Gopal Dhinakar Raj

Molecular prevalence and preponderance of Eimeria spp. among chickens in Tamil Nadu, India

Coccidosis is one of the most commonly prevalent and economically important parasitic diseases of poultry worldwide. Chicken coccidia are protozoan parasites of the genus Eimeria. This study aimed at analysing the molecular prevalence of seven species of Eimeria infecting chickens in Tamil Nadu, India. Tissue samples (caecum, rectum and upper and mid intestines) collected from chickens exhibiting symptoms of coccidiosis were used for DNA extraction, followed by amplification of the internal transcribed spacer (ITS) region of Eimeria genome with genus-specific primers and speciation in nested polymerase chain reaction (PCR) with species-specific primers. Of 43 tissue samples examined, 25 were positive in ITS PCR and all the seven species could be identified. However, the prevalence of each species varied. In broilers, Eimeria necatrix was present in all infected chickens with Eimeria brunetti, Eimeria tenella, Eimeria maxima and Eimeria acervulina present in more than 50% of infected chickens, while Eimeria praecox and Eimeria mitis were only present in 11% to 16%. Although only 7 samples were positive among layers, the prevalence was largely similar, but with a higher prevalence of E. praecox and E. mitis and a lower prevalence of E. tenella. Multiple infections were most common, with 2–6 Eimeria species infecting the same chickens. In order to estimate the preponderance of each infecting species of Eimeria, a random cloning technique was adopted. The genus-specific ITS PCR product was cloned in a TA vector and ten clones were randomly picked and used as template for amplification of all the seven genera of Eimeria. If the specific species of Eimeria is preponderant, then the frequency of the clones showing that species-specific PCR amplification would be higher. Using this method, the most preponderant species present in the rectum, mid and upper intestines of layers was assessed to be E. acervulina, E. brunetti and E. necatrix. E. acervulina was present in 60–90%, E. necatrix in 10–30% and E. brunetti in 10–20% of the clones screened, indicating that these species could be the most preponderant Eimeria species. Intervention strategies should aim at these species. This new method of estimating preponderance of infecting Eimeria species could be used to assess the relative importance of each species at the farm or region level instead of relying only on prevalence estimates.

Rapid detection of Newcastle disease virus replication in embryonated chicken eggs using quantitative real time polymerase chain reaction.

Newcastle disease virus (NDV), an avian paramyxovirus, is an economically important disease of poultry globally. Rapid methods to detect and differentiate the virus are important to curtail the spread of this virus. Nucleic acid based detection methods are routinely employed for diagnosis that suffer from the disadvantage of failure to discriminate viable virus and non-infectious genome. However, virus isolation remains the gold standard for diagnosis of field outbreaks. The sensitivity of virus isolation was combined with nucleic acid based detection methods so that the time taken for confirmatory diagnosis could be considerably reduced while increasing sensitivity. Quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and conventional RT-PCR techniques were compared for the detection of NDV genome replication in 9-11-day-old embryonated chicken eggs (ECE) using the nucleoprotein (NP) gene of the virus as a target. The results suggest that at least two to fourfold increase in cycle threshold (C(t)) values over the baseline C(t) value of samples lacking infectious virus, would indicate live NDV replication. The limit of detection of NDV replication using qRT-PCR was 1×10(4.0) mean embryo infective doses (EID(50)). The earliest time point when live virus replication was detectable by qRT-PCR or RT-PCR was 30h post-inoculation in ECE.

Toll-Like Receptor Responses to Peste des petits ruminants Virus in Goats and Water Buffalo

