Retrospective Single Nucleotide Polymorphism Analysis of Host Resistance and Susceptibility to Ovine Johne's Disease Using Restored FFPE DNA.

Johne's disease (JD), also known as paratuberculosis, is a chronic, untreatable gastroenteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) infection. Evidence for host genetic resistance to disease progression exists, although it is limited due to the extended incubation period (years) and diagnostic challenges. To overcome this, previously restored formalin-fixed paraffin embedded tissue (FFPE) DNA from archived FFPE tissue cassettes was utilized for a novel retrospective case-control genome-wide association study (GWAS) on ovine JD. Samples from known MAP-infected flocks with ante- and postmortem diagnostic data were used. Cases (N = 9) had evidence of tissue infection, compared to controls (N = 25) without evidence of tissue infection despite positive antemortem diagnostics. A genome-wide efficient mixed model analysis (GEMMA) to conduct a GWAS using restored FFPE DNA SNP results from the Illumina Ovine SNP50 Bead Chip, identified 10 SNPs reaching genome-wide significance of p < 1 × 10-6 on chromosomes 1, 3, 4, 24, and 26. Pathway analysis using PANTHER and the Kyoto Encyclopedia of Genes and Genomes (KEGG) was completed on 45 genes found within 1 Mb of significant SNPs. Our work provides a framework for the novel use of archived FFPE tissues for animal genetic studies in complex diseases and further evidence for a genetic association in JD.

Successful restoration of archived ovine formalin fixed paraffin-embedded tissue DNA and single nucleotide polymorphism analysis.

Archived formalin fixed paraffin-embedded (FFPE) tissues are powerful tools in medicine, capable of harboring diagnostic and genetic answers to challenging clinical questions. Successful utilization of DNA derived from FFPE samples is dependent upon repairing DNA damage generated from the fixation process. Methods to repair FFPE DNA have been successful in human medicine for a variety of research and clinical applications, yet remain underutilized in veterinary medicine. Despite the available technology, our study is the first to evaluate the repair of FFPE derived DNA from veterinary species for single-nucleotide polymorphism (SNP) analysis using the Illumina OvineSNP50 BeadChip and Illumina FFPE QC and DNA Restore kit. To accomplish this, 48 ovine FFPE samples were run using the Illumina OvineSNP50 BeadChip with and without restoration. Compared to pre-restore data, we found increased sample call rates, SNP call frequency, and assay metrics for all samples post-restoration. Further, we utilized four sheep with available parallel fresh DNA and FFPE DNA to compare assay metrics and genotype calls between the two starting sample types. Although fresh samples generated increased call rates, we found 99% concordance in allele calls between restored FFPE and fresh DNA for all four samples. Our results indicate successful restoration and genotyping of ovine FFPE samples using this technology, with potential for utilization in other veterinary species.

Mechanism of enzymatic dehairing of skins using a bacterial alkaline protease.

In the conventional dehairing process of leather manufacture, animal skins are subjected to a drastic chemical treatment using lime and sodium sulfide. Sulfide reduces disulfide bonds in keratin present in hair and epidermis and thereby detaches them from skin. Lime, being an alkali, contributes to opening up of collagen fiber structure by cleaving a major portion of the glycosaminoglycans from proteoglycans, the interfibrillar elements of skin connective tissue. Currently, as an alternative to chemical dehairing, enzyme based dehairing processes using proteases avoiding the use of lime and sulfide are being developed because of their environmental benefits. Though both chemical as well as enzymatic dehairing processes are aimed at removing noncollagenous proteins and proteoglycans in addition to fiber opening, the mechanism of enzymatic process is distinct from that of the chemical process. In this study, we attempt to study in detail the mechanism of hair saving enzymatic dehairing process for skins using a bacterial protease against the customary hair burn chemical dehairing process. Quantitative analysis shows that the collagen content remains unaffected in both treatments but there is a marked reduction of proteoglycan constituents from dehaired pelts in the enzymatic process when compared to lime-sulfide process. This is further substantiated by histochemical examination of the sections of dehaired pelts using different stains as well as immunohistochemical studies on the removal of decorin. HPLC profile shows that decorin is extensively degraded by the bacterial protease. This study conclusively demonstrates that proteolytic degradation of decorin and subsequent removal of proteoglycan aggregates play an important role in the opening up of the collagen fiber bundles during enzymatic dehairing.

