Studies on the sequential pathology of Kyasanur Forest Disease (KFD) in Mouse brain: KFD virus induces apoptosis of neurons in cerebrum and hippocampus.

The sequential pathology of Kyasanur forest disease (KFD) in mouse brain was assessed in this study. Kyasanur forest disease virus (KFDV) strain P9605 used in this study was confirmed by real-time reverse transcriptase-polymerase chain reaction targeting the NS5 gene. Mouse Lethal Dose 50 (MLD50) of the virus was determined by in-vivo mice inoculation test. One MLD50 of the KFDV was inoculated intra-cerebrally into 36 mice aged 2-3 weeks. Another group of 36 age-matched mice that served as control group were inoculated with plain media. Six mice each from infected and control groups were euthanized every 24 hrs intervals for six days. Brain tissues were collected in 10% NBF. The collected brain tissues were processed and subjected to histopathological studies by Hematoxylin and Eosin staining. Grossly, the infected mice showed symptoms of dullness, hunched back appearance, weakness, sluggish movements with indication of hind quarter paralysis on day four post-infection. These symptoms got aggravated with complete paralysis of the hind quarters, inability to move and death on 5th and 6th day post-infection. Microscopically, the brain showed apoptosis of neurons, perivascular cuffing, gliosis, congestion, neuropil vacuolation, meningitis, degeneration, and necrotic neurons. The real-time RT-PCR on hippocampus of the KFDV-infected mouse brain showed three-fold higher expression levels of Caspase 3, a crucial mediator of apoptosis. The cerebral cortex, cerebellum and hippocampus that control the motor neuron activities and muscle tone were primarily affected, possibly correlating with the gross symptoms of hind quarter paralysis, ataxia, and other motor neuron dysfunctions noticed. Taken together, these findings reveal that KFDV induces apoptosis of neurons in the cerebrum and hippocampus of KFDV infected mice. Further studies are needed to confirm if the lesions noticed in mice brain simulate the brain lesions in humans since gross motor-neuron symptoms are similar in mice as well as humans.

Evaluation of Safety and Potency of Kyasanur Forest Disease (KFD) Vaccine Inactivated with Different Concentrations of Formalin and Comparative Evaluation of In Vitro and In Vivo Methods of Virus Titration in KFD Vaccine.

We evaluated the safety and potency of the Kyasanur Forest disease (KFD) vaccine inactivated with different formalin concentrations in mice, since the side effects due to higher formalin concentrations have been a major reason for vaccine refusal. Furthermore, with an objective to reduce the use of mice in vaccine testing, we performed quantification of the KFD virus by real-time PCR and compared it with in vivo titration in mice. The KFD vaccine prepared in chicken embryo fibroblast cells was inactivated with 0.04%, 0.06%, and 0.08% concentrations of formalin. The vaccine inactivated with 0.04% and 0.06% formalin failed the safety test, whereas the KFD vaccine inactivated with 0.08% formalin was safe and potent with a log protective index of 5678 in mice. This reduced formalin content may induce no/lesser side-effects of pain/swelling which may increase the vaccine acceptance. The real-time PCR on individual KFD vaccine harvests interpreted that when the CT value of each harvest is <20, the vaccine will have sufficient viral particles to pass the potency test. Comparison of the real-time PCR on tenfold dilutions of the pooled harvests with in vivo mice inoculation test revealed that the 1MLD50 of the vaccine lies in the tenfold dilution that yields CT values between 31 and 34.

Foot and Mouth Disease (FMD) incidence in cattle and buffaloes and its associated farm-level economic costs in endemic India.