Ovine rinderpest or goat plague is an economically important and contagious viral disease of sheep and goats, caused by the Peste des petits ruminants virus (PPRV). Differences in susceptibility to goat plague among different breeds and water buffalo exist. The host innate immune system discriminates between pathogen associated molecular patterns and self antigens through surveillance receptors known as Toll like receptors (TLR). We investigated the role of TLR and cytokines in differential susceptibility of goat breeds and water buffalo to PPRV. We examined the replication of PPRV in peripheral blood mononuclear cells (PBMC) of Indian domestic goats and water buffalo and demonstrated that the levels of TLR3 and TLR7 and downstream signalling molecules correlation with susceptibility vs resistance. Naturally susceptible goat breeds, Barbari and Tellichery, had dampened innate immune responses to PPRV and increased viral loads with lower basal expression levels of TLR 3/7. Upon stimulation of PBMC with synthetic TLR3 and TLR7 agonists or PPRV, the levels of proinflammatory cytokines were found to be significantly higher while immunosuppressive interleukin (IL) 10 levels were lower in PPRV resistant Kanni and Salem Black breeds and water buffalo at transcriptional level, correlating with reduced viralloads in infected PBMC. Water buffalo produced higher levels of interferon (IFN) α in comparison with goats at transcriptional and translational levels. Pre-treatment of Vero cells with human IFNα resulted in reduction of PPRV replication, confirming the role of IFNα in limiting PPRV replication. Treatment with IRS66, a TLR7 antagonist, resulted in the reduction of IFNα levels, with increased PPRV replication confirming the role of TLR7. Single nucleotide polymorphism analysis of TLR7 of these goat breeds did not show any marked nucleotide differences that might account for susceptibility vs resistance to PPRV. Analyzing other host genetic factors might provide further insights on susceptibility to PPRV and genetic polymorphisms in the host.

Formulation of Newcastle disease virus coupled calcium phosphate nanoparticles: An effective strategy for oculonasal delivery to chicken

In this report, calcium phosphate (CaP) nanoparticles were synthesized by continuous flow method using β-cyclodextrin (β-CD) as a medium and functionalized with amino propyl triethoxy silane (APTES). The blood biocompatibility of the nanoparticles was assessed using the whole blood haemolysis, erythrocytes haemolysis and erythrocyte aggregation tests. Based on the results, we found that the synthesized β-CD-CaP nanoparticles did not cause any remarkable toxic effect. The 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT) assay of chicken peripheral blood mononucleated cells (PBMCs) incubated with these nanoparticles indicated that these particles did not exert any significant cytotoxicity. The aminosilane functional group modified β-CD-CaP was used as tool for coupling of Newcastle disease virus (NDV). The NDV conjugated nanoparticles were confirmed by using Fourier transformed infrared spectroscopy, X-ray diffraction patterns, Raman spectroscopy differential scanning calorimetry and energy-dispersive X-ray spectroscopy. Immunogenicity trials in chickens proved that β-CD-CaP-NDV used as a vaccine was better than the commercial vaccine when given oculonasally during the first 2 weeks post vaccination. The birds vaccinated with the above nano-NDV vaccine were completely protected against virulent NDV challenge. This study confirms that the oculonasal β-CD-CaP-NDV delivery of vaccines is a potential method for enhancing the immune responses of existing commercial vaccines.

Real-time PCR-based quantification of Eimeria genomes: a method to outweigh underestimation of genome numbers due to PCR inhibition

Eimeria species parasites can cause the disease coccidiosis in all livestock species, most notably poultry. Traditional diagnostics such as faecal microscopy have now been supplemented by molecular assays including genus-specific and species-specific quantitative polymerase chain reaction (qPCR), although DNA extracted from faecal samples is commonly affected by PCR inhibition. This was confirmed when genomic DNA extracted from chicken faeces inhibited the threshold cycle value of internal positive control (IPC) DNA amplification by 15.33%. Hence, the objective of the present study was to use IPC qPCR to determine PCR inhibition in a series of experimental samples and use the increase in IPC qPCR threshold cycle value as an individual (sample-specific) correction factor for an established 5S rDNA qPCR used to estimate total Eimeria genome numbers. IPC-corrected genome counts were correlated with conventional oocyst per gram counts and compared with non-corrected counts, revealing a 0.1769 increase in correlation coefficient to outweigh underestimation of oocyst counts. Though the sample size used in this study is small, this limitation would be offset by the sample-specific correction factor determined using the IPC along with each sample.

