Development of an enzyme immunoassay using recombinant invariant surface glycoprotein (rISG) 75 for serodiagnosis of bovine trypanosomosis.

Trypanosomosis or surra is caused by the haemoflagellate parasite, Trypanosoma evansi and is an important disease of animals, including domestic and wild herbivores and carnivores, in tropical countries. The invariant surface glycoproteins (ISGs) are blood stream stage specific and are uniformly distributed over the entire surface of the trypanosomes. In the present study, the extracellular domain (ED) region of ISG-75 from T. evansi, consisting of 1320 nt, encoding a polypeptide of 440 amino acids, has been heterologously expressed in Escherichia coli. Further, the immunoreactivity of recombinant ISG-75 (rISG-75) was characterized in immunoblot and ELISA using T. evansi hyper immune sera raised in experimental animals. The protein was found immunoreactive when compared with a panel of antigens (VSG RoTat 1.2 and whole cell lysate) using bovine serum samples from field. The diagnostic potential of rISG-75 was evaluated in ELISA with large number of bovine field serum samples. The optimum sensitivity and specificity were 98.47 and 99.1, respectively. The present finding showed that the expressed protein has potential use in the serodiagnosis of trypanosomosis.

Prevalence of Peste-des-petits-ruminant virus antibodies in cattle, buffaloes, sheep and goats in India.

The present study describes the prevalence of Peste-des-petits-ruminant virus (PPRV) antibodies in cattle, buffaloes, sheep and goats carried out during the period 2011 using the serum samples randomly collected from different villages of five states of India. A total of 1,498 serum samples [n = 605 (cattle); n = 432 (buffaloes); n = 173 (sheep); n = 288 (goats)] were collected from 52 districts in five states (Andhra Pradesh, Gujarat, Jammu and Kashmir, Maharashtra and Rajasthan) of India and were screened for PPRV-specific antibodies by using PPR monoclonal antibody-based competitive ELISA kit. Analysis of 1,498 samples indicates that an overall seroprevalence of 21.83 % with 11.07 % in cattle, 16.20 % in buffaloes, 45.66 % in sheep and 38.54 % in goats. This report presents the results of PPRV-specific antibodies in situations where the subclinical, inapparent or nonlethal or recovery of infection was suspected in cattle, buffaloes, sheep and goats. The presence of PPRV antibodies demonstrate that bovines are exposed to PPRV infection and it implies the importance of cattle and buffaloes as subclinical hosts for the virus besides widespread presence of the disease in sheep and goats. Further, the study showed that the prevalence of PPRV antibodies in apparently healthy livestock under natural situation, 21.83 % of the animals were protected from PPRV re-infection. This inturn help in the implementation of disease control strategies such as vaccination in that particular geographical area.

PCR based diagnosis of trypanosomiasis exploring invariant surface glycoprotein (ISG) 75 gene.

The invariant surface glycoprotein (ISG-75) gene of Trypanosoma evansi buffalo isolate from Karnataka state in India was sequenced and analyzed to elucidate its relationship with other isolates/species. The sequenced ISG-75 gene was also explored to device a polymerase chain reaction (PCR) strategy for the diagnosis of trypanosomiasis in carrier animals. The six cloned ISG gene sequences revealed the open reading frame (ORF) of 1572 and 1527 nucleotide (nt) encoding a polypeptide of 523 and 508 amino acids (aa) respectively and belongs ISG-75 gene family. Sequence analysis revealed 91-100% and 65-99% similarity at nt and aa levels, respectively with other isolates/species and belongs to the RoTat 1.2 strain. The diagnostic PCR based on ISG-75 sequence amplifies a 407 bp product specifically from the different T. evansi isolates and could detect 0.04 pg and 1.2 ng of DNA from purified trypanosomes and T. evansi infected rat blood samples respectively. Subsequently the PCR detected 0.02 and 0.27 trypanosomes ml(-1) respectively, from purified trypanosomes and T. evansi (buffalo isolate) infected rat blood. By the developed PCR assay trypanosomal nucleic acid was detected in experimental rats and buffalo on 24 h post infection (p.i.) and 3rd day post infection (d.p.i.), respectively. The developed ISG-75 gene based PCR assay could be useful in detection of carrier status of trypanosomiasis in animals.

