and polymerase chain reactions (PCRs). Results: Out of total 121 animals, 17 (14.04%) animals were positive reactors to SICCT test while only one (0.82%) animal for γ-IFN assay. By PCR, Mycobacterium TB complex was detected in 19 (15.70%) animals out of which 4 (3.30%) animal were also positive for M. bovis. Conclusions: Diagnosis of bovine TB can be done in early stage in live animals with multiple approaches like skin test followed by a molecular technique like PCR which showed promising results.

Evaluation of lateral flow assay as a field test for investigation of brucellosis outbreak in an organized buffalo farm: a pilot study.

Aim: The aim was to evaluate lateral flow assay (LFA) as a field test for investigation of brucellosis outbreak in organized buffalo farm. Materials and Methods: A total of 153 serum samples were tested to detect the presence of brucella antibodies by LFA and three other serological tests i.e. rose bengal plate test (RBPT), protein G based indirect enzyme-linked immunoassay (iELISA), and competitive ELISA (cELISA). The performances of LFA and other serological tests were evaluated using OIE complaint cELISA as the gold standard. Results: Serological tests revealed 50% of the animals were seropositive for Brucella antibodies and correlated with clinical history of abortions, infertility, and productive failures. The newly developed assay showed 87.1% and 92.6% sensitivity and specificity, which was even higher than the specificity of RBPT. Conclusions: The investigation proved the potential usefulness of LFA for field diagnosis of brucellosis in the regions where laboratory facilities are limited.

Epidemiological and immunopathological studies on Porcine parvovirus infection in Punjab

Aim: The aim of this study was to get the first-hand knowledge about the seroprevalence of Porcine parvovirus (PPV) in Punjab and a diagnosis of PPV from abortion cases of swine using gross, histopathological, and immunohistopathological techniques to observe the tissue tropism of the virus strain. Materials and Methods: Tissue samples from the reproductive tract of pig (n=32), placental tissue (n=10), and aborted fetuses (n=18) were collected from Postmortem Hall of the Department of Veterinary Pathology, GADVASU, field outbreaks and from butcher houses in and around Ludhiana. These samples were processed for histopathological and immunohistochemical (IHC) studies. For seroprevalence study, 90 serum samples of different sex and age were collected from 15 swine farms of Punjab and were subjected to indirect enzyme linked immunosorbent assay using commercial kit. Results: Overall, seroprevalence of PPV was found to be 41.1%. Sex and age related difference in the prevalence was noted. In abortion cases grossly congested and emphysematous lungs, congested internal organs with fluid in abdominal cavity and congestion in brain, changes were noted in fetuses, while diffuse hemorrhages and edema was observed in placental tissue. Histopathologically, the most frequent fetal lesions in aborted fetuses were noted in lungs, liver, and brain. IHC staining revealed PPV antigens in sections of heart, liver, lung, spleen, brain, lymph node of fetuses, placenta, and uterus of sow. Gross, histopathological, and IHC examination of the samples confirmed 5 fetus, 2 placenta and 3 female reproductive samples positive for parvovirus infection. Conclusions: Seroprevalence results may serve as a support either in prevention or control of the disease. IHC is the sensitive technique for diagnosis of PPV associated with the reproductive tract of swine and was found to supplement the gross and histopathological alterations, respectively, associated with the disease.

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.

Diagnosis of Bovine Tuberculosis in Lactating Cattle and Buffaloes by Comparative Intradermal Tuberculin Test and Bovine Gamma-Interferon Immunoassay

Two hundred lactating animals (158 cattle and 42 buffaloes) of organized and unorganized farms were investigated for the bovine TB using comparative intradermal tuberculin test (CITT) and IFN-γ assay. CITT was performed using avian and bovine PPD and IFN-γ assay by Mycobacterium bovis gamma interferon test kit. Overall, 14.5% and 11.5% animals were found positive by CITT and IFN-γ assay, respectively. However, 22.5% animals were detected TB positive through combination of both the tests. So, both CITT and IFN-γ assay, when used together lead to more accurate screening for bovine TB in dairy herd.

Seroprevalence of Paratuberculosis in Rural Bovine Herds from Different Agro-Climatic Zones of Punjab

Paratuberculosis (Johnes disease) is an OIE listed notifiable economically important contagious mycobacterial disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis. The present study reports the seroprevalence of paratuberculosis in bovines of the rural area from five different agro-climatic zone of Punjab. A total of 736 serum samples from both cattle and buffalo herds were evaluated for the presence of antibodies to MAP using a commercially available paratuberculosis screening enzyme-linked immunosorbent assay (ELISA) kit. Twenty three animals were positive and the seroprevalence was found as 3.125%. Highest prevalence was recorded in western plain zone (6.66%) followed by western zone (3.07%), central zone (2.68%), sub mountain undulating zone (2%) and nil in undulating plain zone

Molecular and immunohistochemical detection of Mycobacterium bovis in formalin-fixed tissues from animals with spontaneous bovine tuberculosis

Bovine tuberculosis (TB) is an infectious and emerging but neglected disease of cattle, caused by Mycobacterium bovis. Diagnostic techniques including histopathology, acid fast staining, immunohistochemistry, polymerase chain reaction (PCR) and real-time PCR were compared for extent of accurate diagnosis of bovine TB. Study was conducted on 16 archival tissue samples collected from suspected cases of bovine tuberculosis. Histopathological studies revealed both well-organized and poorly-organized granulomas in the lungs and lymph nodes. Acid fast bacilli were detected by Ziehl Neelsens staining in 13 (81.25%) of the 16 suspected cases. Immunohistochemistry using specific polyclonal antibody against M. bovis detected mycobacterial antigen extracellularly in caseous areas as well as intracellularly in macrophages and giant cells. DNA was extracted from formalin-fixed, paraffin-embedded tissues and subjected to IS6110 PCR using T4/T5 and INS1/INS2 primers specific for Mycobacterium tuberculosis complex (MTC) with an expected band size of 123 bp and 245 bp, respectively. The IS6110 PCR assay was positive in 12 out of 16 cases (75%). IS6110 real-time PCR using IS6110_T primers confirmed 6 (37.5%) samples positive for M. bovis. The present investigation indicated that real-time PCR using IS6110_T primers had increased specificity over conventional IS6110 PCR assay and can be used for detecting specific M. bovis infection in animals suspected for bovine tuberculosis.