The study investigated the important epidemiological parameters and farm-level economic costs of FMD incidence in cattle and buffaloes during 2013-14 to 2015-16 in various states of India. Multistage random sampling procedure was adopted for the primary survey and data was collected through face-to-face personal interview from 18,609 cattle and buffalo rearing farm households from 123 districts across twelve states and one Union Territory. Besides epidemiological parameters, different farm-level direct and indirect loss associated with FMD was assessed at disaggregated level (states) by employing deterministic mathematical models. Highest number of affected villages and disease incidence was observed in non- FMD control programme (FMD-CP) implemented Madhya Pradesh and Assam states, respectively whereas negligible incidence was in FMD-CP implemented Punjab state. The disease incidence was high during 2013-14 and declined during 2014-15 and 2015-16, respectively implied severe incidence scenario (2013-14) succeeded by moderate (2014-15) and mild (2015-16) scenarios. The crossbred and high productive animals were severely affected than local breeds whereas on sexwise and agewise comparison revealed higher incidence in females and adult animals. During severe incidence scenario, milk loss/animal ranged from USD 6.87-47.44, 18.42-125.88, 16.33-91.43, and 27.17-123.62; mortality loss/animal ranged from USD 32.61-804.27, 30.76-577.7, 65.36-502.2, and 188.04-413.7; distress sale loss/animal ranged from USD 3.22-188.63, 64.34-519.3, 214.47-341.8, and 209.11-450.3; and opportunity cost of labour/animal from USD 5.49-54.29, 5.49-67.78; 7.95-31.37 and 9.83-72.38 in indigenous cattle, crossbred cattle, local and improved buffalo, respectively. The estimated draught power loss/animal varied from USD 39.46-142.94 with least being in Madhya Pradesh and highest in Assam states whereas the median treatment cost/animal was USD 9.18 and USD 27.07 in indigenous cattle and upgraded buffaloes, respectively. The total farm-level economic loss projected due to FMD in cattle and buffaloes in India was USD 3159 million (INR 221,110 million), USD 270 million (INR 18,910 million) and USD 152 million (INR 10,610 million), respectively during the severe, moderate and mild incidence scenarios at 2015-16 constant prices. The loss varied across the states, and in severe incidence scenario, the country might lose USD 3.2 billion/year and hence, the bi-annual vaccination schedule need to be strictly implemented in all the states. Besides timely vaccination coverage, managing unabated animal movement, educating and motivating the farmers to vaccinate their animals might reduce the incidence and consequential losses to various stakeholders in endemic states like India.

Bio-inspired materials for defluoridation of water: A review.

The polluted water sources pose a serious issue concerning the various health hazards they bring along. Due to various uncontrolled anthropogenic and industrial activities, a great number of pollutants have gained entry into the water systems. Among all the emerging contaminants, anionic species such as fluoride cause a major role in polluting the water bodies because of its high reactivity with other elements. The need for water remediation has led the research community to come up with several physicochemical and electrochemical methods to remove fluoride contamination. Among the existing methods, biosorption using bio and modified biomaterials has been extensively studied for defluoridation, as they are cheap, easily available and effectively recyclable when compared to other methods for defluoridation. Adding on, these materials are non-toxic and are safe to use compared to many other synthetic materials that are toxic and require high-cost design requirements. This review focuses on the recent developments made in the defluoridation techniques by biosorption using bio and modified biomaterials and defines the current perspectives of fluoride removal specifically using biomaterials.

Serosurveillance of foot and mouth disease in Karnataka state, India: a 3 years study.

A prospective serological investigation was conducted to determine the prevalence and distribution of foot-and-mouth disease (FMD), as well as to monitor the effectiveness of the FMD control programme (FMD-CP) through vaccination in Karnataka, India. Random serum samples were collected every year between May and August before the start of vaccination in 2011, and subsequently following two phases of vaccination in 2012 and 2013. Infection status (seroprevalence) was inferred by subjecting the sera to indirect r3AB3 non-structural protein-ELISA, using kits developed by the Project Directorate on FMD, India. The seromonitoring of FMD-CP was carried out by detecting antibodies deemed to be protective in the pre- and post-vaccinal sera, using liquid-phase blocking-ELISA for structural proteins. The results revealed significant decrease in seroprevalence from 58 to 21 %, providing more definitive data supporting our earlier findings obtained through clinical observations (Hegde et al. in Virusdisease 25:504-509, 2014), and detecting active infection in some of the populations which were considered to be free based on passive surveillance. On the other hand, after four rounds of vaccination, a gradual and significant increase from 4.5 to 59 % of animals carrying antibody levels deemed to be protective was observed against all the three serotypes. The findings of this study could be useful for further strategizing to strengthen the ongoing FMD-CP in Karnataka State, India.

Serotyping of foot and mouth disease virus and Pasteurella multocida from Indian gaurs (Bos gaurus), concurrently infected with foot and mouth disease and haemorrhagic septicaemia.