Development of a probe based real time PCR assay for detection of bovine herpes virus-1 in semen and other clinical samples.

This study describes development of a TaqMan probe based real time PCR assay that can detect BoHV-1 of as low as 0.001 TCID50/0.1 ml in clinical samples, its comparative evaluation with indirect ELISA and virus isolation for detection of Bovine herpes virus-1 (BoHV-1) in semen and swab clinical samples. For this study, we collected samples from 212 animals (cattle and buffaloes) comprising 91 bulls and 121 females. Avidin-biotin ELISA employed on serum samples from 212 animals revealed 74 as seropositive for BoHV-1. On inoculation of semen/swabs on MDBK cell line, nine samples yielded cytopathic changes characteristic of herpes viruses. The isolates were confirmed by VNT and a conventional PCR. A real time PCR assay was standardised by designing a new set of TaqMan probe and primers targeting a 71 bp region on gB gene of the virus. The assay detected viral antigen in 21 seropositive and 14 seronegative animals, emphasizing the relevance of serology in BoHV-1 diagnosis, particularly in breeding stations. Further, real time PCR assay was 100 % sensitive and 87.19 % specific compared to virus isolation in detection of the BoHV-1 in clinical samples. The assay was validated at reputed national laboratories, with a sensitivity of ≥99 %.

Comparative immunogenicity of two peste des petitis ruminants (PPR) vaccines in South Indian sheep and goats under field conditions.

Peste des petitis ruminants (PPR) is an economically important endemic viral disease of sheep and goats in India, where several different homologous PPR vaccine candidates have been developed. We evaluated the serological response to two vaccine strains, Arasur/87 and Sungri/96, in South Indian cross-bred and native sheep and goats reared under organized and unorganized settings. Animals seronegative (percent inhibition or PI <40) by competitive enzyme-linked immunosorbent assay (c-ELISA) were immunized with either of the vaccine strains or placebo. Sera collected on 21, 60 and 90 days post-vaccination were subjected to c-ELISA and serum neutralization test (SNT). Seropositivity (PI >40), seroconversion (fourfold increase in SNT titres) and seroprotection (SNT titre of ≥8 deemed to be protective) ranged from 66.7 to 84.0 %, 56.0 to 69.2 %, and 60.0 to 76.0 %, respectively. However, no significant difference was observed between responses to the two vaccine strains. These results support the premise that the two vaccine strains are equally efficacious.

Prevalence of Neospora caninum antibodies in dairy cattle and water buffaloes and associated abortions in the plateau of Southern Peninsular India.

A seroprevalence study of bovine neosporosis was conducted among 1,927 dairy cattle and 341 water buffaloes from Karnataka and Andhra Pradesh states in plateau of southern peninsular India by employing competitive enzyme-linked immunosorbent assay. Overall, 12.61 and 9.97 % sera samples were found positive for the presence of Neospora caninum antibody, respectively, among cattle and water buffaloes. Out of 1,927 sera samples from cattle, 912 and 1,015 samples were collected from unorganized and organized herds, respectively. The cattle screened were of upgraded Holstein-Friesian and water buffaloes were of graded Surti breed. Significantly (p < 0.05) higher prevalence was found in the cattle in unorganized herds (16.66 %) in comparison to organized herds (8.96 %). The highest seroprevalence was recorded in the age group of 4 years and above in both type of cattle herds and water buffaloes. There was a significant variation of seroprevalence (p < 0.05) observed between different age groups of cattle. The rate of seroprevalence increased with the increment in the age of the animals suggesting a possibility of horizontal mode of transmission of the infection from the environment. The percentage of abortion history was more in seropositive group (51.65 %) in comparison to the seronegative group (5.84 %) and the seropositive cattle were 8.84 times more likely to experience abortion than the seronegative cattle. The occurrence of abortion among different age groups varied significantly (p < 0.05). The findings revealed the presence of neosporosis in the southern peninsular India among cattle and water buffaloes and a strong association between the seroprevalence and abortion.