Molecular Cloning and Tissue-Specific Expression of Toll-Like Receptor 5 Gene from Turkeys

SUMMARY. Toll-like receptors (TLRs), a family of transmembrane and cytosolic proteins, detect microbial patterns, initiating innate immune responses in various organisms. Although they are abundant, genetic characterization and functional differences of TLRs in economically important avian species such as chickens and turkeys have not been investigated in detail. In this study, the putative TLR5 coding region from turkey genome was sequenced, and its homology to other vertebrate species was analyzed. Secondary structure analysis revealed protein motifs typical of the chicken TLR5 protein structure, with 97% amino acid identity between them. mRNA expression profiling in adult turkeys revealed abundant TLR5 expression in a broad range of tissues. Stimulation with the TLR5 ligand flagellin resulted in the production of the inflammatory mediators interleukin (IL)-1&bgr;, IL-6, and nitric oxide in peripheral blood mononuclear cells. To our knowledge, this is the first complete turkey TLR5 coding DNA sequence reported in sequence databases.

Expressed sequence tags from Eimeria brunetti—preliminary analysis and functional annotation

As a first attempt to generate sequence information from the protein-coding genes of the genomically unknown parasite, Eimeria brunetti, a cDNA library was generated from purified sporozoites in the λTriplEx2™ vector. Analysis of 283 expressed sequence tags (ESTs) from the cDNA library constructed revealed 12 contigs (26 ESTs) and 257 singletons. BLASTx analysis revealed that 50 transcripts had significant matches to known proteins, whereas the remaining 233 had no significant matches, probably representing novel genes. Based on Gene Ontology classification, the transcripts were categorized as biological process (46 ESTs), molecular function (37 ESTs), and cellular component (19 ESTs). The transcripts analyzed show maximum homology to the apicomplexan parasite Toxoplasma gondii. Despite the small number of transcripts, this is the first transcriptome analysis of E. brunetti and provides preliminary data that will increase understanding of parasite biological function.

Isolation and molecular characterization of canine distemper virus from India

Ocular swabs from canine distemper virus (CDV) suspected live or brain tissue from dead dogs were tested for the presence of CDV nucleoprotein (N) gene using reverse transcriptase polymerase chain reaction (RT-PCR). Partial “N” gene sequencing of the RT-PCR-positive samples and the local vaccine virus revealed that the Ind/Andaman 01/07 virus was highly divergent from the rest of the CDV isolates and from the vaccine strain. Quantitative real-time PCR (qRT-PCR) using SYBR Green I chemistry for CDV haemagglutinin “H” gene quantification showed Ct values ranging from 29.76–30.67 in the RT-PCR-positive samples. Two of the positive samples, designated Ind/TN 01/07 and Ind/Andaman 01/07 were used for virus isolation in B95a cell line. Characteristic cytopathic changes such as rounding of cells, syncytia formation, and ballooning were seen from the first passage onwards. Specific cytoplasmic fluorescence was seen in infected cells with a commercial reference serum against CDV. To the best of our knowledge, this is the first report of CDV isolation from clinical cases in India.

Detection of Rabies Virus Antigen or Antibody Using Flow Cytometry

Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem. Rabies diagnosis must be rapid and conclusive. Detection and quantification of antirabies antibodies is used for assessment of the effectiveness of rabies vaccines. Hence, computer‐automated detection of fluorescence using flow cytometry was attempted to reduce the work time required to undertake the conventional rapid fluorescent focus inhibition test (RFFIT).

Bactericidal paper trays doped with silver nanoparticles for egg storing applications

In this study, a cost-effective way to deposit the silver nanoparticles (AgNPs) on paper egg trays was developed, which proved suitable for prolonged storage of table eggs for house-hold use without deterioration of egg quality. Silver nanoparticles were synthesized based on chemical reduction approach and mixed with gelatin–chitosan mixer used as a colloidal stabilizer as well as fixing agent. AgNPs-doped paper egg trays were characterized by TEM, SEM, FTIR, EDX and XRD. AgNPs containing egg trays were tested for its bactericidal effect against commonly found bacteria on egg shells, E. coli, S. aureus, Streptococcus spp and Salmonella spp. Storing of eggs in the AgNPs-deposited paper egg trays improved the shelf-life of the eggs by more than 14 days compared to controls (eggs stored in conventional trays). In conclusion, the developed paper trays possessed strong antimicrobial activity and it could be an effective storage material for eggs.