We report the serotyping of foot-and-mouth disease virus (FMDV) and Pasteurella multocida from Indian gaurs which were concurrently infected with foot-and-mouth disease (FMD) and haemorrhagic septicaemia. Bannerghatta biological park (BBP), a national park located in the outskirts of Bengaluru city, Karnataka, India, is bordered by several villages. These villages witnessed massive outbreaks of FMD which spread rapidly to the herbivores at BBP. Post-mortem was conducted on carcasses of two Indian gaurs that died with symptoms of FMD. The salient gross findings included extensive vesicular lesions on the tongue, gums, cheeks, upper palate and hooves. Haemorrhagic tracheitis and ecchymotic haemorrhages on the heart were characteristic. The vesicular lesions of oral cavity were positive for 'O' type of FMD virus by sandwich enzyme-linked immuno sorbent assay (ELISA). The heart blood and spleen samples yielded growth of pure cultures of P. multocida. The isolates were typed as P. multocida type B using KTSP61 and KTT72 primers yielding specific amplicons of 620 bp. The phylogenetic analysis of the isolates was carried by sequencing of 1.4-Kbp nucleotides on the 16S ribosomal RNA (rRNA) gene of the isolates.

Limit of detection of genomic DNA by conventional PCR for estimating the load of Staphylococcus aureus and Escherichia coli associated with bovine mastitis.

Detection of mastitis-associated bacteria can be accomplished by culturing or by molecular techniques. On the other hand, rapid and inexpensive methods to enumerate bacterial load without culturing can be better achieved by molecular methods. Staphylococcus aureus and Escherichia coli are the predominant bacterial pathogens associated with bovine mastitis. Here, we describe the application of conventional PCR for the limit of detection (LOD) of genomic DNA of S. aureus and E. coli based on single-copy genes. The selected genes were thermonuclease (nuc), aureolysin (aur), and staphopain A (scpA) for S. aureus and ß-D-glucuronidase A (uidA), cytochrome d oxidase (cyd), and rodA (a gene affecting cell shape and methicillin sensitivity) for E. coli. The LOD was 5.3, 15.9, and 143 pg for aur, nuc, and scpA genes, corresponding to S. aureus genomic copies of 1.75 × 10(3), 5.16 × 10(3), and 4.71 × 10(4), respectively. The LOD was 0.45, 12.3 and 109 pg for uidA, rodA and cyd genes, corresponding to E. coli genome copies of 8.91 × 10(1), 2.43 × 10(3), and 2.16 × 10(4), respectively. Application of uidA and aur PCRs to field strains revealed that as low as approximately 100 genome copies of E. coli and 1000-10,000 copies of S. aureus could be detected. This study is the first to report LOD of genomic DNA using conventional PCR for aur and scpA genes of S. aureus, and rodA and cyd genes of E. coli. The results should be useful for developing assays to assess bacterial load in milk and to determine the load that contributes to subclinical or clinical mastitis.

A multi-species indirect ELISA for detection of non-structural protein 3ABC specific antibodies to foot-and-mouth disease virus.

Reliable diagnostic tests that are able to distinguish infected from vaccinated animals are a critical component of regional control programs for foot-and-mouth disease (FMD) in the affected countries. Non-structural protein (NSP) serology based on the 3ABC protein has been widely used for this purpose, and several kits are commercially available worldwide. This report presents the development of a 3ABC-antigen-based indirect ELISA, employing a peroxidase-conjugated protein G secondary antibody that can detect antibodies from multiple species. Recombinant 3ABC protein was expressed in insect cells and purified using affinity column chromatography. Using this protein, an indirect ELISA was developed and validated for the detection of NSP antibodies in serum samples collected from animals with different status of FMD. Diagnostic sensitivity and specificity were found to be 95.8 (95 % CI: 92.8-97.8) and 97.45 % (95 % CI: 94.8-99.0), respectively. The in-house ELISA compared well with the commercially available prioCHECK FMDV NS-FMD kit, with a high agreement between the tests, as determined by the kappa coefficient, which was 0.87. The in-house ELISA showed higher sensitivity for detecting vaccinated and subsequently infected animals, when compared to the reference test. Both of the tests were able to detect NSP antibodies as early as 7-8 days after experimental infection.

Epidemiology of foot and mouth disease in Karnataka state, India: a retrospective study.