Expressed truncated N-terminal variable surface glycoprotein (VSG) of Trypanosoma evansi in E. coli exhibits immuno-reactivity.

The variant surface glycoprotein (VSG) of trypanosome is an important part of its body surface coat, which is expressed in early, middle and late stages of infection contributing a major diagnostic value. In the present study, the 5' end of the partial VSG gene sequences (681 bp) encoding N-terminal protein of RoTat 1.2 VSG (227 amino acid) was amplified, cloned into pET32a vector, and expressed in prokaryotic system. The fused His-tagged expressed VSG protein (43 kDa) of the Trypanosoma evansi was characterized in SDS-PAGE and immunoblotting using hyperimmune/immune sera raised against buffalo, dog, lion and leopard isolates of T. evansi. The expressed protein remained immunoreactive with all the sera combinations. The animals immunized with whole cell lysate or recombinant protein showed similar antibody reactions in ELISA and CATT (Card Agglutination Test for Trypanosomiasis). This study suggests the expressed recombinant truncated VSG is having its importance for its possible use in sero-diagnosis of surra.

Phylogenetic analysis of NS5B gene of classical swine fever virus isolates indicates plausible Chinese origin of Indian subgroup 2.2 viruses.

Twenty-three CSFV isolates recovered from field outbreaks in various parts of India during 2006-2009 were used for genetic analysis in the NS5B region (409 nts). Seventeen of these were studied earlier [16] in the 5'UTR region. Phylogenetic analysis indicated the continued dominance of subgroup 1.1 strains in the country. Detailed analysis of a subgroup 2.2 virus indicated the plausible Chinese origin of this subgroup in India and provided indirect evidence of routes of CSFV movement within South East Asia region.

PCR-based diagnosis of surra-targeting VSG gene: experimental studies in small laboratory rodents and buffalo.

Trypanosoma evansi, the causative organism of 'surra' expresses its variable surface glycoprotein (VSG) at early, middle and late stages of infection in animals. The variable antigenic nature of VSG caused by switching its expression type favours evasion from the host immune response and leads to chronic and persistent infection. Developing a polymerase chain reaction (PCR)-based diagnostic tool targeting the VSG gene is expected to be highly specific and sensitive for diagnosis of surra. Hence, in the present study, we have designed EXP3F/4R primer pair and amplified the 1.4 kb of VSG gene of T. evansi and studied the phylogenetic relationship by in silico analysis. The PCR method was standardised using another set of primer, DITRYF/R, and 400 bp was amplified from blood and tissue samples of experimentally infected animals. Applying the PCR method, we were able to detect as low as 0.15 trypanosomeml(-1). Considering the number of parasite-to-DNA concentration, the PCR method has a sensitivity of 0.015 pg ml(-1). The PCR could detect the presence of the parasite as early as 24h post-infection (p.i.) and 72 h p.i., respectively, in experimentally infected rats and buffalo. No amplification was observed with DNA of Babesia bigemina and Theileria annulata, indicating the primers are specific for T. evansi. The PCR method could detect the dog, lion and leopard isolates of T. evansi. Similarly, amplifying the DNA from the experimentally infected tissues was also found to be sensitive. Thus, the findings of this study favour the application of PCR over the parasitological methods for the detection of the early and/or chronic stage of surra in domestic and wild animals.

Genetic typing of recent classical swine fever isolates from India.

Seventeen classical swine fever virus (CSFV) isolates recovered during the period of 3 years (2006-2008) from India were subjected to nucleotide sequencing in the 5' untranslated region (UTR). For genetic typing, 150 nucleotides within this region were used. For better epizootiological understanding, 39 nucleotide sequences of the above region, including 13 Indian CSFV sequences, available either in the Genbank or published literature were also included in the study. Based on the phylogenetic analysis, the Indian isolates could be grouped in to two subgroups, viz., 1.1 and 2.2. The study also revealed predominance of subgroup 1.1 and involvement of viruses of more than one subgroup in an outbreak.

Sequence analysis of the S10 gene of six Bluetongue Virus isolates from India.

