Soumendu Chakravarti

Evidence of mixed infection of peste des petits ruminants virus and bluetongue virus in a flock of goats as confirmed by detection of antigen, antibody and nucleic acid of both the viruses.

A mixed infection with peste des petits ruminants virus (PPRV) and bluetongue virus (BTV) occurred in goats which exhibited symptoms characteristic of PPR. A number of samples were collected from ailing or dead goats for labrotory diagnosis. Antibody to BTV and PPRV was detected in sera samples by competitive ELISA. No PPRV antigen was detected in tissue samples like lung and spleen, however, presence of PPRV antigen in some sera samples was confirmed by sandwich ELISA. All the blood samples collected from the ailing animals were found positive for BTV antigen by a sandwich ELISA. BTV- and PPRV nucleic acids were amplified from the pooled blood and tissue samples respectively by RT-PCR assays. The identity of the amplicons was confirmed by cloning and sequencing. All these tests confirm that the goats were infected with PPRV and BTV simultaneously. Isolation of viruses from the clinical samples is underway.

Molecular typing of canine parvovirus strains circulating from 2008 to 2012 in an organized kennel in India reveals the possibility of vaccination failure

Canine parvovirus-2 (CPV-2), which emerged in 1978, is considered as the major viral enteric pathogen of the canine population. With the emergence of new antigenic variants and incidences of vaccine failure, CPV has become one of the dreaded diseases of the canines worldwide. The present study was undertaken in an organized kennel from North India to ascertain the molecular basis of the CPV outbreaks in the vaccinated dogs. 415 samples were collected over a 5year period (2008-2012). The outbreak of the disease was more severe in 2012 with high incidence of mortality in pups with pronounced clinical symptoms. Molecular typing based on the VP2 gene was carried out with the 11 isolates from different years and compared with the CPV prototype and the vaccine strains. All the isolates in the study were either new CPV-2a (2012 isolates) or new CPV-2b (2008 and 2011 isolates). There were amino acid mutations at the Tyr324Ile and at the Thr440Ala position in five isolates from 2012 indicating new CPV mutants spreading in India. The CPV vaccines used in the present study failed to generate protective antibody titer against heterogeneous CPV antigenic types. The findings were confirmed when the affected pups were treated with hyper-immune heterogeneous purified immunoglobulins against CPV in dogs of different antigenic types.

Role of heavy water in biological sciences with an emphasis on thermostabilization of vaccines.

Preservation of vaccines, viruses and other biologicals is one of the onerous tasks in maintaining the quality of the products from manufacture until they reach the end users. Live-attenuated viral vaccines, serum immunoglobulins, plasma fractions and clinical samples, including tissues and body fluids, are all materials that usually require cold-chain maintenance during storage and distribution. A number of stabilizers are currently used to help retain the quality of these materials, in particular vaccines, during transit. Deuterium oxide (heavy water; D2O) has previously been reported to have a protective effect on biomolecules (proteins and nucleic acids), cells and simple multicellular organisms against thermal shock. Of late, the potential of D2O has been demonstrated in stabilization of the oral polio and yellow fever 17D vaccines. This review is the outcome of a thorough search and scan of the literature in a quest to explore the potential use of heavy water in the stabilization of veterinary biologicals. The literature search revealed this potential of heavy water as exemplified by successful stabilization of oral polio and yellow fever vaccines. Through this review, the authors wish to inform animal health researchers and disseminate their knowledge on the use of heavy water in biomolecule stabilization and its potential application in the stabilization of veterinary vaccines and other biologicals.

Polymerase spiral reaction (PSR): a novel, visual isothermal amplification method for detection of canine parvovirus 2 genomic DNA

Canine parvovirus-2 (CPV-2), which is ubiquitously distributed worldwide, causes severe and often fatal gastroenteritis in dogs. Accurate, differential and rapid diagnosis of canine parvoviral enteritis remains a challenge for clinicians. A recently developed isothermal amplification technique, polymerase spiral reaction (PSR), was optimized for the first time for a viral pathogen with reference recombinant plasmid standards from different CPV-2 antigenic variants (CPV-2, CPV-2a, CPV-2b and CPV-2c) and subsequently validated using clinical samples. Addition of chromogenic substrate SYBR Green I after the completion of the reaction resulted in bright green fluorescence in positive samples, while negative samples and a no-template control remained orange. These results were further substantiated through visualization of a laddering pattern of the PSR-amplified product in an agarose gel in positive cases and the absence of this pattern in no-template control and negative samples. The PSR assay was found to be highly specific, as it did not react with other putative canine pathogens (canine adenovirus 1 and canine distemper virus). The sensitivity of the newly developed PSR technique was compared with that of conventional PCR, real-time PCR and LAMP, using a serial tenfold dilution of canine parvovirus DNA. The detection limit of PSR was found to be at the femtogram level, which is comparable with that of real-time PCR and LAMP, which are ten times more sensitive than conventional PCR. The assay was validated using 90 clinical samples, of which 54 were found positive, while only 45 samples were positive in conventional PCR. This novel assay, which is fully compliant with the ‘ASSURED’ concept for disease diagnosis, provides a simple, rapid, specific, sensitive and cost-effective method for diagnosis of canine parvoviral enteritis in veterinary clinics.

Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus.

Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally.Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally.