Ecofriendly lime and sulfide free enzymatic dehairing of skins and hides using a bacterial alkaline protease.

The ever-increasing attention to the environmental impact of leather industry has necessitated the development of enzyme-based processes as potent alternatives to pollution causing chemicals. In this study, a hair saving process is developed for dehairing of skins and hides using a bacterial alkaline protease preparation, completely eliminating the use of lime and sulfide. To evaluate the efficacy of the enzymatic process, comparative studies have been carried out with two controls; a conventional lime-sulfide process and enzyme-assisted process using commercial dehairing enzyme with reduced quantities of lime and sulfide. The developed process requires a shorter duration of 6h for complete dehairing of skins and hides than control groups and also, it avoids the use of silicate carriers since the enzymatic dehairing is carried out by dip method. Histological and scanning electron microscopic analyses of the dehaired pelts obtained from enzymatic process reveal complete removal of hair and epidermis with moderate opening up of fiber structure in both dermis and corium. Moreover, the collagen is not damaged and resulting in a leather of good quality. The developed process has resulted in a remarkable reduction of effluent load in terms of biochemical oxygen demand, chemical oxygen demand, total dissolved solids and total suspended solids. Physicochemical studies conclusively show that the leathers produced by enzymatic process are equivalent to or better than that obtained by control systems. Thus, the developed enzymatic process offers immense potential for greener mode of dehairing of skins and hides in leather industry coupled with environmental excellence.

Adaptation of a velogenic Newcastle disease virus to vero cells: assessing the molecular changes before and after adaptation.

A velogenic Newcastle disease virus isolate was passaged 50 times in Vero cell culture and the virus was assessed for the molecular changes associated with the passaging. At every 10th passage, the virus was characterized conventionally by mean death time (MDT) analysis, intracerebral pathogenicity index (ICPI) and virus titration. At increasing passage levels, a gradual reduction in the virulence of the virus was observed. Molecular characterization of the virus included cloning and sequencing of a portion of the fusion gene (1349 bp) encompassing the fusion protein cleavage site (FPCS), which was previously amplified by reverse transcription-polymerase chain reaction. Sequence analysis revealed a total of 135 nucleotide substitutions which resulted in the change of 42 amino acids between the velogenic virus and the 50th passage virus. The predicted amino acid motif present at the cleavage site of the virulent virus was (109)SRRRRQRRFVG(119) and the corresponding region of the adapted adapted virus was (109)SGGRRQKRFIG(119). Pathogenicity studies conducted in 20-week-old seronegative birds revealed gross lesions such as petechial haemorrhages in the trachea, proventricular junction and intestines, and histopathological changes such as depletion and necrosis of the lymphocytes in thymus, spleen, bursa and caecal tonsils in the birds injected with the velogenic virus and absence of the lesions in birds injected with the adapted virus. The 50th-passage cell culture virus was back-passaged five times in susceptible chickens and subjected to virulence attribute analysis and sequence analysis of the FPCS region, with minor difference found between them.

Detection of rabies virus genes by in-situ polymerase chain reaction.

Rabies is diagnosed by FAT in the impression smears of brain tissues. In this study, an attempt was made to diagnose rabies using in situ polymerase chain reaction (ISPCR). A digoxigenin-labelled double-stranded probe specific for a portion of the 'N' gene of rabies virus was used. Positive signals were identified as blue dots in the intraneuronal and neuropil areas.

Xenobiotic-metabolizing enzymes in canine mammary tumours.