Bluetongue Virus (BTV) genome segment 10 (S10)-based phylogenetic studies are important in understanding the BTV evolution. S10 gene-based phylogenetic analysis grouped six different BTV isolates (BTV serotype 1, 18 and 23) from India in subclade A1 and showed closer relationship with BT viruses from Mediterranean Basin. Indian BTV serotypes 18 and 23 formed a single cluster distinct from BTV serotype 1 isolates and were evolved from BTV from China, Indonesia and Australia. The overall S10 sequences of BTV isolates from India were largely conserved (>95.7% homology) and were distinct from other BT viruses of the world.

Seroepidemiology of peste des petits ruminants in sheep and goats of southern peninsular India.

This paper presents the results of a seroepidemiological study carried out between July 2006 and March 2007 to detect the presence of antibodies to peste des petits ruminants (PPR) virus in randomly collected serum samples from sheep and goats in southern peninsular India. The authors used a competitive enzyme-linked immunosorbent assay with a monoclonal antibody developed against a neutralising epitope of the haemagglutinin (HA) protein of the virus. A total of 1,492 sheep sera and 2,068 goat sera collected from the six southern Indian states were screened. It was determined that 41.35% of the sheep sera and 34.91% of the goat sera were positive for the presence of antibody. The study indicated an extensive endemicity of the disease in these states, which is attributed to the agro-climatic conditions and the migration of livestock.

Leptospira infection in animals and humans: a potential public health risk in India.

Leptospirosis is recognised as one of the commonest zoonotic infections in the world. In India, where animals provide the draught power for agriculture, which is the main profession of the population, the incidence of leptospirosis among animals and humans is high. In this paper, the isolation of pathogenic leptospires from human and animal hosts from several parts of India is reported. Because there are only limited facilities for serotyping within the country, most of the isolates were typed to the serogroup level only. In addition, the potential of leptospirosis to be a serious public health problem in India is discussed.

Brucellosis in high risk group individuals.

PURPOSE: The purpose of this study was to investigate the seroprevalence of brucellosis among high-risk group individuals, consisting of veterinarians and para-veterinarians, shepherds, butchers and animal owners. METHODS: The present work was carried out at Project Directorate on Animal Disease Monitoring and Surveillance, Bangalore, by using the recently developed indirect enzyme-linked immunosorbent assay (ELISA) for antibodies to Brucella abortus. RESULTS: The results were compared with the conventional serological tests, Rose Bengal plate test and standard tube agglutination test. The result showed that the indirect ELISA was more sensitive than the conventional tests. Of 618 tested, the disease of prevalence was at 41.23% in veterinary inspectors, 30.92% in veterinary assistants, 12.37% in veterinary officers, 6.18% in veterinary supervisors, 6.18% in Group D workers, 2.06% in shepherds and 1.03% in butchers. CONCLUSIONS: This study results highlight the immediate necessity to institute control measures to control Brucellosis.

Development and standardization of a piezo electric immunobiosensor for foot and mouth disease virus typing.

An immunobiosensor using a piezo electric (PZ) crystal was developed and standardized for foot and mouth disease (FMD) diagnosis and virus typing. A 6MHz quartz crystal was used as the frequency determining element. Foot and mouth disease virus (FMDV) type specific antibody raised in rabbits/monoclonal antibody was coated on the crystal surface and the resonance measured. One microlitre of the 10% aqueous suspension of the clinical sample (tongue or foot epithelium) was applied on both surfaces of the crystal and the resonance recorded. A difference in resonance of more than -2.5Hz was obtained in positive samples (homologous antigen and antibody). The test was standardized initially using various dilutions of FMD tissue culture antigen. Repeatability and sensitivity were also tested and it was found that the crystals could be washed and reused eight times. The test could be used for FMDV type specifically and no cross-reaction between FMDV types was observed. The shelf-life of the antibody-coated crystal stored at room temperature was 18 weeks. Application of the biosensor test to the FMDV clinical samples confirmed virus typing results when compared with enzyme-linked immunoabsorbent assay (ELISA) and it could also detect virus in ELISA negative samples and mixed virus infections.

Protective immune response to 16 kDa immunoreactive recombinant protein encoding the C-terminal VP1 portion of Foot and Mouth Disease Virus type Asia 1.