A retrospective study on the epidemiology of foot and- mouth disease (FMD) in Karnataka, India between the years 1977 and 2012-13 based on the data collected through passive and active surveillance was undertaken. A total of 11,159 outbreaks with 0.271 million cases of FMD were recorded from 30 different revenue districts of Karnataka. There was a significant difference between the years for the annual incidence of FMD (P = <0.001, F = 19.10) and also between the months (P = <0.001, F = 4.22). Cattle and buffaloes were the predominant species affected being involved in all of the outbreaks reported. A significant correlation was observed between livestock density and the number of outbreaks reported (r = 0.70, p < 0.02), and number of cases (r = 0.76, p < 0.01) for all the agro-climatic zones. The Central dry zone (n = 2257, 19.89 %) reported the highest number of outbreaks followed by the Northern dry zone (n = 1881, 16.58 %) and the Southern transition zone (n = 1761, 15.52 %), and attack rates were concentrated in the North/Northeastern/Central dry and transition zones. A large majority of the outbreaks were caused by serotype O (64.04 %), followed by Asia 1 (19.87 %) and A (12.27 %). Serotype C was not reported since 1993 in the state. In recent years, serotype O has dominated (82.59 %), with the rest of the outbreaks being almost equally caused by A (9.01 %) and Asia 1 (8.40 %). The study highlights the significance of the O serotype and cattle as the main indicator species in the epidemiology of FMD in Karnataka, India. The findings from this study can be used as baseline epidemiological data for further research to identify endemic and epidemic areas for the development of a sustainable programme for the progressive control of FMD in the state of Karnataka as well as other endemic settings.

Incidence of subclinical mastitis and prevalence of major mastitis pathogens in organized farms and unorganized sectors.

Subclinical mastitis (SCM) represents a major proportion of the burden of mastitis. Determining somatic cell count (SCC) and electrical conductivity (EC) of milk are useful approaches to detect SCM. In order to correlate grades of SCM with the load of five major mastitis pathogens, 246 milk samples from a handful of organized and unorganized sectors were screened. SCC (>5 × 10(5)/mL) and EC (>6.5 mS/cm) identified 110 (45 %) and 153 (62 %) samples, respectively, to be from SCM cases. Randomly selected SCM-negative samples as well as 186 samples positive by either SCC or EC were then evaluated for isolation of five major mastitis-associated bacteria. Of the 323 isolates obtained, 95 each were S. aureus and coagulase-negative staphylococci (CoNS), 48 were E. coli and 85 were streptococci. There was no association between the distribution of organisms and (a) the different groups of SCC, or (b) organised farms and unorganised sectors. By contrast, there was a significant difference in the distribution of CoNS, and not other species, between organized farms and unorganized sectors. In summary, bacteria were isolated irrespective of the density of somatic cells or the type of farm setting, and the frequency of isolation of CoNS was higher with organized farms. These results suggest the requirement for fine tuning SCC and EC limits and the higher probability for CoNS to be associated with SCM in organized diary sectors, and have implications for the identification, management and control of mastitis in India.

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.

Comparative efficacy of peste des petits ruminants (PPR) vaccines.

Peste des petits ruminants (PPR) is a highly contagious, economically important viral disease of sheep and goats with high morbidity and mortality rates. In order to control the disease effectively, highly sensitive diagnostic tests coupled with potent vaccines are important pre-requisites. At present, there are three live attenuated PPR vaccines available in India including Sungri 96, Arasur 87 and Coimbatore 97. Indian Veterinary Research Institute (IVRI) Mukteswar developed the PPR Sungri 96 (isolate of goat origin) vaccine; while Tamil Nadu Veterinary and Animal Sciences University (TANUVAS) developed the Arasur 87 (isolate of sheep origin) and Coimbatore 97 (isolate of goat origin). In this study, the potency of these vaccines including a fourth vaccine from Institute of Animal Health and Veterinary Biologicals, Bangalore (IAH&VB) were tested as per the office International des Epizooties (OIE) guidelines by challenge studies in sheep and goats and their efficacies were evaluated using PPR C-ELISA. Potency tests of these vaccines in sheep and goats revealed that three of the vaccines were potent; however, the IAH &VB vaccine was comparatively less potent. The three vaccines could presumably be used for mass vaccination of both sheep and goats while contemplating PPR control program.

Standardization of large scale production of homologous live attenuated PPR vaccine in India.

Live attenuated homologous vaccine against peste des petits ruminants of sheep and goats was produced on a large scale basis in roller culture bottles using seed virus developed at the Indian Veterinary Research Institute, Muktheswar, India. Vero cells between 130-150 passages with six percent foetal calf serum were used for the production of vaccine. The cells were infected with 0.01 multiplicity of infection and harvested when the cytopathic effect was 80%. The vaccine was freezedried in order to maintain the stability of the vaccine. Identity test and titration was performed and the vaccine titre was monitored to be minimum of 10(5)/100 doses. In-house sterility tests and quality control tests using experimental animals and small ruminants were performed. The vacuum and moisture content of the vaccine were also regulated to be within the normal limits.