Mammary tumours are the most common neoplasms in female dogs. The present study was designed to evaluate the relationship between different clinical stages with activities of phase I and phase II carcinogen-metabolizing enzymes in canine mammary tumours. The levels of cytochrome P450 and cytochrome b5 and the activities of glutathione S-transferase (GST), gamma-glutamyl transpeptidase (GGT), DT-diaphorase (DTD) and NADPH diaphorase in tumour tissues of 25 bitches was estimated. Enhanced levels of cytochrome P450 and b5 and phase II enzyme activities were observed in tumour tissues compared to the corresponding uninvolved adjacent tissues. The magnitude of the changes in phase I and phase II enzyme status was, however, more pronounced in stages I and II compared to stages III and IV. The results suggest that the balance between phase I carcinogen activation and phase II detoxification systems may play an important role in canine mammary tumour development.

Protection of the reproductive tract of young chicks by Newcastle disease virus-induced haemagglutinationinhibition antibodies.

The present study was conducted to assess the haemagglutination-inhibition (HI) titres required to protect the chicken reproductive tract against direct damage caused by Newcastle disease virus (NDV). Precociously induced oviduct and uterus by oestrogen treatment of young chicks were used to assess the damage or protection against the damage by analysis of ciliostasis or histopathological lesions. Unvaccinated day-old female white leghorn chickens were used as the maternally derived antibody (MDA) group. Chickens were vaccinated with either a live lentogenic vaccine on day 14 of age or, along with it, an inactivated vaccine at day 36 of age, to generate birds with a range of primary or secondary response induced HI antibodies. Birds with different HI antibody levels were challenged with virulent NDV. It was found that a HI antibody titre of 128 and above was protective against direct damage of the reproductive tract, while the 32-64 titre range was protective when derived through secondary vaccination only.

Altered oxidant-antioxidant profile in canine mammary tumours.

Mammary tumours are the most common neoplasms in female dogs. Oxidative stress arising due to overproduction of reactive oxygen species, coupled with altered antioxidant capacities has been implicated in the pathogenesis of all types of cancers. However, the extent of lipid peroxidation and the status of antioxidants in canine mammary tumours have not been investigated. The present study was designed to evaluate the oxidant-antioxidant profile in canine mammary tumours. Lipid peroxidation as evidenced by the formation of thiobarbituric acid-reactive substances, lipid hydroperoxides, and conjugated dienes, as well as the status of the antioxidants superoxide dismutase, catalase, reduced glutathione, glutathione peroxidase, glutathione S-transferase and vitamin C, in tumour tissues of 25 bitches was estimated. Lipid peroxidation in tumour tissues was enhanced compared to the corresponding adjacent uninvolved tissues. This was accompanied by significant elevation in both enzymatic and non-enzymatic antioxidants. This study suggests that upregulation of antioxidants induced by lipid peroxidation confers a selective growth advantage to tumour cells over their adjacent normal counterparts.

Molecular characterisation of lineage IV peste des petits ruminants virus using multi gene sequence data.

Peste des petits ruminants is responsible for an economically important plague of small ruminants that is endemic across much of the developing world. Here we describe the detection and characterisation of a PPR virus from a recent outbreak in Tamil Nadu, India. We demonstrate the isolation of PPR virus from rectal swab and highlight the potential spread of disease to in-contact animals through faecal materials and use of faecal material as non-invasive method of sampling for susceptible wild ruminants. Finally we have performed a comprehensive 'multi-gene' assessment of lineage IV isolates of PPRV utilising sequence data from our study and publically available partial N, partial F and partial H gene data. We describe the effects of grouping PPRV isolates utilising different gene loci and conclude that the variable part of N gene at C terminus gives the best phylogenetic assessment of PPRV isolates with isolates generally clustering according to geographical isolation. This assessment highlights the importance of careful gene targeting with RT-PCR to enable thorough phylogenetic analysis.

Induction of apoptosis by fungal culture materials containing cyclopiazonic acid and T-2 toxin in primary lymphoid organs of broiler chickens.