Recombinant protein of Foot and Mouth Disease Virus (FMDV) type Asia 1 corresponding to the C-terminal half of VP1 was expressed in Escherichia coli. As an alternative to the synthetic peptide, this selected C-terminal region was used as a protein vaccine in guinea pigs in order to study the immune response with various adjuvant formulations: immune stimulatory complexes (ISCOMs), Montanide ISA 206, Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS) and cytokine mixture. A primary dose of 40 microg/animal followed by a booster of the same dose was injected after a 21-day interval. The sera were collected at intervals of 21, 42 and 63 days after the booster. The humoral response to vaccine was monitored by sandwich enzyme-linked immunosorbent assay (ELISA) and a serum neutralization test (SNT). The guinea pig sera showed high titers both in ELISA and SNT, which could be protective. Further, irrespective of the adjuvant preparation used, the vaccine conferred protection against the challenge virus 105 days post-vaccination in 13 of 15 animals (86%). The results indicated that a combination of recombinant protein ISCOMs and Montanide ISA 206 would be a better choice for achieving early protective titers and longer lasting immunity and that the C-terminal half of the VP1 protein may be tried as a safe vaccine for secondary immunization.

E. coli expressed proteins as diagnostic reagents for typing of foot-and-mouth disease virus.

Truncated proteins corresponding to the C-terminal half of VP1 of four vaccine strains and two field variants of foot-and-mouth disease virus (FMDV) were expressed in E. coli. The expressed proteins were affinity purified and their type specific reactivity was confirmed by immunoprecipitation with anti-virus antibodies. Antibodies were raised against the purified proteins in guinea pigs and the type specificity of the anti peptide antibodies was confirmed by antigen capture reverse transcription polymerase chain reaction (Ag-RT/PCR) where the sera against a particular type captured the homologous virus. Antibodies were purified by immuno-affinity chromatography and tested for specificity by various serological tests. Using the purified proteins and the antibodies raised against them, tests like ELISA, Ag-RT/PCR, and latex agglutination test (LAT) were standardized. Application of the reagents in various tests was studied by screening a few field samples and by nucleotide sequencing. Specific reactivity of antibodies raised against expressed protein was seen with both vaccine virus and field samples. Thus E. coli expressed proteins and antibodies to them may form an alternative and cheap source of diagnostic reagents. The studies showed that antibodies against peptides were mono-specific and therefore may be used in LAT for rapid typing of FMDV and Ag-RT/PCR for typing ELISA negative field samples.

Immuno affinity purification of foot and mouth disease virus type specific antibodies using recombinant protein adsorbed to polystyrene wells.

The specificity of foot and mouth disease virus (FMDV) serological tests depends largely on the quality and purity of the antibodies used. Such type specific antibodies can be generated by hybridoma technology. Alternatively, the specific antibodies can be selected from polyclonal serum by immunoaffinity chromatography using recombinant protein/peptide bound affinity matrices. Based on this approach, we purified selectively antibodies against the major epitopes of VP 1 of FMDV serotype Asia 1 using recombinant protein adsorbed to polystyrene wells. Optimum buffer conditions were standardised for efficient elution. Buffer consisting of 4 M MgCl2 with 75 mM HEPES pH 6.5 was found to be optimum with respect to elution efficiency of bound antibodies and integrity of antigen. The specific reactivity of eluted antibodies was confirmed by dot-enzyme linked immunosorbent assay (dot-ELISA) and antigen capture reverse transcription polymerase chain reaction (Ag/RT-PCR). The effect of temperature and repeated elution on the stability of coated protein were studied.

A note on outbreaks caused by mixed foot-and-mouth disease virus infections.

Two outbreaks of foot-and-mouth disease (FMD) in vaccinated cattle were investigated wherein a mixed infection due to FMD virus (FMDV) types O and Asia 1 was detected by sandwich enzyme-linked immunosorbent assay (ELISA) and confirmed by antigen capture polymerase chain reaction (PCR). The clinical picture and the epidemiological data on these outbreaks are presented. The isolated virus strains were compared to the respective vaccine strains by means of monoclonal antibody (MAb) profiling and nucleotide sequence analysis. The probable cause of the mixed FMDV infection and its significance in disease control are discussed.