Novel Polymerase Spiral Reaction (PSR) for rapid visual detection of Bovine Herpesvirus 1 genomic DNA from aborted bovine fetus and semen

Bovine herpesvirus-1 (BHV-1) is a major viral pathogen affecting bovines leading to various clinical manifestations and causes significant economic impediment in modern livestock production system. Rapid, accurate and sensitive detection of BHV-1 infection at frozen semen stations or at dairy herds remains a priority for control of BHV-1 spread to susceptible population. Polymerase Spiral Reaction (PSR), a novel addition in the gamut of isothermal techniques, has been successfully implemented in initial optimization for detection of BHV-1 genomic DNA and further validated in clinical samples. The developed PSR assay has been validated for detection of BHV-1 from bovine semen (n=99), a major source of transmission of BHV-1 from breeding bulls to susceptible dams in artificial insemination programs. The technique has also been used for screening of BHV-1 DNA from suspected aborted fetal tissues (n=25). The developed PSR technique is 100 fold more sensitive than conventional PCR and comparable to real-time PCR. The PSR technique has been successful in detecting 13 samples positive for BHV-1 DNA in bovine semen, 4 samples more than conventional PCR. The aborted fetal tissues were negative for presence of BHV-1 DNA. The presence of BHV-1 in bovine semen samples raises a pertinent concern for extensively screening of semen from breeding bulls before been used for artificial insemination process. PSR has all the attributes for becoming a method of choice for rapid, accurate and sensitive detection of BHV-1 DNA at frozen semen stations or at dairy herds in resource constrained settings.

Abortions in an organized dairy farm from North India reveal the possibility of breed susceptibility to Bovine Brucellosis

The present study was undertaken over a three year period (2012–2014) in an organized dairy farm located in North India to ascertain Brucella abortus as the putative cause of abortion. The dairy farm maintained cattle of Frieswal, Crossbred and Sahiwal breeds and followed calf-hood vaccination with Brucella abortus Strain 19 live vaccine in all the heifers. Even with the recommended vaccination schedule and good managemental practices in place, 88 cases of abortions clinically suspected of bovine brucellosis (40 from Frieswal breed, 17 from Crossbred cattle and 31 from Sahiwal breed) were reported from this farm. From these abortion cases, bacteriological isolation was possible in only four dams while 16 dams were found to be serologically positive in Serum Tube Agglutination Test (STAT). Molecular screening by PCR assay (specific for the bcsp31 gene of B. abortus) revealed that 24 dams were positive, out of which 20 were from Frieswal breed and rest four were from Crossbred herd. Prominently, all Sahiwal dams were found to be negative in bacteriological isolation and also in PCR assay. These results thus indicate towards the possibility of breed predisposition to abortions due to B. abortus infection. Statistical analysis by Fischer exact test (p < 0.01) too substantiated that breed susceptibility exists among these PCR positive cases. This study is novel as breed variation in abortions due to B. abortus in cattle is being documented for the first time. Seven representative PCR amplicons generated during the study were also sequenced and submitted to NCBI GenBank. Moreover, this study also accentuates the importance of PCR screening especially in vaccinated herd and raises concerns on over-dependence of serological assays when intensive vaccination is practised without any concomitant DIVA strategy. Thus, besides assisting in planning pragmatic control strategies against bovine brucellosis these findings are also imperative from ‘One Health’ context, also.

Canine Monocytic Ehrlichiosis among working dogs of organised kennels in India: A comprehensive analyses of clinico-pathology, serological and molecular epidemiological approach

Abstract Canine Monocytic Ehrlichiosis (CME) is a serious tick-borne rickettsial disease affecting canine populations globally. Besides few reports from stray and pet dogs from localised geographical regions (cities/towns/small states), a comprehensive study on prevalence of Ehrlichia canis (E. canis) among working dogs from different geo-climatic zones of India was pertinently lacking. Study of CME among these dog populations was thus carried out, encompassing clinical aspects and different diagnostic methodologies viz., microscopy, serology and molecular biology. During the two-year study period, clinical specimens from 225 cases suspected of canine ehrlichiosis were examined for clinical pathology and presence of the haemoparasites. Overall prevalence of ehrlichiosis by microscopic examination, commercial dot-ELISA kit and nested PCR assay was estimated to be 1.3%, 19.1% and 5.8%, respectively, which were found to be statistically significant by McNemar Chi squared test (p<0.05). It was also observed that possibly due to widespread use of doxycycline therapy in field, CME presently does not remain a potential threat which it uses to pose earlier. However, concurrent infections of E. canis and Babesia gibsoni were found to be mostly fatal. Keeping in view of high number of apparently healthy dogs (24) out of total positive cases (46) observed during the study, it is recommended that prevalence studies on CME should also involve screening of apparently healthy dogs. Phylogenetic analysis carried on partial sequencing of 16S rRNA of E. canis strains revealed that all of the Indian strains clustered in a single clade with other E. canis species from India and rest of the world. Molecular divergence was observed among the sequences of Brazilian and American isolates which were also included in the present study. These findings have thus opened a new paradigm for planning of pragmatic control strategies against CME.

Investigation on Peste des Petits Ruminants Outbreak in Goats of Bareilly District of Uttar Pradesh, India

Peste des petits ruminants (PPR) is an economically important viral disease of small ruminants, endemic in India. The present study reports on socio-epidemiological aspect of an outbreak of PPR in goats of Bareilly district of Uttar Pradesh in Northern part of India. During a visit to the affected villages and also from animals brought to a local clinic, it was observed that goats had clinical signs such as pyrexia, oculo-nasal discharges, dyspnea and diarrhoea indicative of PPR. However, oral lesions were not so prominent. In total, 96 clinical samples (ocular and nasal swabs) were collected from the affected goats suspected for PPR. The PPR virus (PPRV) antigen and nucleic acid were confirmed by sandwich-ELISA and ‘N’ gene-based RT-PCR, respectively. Sequence and phylogenetic analysis of partial ‘N’ gene PCR amplicons revealed that the PPRV belonged to lineage IV. The present investigation describes the importance of understanding the role of socio-epidemiological factors on PPR outbreak for efficient implementation of control and eradication strategies.