Thirty-six, twenty-eight-day-old broiler chicks were randomly distributed into three groups of 12 birds each. Two groups were fed diets containing 10 ppm cyclopiazonic acid (CPA) and 1ppm T-2 toxin, respectively, to determine the mechanism of cell death in spleen and thymus at 6, 12, 24, and 36 h of post-treatment. The other group served as control. T-2 toxin treated group showed significant (P < 0.01) induction of apoptosis in thymus with peak induction at 24 h post-treatment where as, no significant differences were observed between the control and CPA groups. The CPA toxin treated group showed significant (P < 0.01) induction of apoptosis in spleen with peak induction at 24 h post-treatment. No significant differences were observed between the control and T-2 toxin group even though the latter showed a slight increase in the quantity of apoptotic cells at 36 h post-treatment in spleen. The semi-thin sections stained with toluidine blue from the spleen of CPA treated group exhibited crescent margination of chromatin against the nuclear envelope and shrinkage of lymphoid cells without any surrounding inflammation, the characteristics of apoptosis. The apoptotic thymocytes from T-2 fed birds appeared shrunken with condensed nucleus and showed crescent margination of chromatin against the nuclear envelope without any surrounding inflammation when compared with well-defined nuclei with dispersed chromatin in normal thymocytes. Ultrastructurally, splenocytes of the CPA treated group and thymocytes of the T-2 toxin treated birds showed apoptotic bodies characterized by crescent margination of the chromatin against the nuclear envelope. The study indicates that one route of the CPA and T-2 toxin induced cell death in lymphoid organs of broiler chicken is by apoptosis.

Immunopathological effect of the mycotoxins cyclopiazonic acid and T-2 toxin on broiler chicken.

Forty, newly hatched, unsexed broiler chicks were fed diets containing 10 ppm cyclopiazonic acid (CPA) and 1 ppm T-2 toxin (T2) either individually or in combination for 28 days to study the immunopathological effects. Lymphoid organs revealed lymphocytolysis and lymphoid depletion in all toxin fed birds. Thymic and splenic CD+4 and CD+8 lymphocytes decreased significantly (p<0.01) in toxin fed birds when compared to the control. Thymic CD+8 lymphocytes of T2 and CPA-T2 showed significant (p<0.01) decrease from that of CPA and control groups. Splenic CD+4 and CD+8 lymphocytes showed significant (p<0.01) decrease in CPA and CPA-T2 fed groups when compared to the control. The T2 group did not differ significantly from that of control. The stimulation index (SI) of splenocytes to concavalin A revealed significant (p<0.01) decrease in all toxin fed birds. Significant (p<0.01) decrease were observed for the haemagglutination inhibition (HI) titres to Newcastle disease virus vaccine F strain (NDV) of birds fed CPA, T2 and in combination. Significant (p<0.01) interaction was found for lymphocyte subsets, SI and HI titres to NDV. The study indicated the immunosuppressive effect of these toxins either alone or in combination in broiler chicks.

An in vitro and in vivo evaluation of the virulence of Newcastle disease virus and vaccines for the chicken reproductive tract.

The virulence of two vaccine strains and two field strains of Newcastle disease virus (NDV) for the female reproductive tract of chickens was assessed using oviduct organ cultures (OOC) prepared from precociously-induced oviducts in young chicks by oestrogen treatment. Ciliostasis, haemagglutination and virus isolation from infected OOC supernatants, histopathology and immunoperoxidase test results indicated the pathogenic nature of both vaccine and virulent NDVs for the precocious oviducts. The virulent viruses, mesogenic and lentogenic vaccines caused damage in that order of magnitude and the uterus had a higher susceptibility than oviducts. One virulent and the mesogenic strain of NDV were used for in vivo trials. The pathogenicity was assessed in oestrogen-treated infected chickens using histopathology and immunoperoxidase test. The vaccine virus produced transient damage up to 6 days post-infection, while the damage with the virulent isolate persisted for at least 9 days post-infection. This technique could be a pointer to possible variations in virulence of NDV vaccine and field strains, and warrants further investigation. The potential value of OOC from young chickens for testing the possibility of NDV vaccines causing damage by themselves and offering protection against damage of the reproductive tract caused by virulent isolates is emphasized.