Molecular detection of respiratory avian mycoplasmosis associated bacterial and viral concurrent infections in the poultry flocks.

Respiratory tract infections are of serious concern to the poultry industry. The present study was aimed to delineate the extent of respiratory avian mycoplasmosis associated bacterial and viral concurrent infections in the poultry flocks. A total of 146 poultry flocks of Haryana and Rajasthan, India, suspected for chronic respiratory disease (CRD) were screened for avian mycoplasmas, Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and avian pathogenic Escherichia coli (APEC) by conventional polymerase chain reaction (PCR) assays. A total of 49.31% (72/146) flocks were found positive for Mycoplasma infection. Of the Mycoplasma-positive flocks, 80.55% (58/72) represented pathogenic avian mycoplasmas (MG and/or MS), while 19.44% (14/72) flocks were positive for commensal avian mycoplasmas (other than MG and MS). A correlation was deduced between avian mycoplasmosis and bacterial and/or viral co-infections. The results revealed that 17.24% (10/58) flocks had only avian mycoplasmosis infection. However, in the remaining flocks, the avian mycoplasmosis was associated either with APEC infection [17.24% (10/58)], IBV infection [43.10% (25/58)], or both APEC and IBV infections [22.41% (13/58)], respectively. Further epidemiological studies on respiratory avian mycoplasmosis associated concurrent infections with other pathogens are recommended to assess circulating strains, risk factors, and economic losses.

Studies on intra-ocular vaccination of adult cattle with reduced dose Brucella abortus strain-19 vaccine.

Brucella abortus vaccines play a central role in bovine brucellosis control with tremendous success worldwide for decades. The study was aimed to evaluate the efficacy of reduced dose (5.0 × 10 9 cfu) of S19 vaccine in adult cattle and its shedding in the milk of vaccinated cattle using molecular techniques. The OIE recommended tests (RBPT, SAT, and iELISA) for brucellosis screening in cattle were used. Seronegative cattle (n = 90) of different age groups (young, old heifers & milking cows, n = 30 each) were selected for the vaccine trials. Antibody titers were recorded at 7th, 21st, 30th, 60th, 90th and 120th days post-vaccination (DPV) to monitor the immune responses following vaccination and at 150th, 180th, 210th and 240th DPB following booster-dose to an intraocular group. The humoral immune responses observed by RBPT and ELISA, proved that antibody titers persisted in s/c group compared to the i/o group in all categories. The IFN-γ stimulation (CMI) due to reduced dose vaccination was noticed early as 30th in all groups and declined after 90th DPV, with higher IFN-γ stimulation among the s/c group. The Bcsp31 and IS711 targeted PCR detected the presence of Brucella DNA in milk samples (n = 120) from the vaccinated cows (n = 30) and confirmed by qPCR (TaqMan assay) at 30th, 60th, 90th and 120th DPV. A Significant number, 70% (7/10) was detected in s/c by qPCR. BCSP31 sequence was deposited at NCBI GenBank (accession no. MK881173-6). PCR and qPCR techniques could provide a reliable diagnosis of brucellosis from milk. The intraocular route remains the safer route for vaccinating adult cattle than subcutaneous.

Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays.

Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically important disease of poultry industry. The present study was aimed to develop duplex PCR as a rapid, specific and economical method for accurate detection of MG and MS in poultry and its comparison with single (monoplex) MG/MS PCR. During present investigation, a total of 146 poultry flocks having clinical history of respiratory disease were screened. Pooled tissue samples (trachea, lungs and air sacs) from 4-5 birds of each flock were collected during necropsy at disease investigation laboratories, Hisar, Haryana, India. The single and duplex PCR assays were standardized using primers of intergenic spacer region (IGSR; 16S-23S rRNA) for MG and hemagglutinin vlhA gene for MS, with expected amplicon size of 812 bp and 1200 bp products, respectively. In single PCR, 6.85%, 2.74% and 2.74% tissue samples were found positive for MG, MS and both MG and MS, respectively. However, duplex PCR showed, 7.53%, 2.74% and 1.37% positivity for MG, MS and both MG and MS, respectively. Taking the results of monoplex PCR as a gold standard, sensitivity and specificity of the developed duplex PCR was found to be 94.44% and 100%, respectively. Moreover, Cohen's kappa statistic (k = 0.97) measured a 'perfect' agreement between monoplex and duplex PCR assays. The positive and negative predictive values of duplex PCR was found to be 1.0 and 0.9922, respectively at 95% confidence interval (CI), as compared to monoplex PCR. The simultaneous use of two genes in a duplex PCR was more rapid and economical than two separate single PCR reactions.

Epidemiology of bovine brucellosis in Hisar, India: identification of risk factors and assessment of knowledge, attitudes, and practices among livestock owners.

Brucellosis caused by facultative intracellular bacteria, Brucella, remains a global threat to both animal and human health. In this study we aimed to identify potential risk factors of bovine brucellosis and to assess the knowledge, attitudes, and practices (KAPs) of livestock keepers in Hisar, India. A standardized questionnaire was used to collate information regarding potential risk factors of bovine brucellosis and livestock owners' KAPs. A total of 127 livestock keepers were involved. Serum samples from their animals (n = 635) were tested for the presence of antibodies against Brucella by Rose Bengal Plate Test (RBPT) and indirect enzyme-linked immunosorbent assay (iELISA). Out of these, 78 (61.4%) of the herds had at least one seropositive animal, and 302 (47.6%) of the cattle were seropositive. Univariate and multivariate analysis revealed significant associations between intensive farm type (OR = 4.6; 95% CI, 1.6-16.7; P = 0.009), hygienic disposal of aborted fetuses (OR = 0.3; 95% CI, 0.08-0.9; P = 0.04) and herd seropositivity for brucellosis. The majority, 96 (75.6%) of the respondents, were males aged 18-50, and 82 (64.6%) owned a small-backyard farm. Only 51 (40.2%) of the participants knew about brucellosis; out of them, 54.9% (28/51) could not identify clinical signs of brucellosis. Six (11.8%) participants indicated abortion as the most noticeable clinical sign, and 45.1% indicated that consumption of raw milk is associated with high risk of contracting brucellosis. A large proportion of respondents confirmed that milk from their animals was regularly consumed (86.6%) and sold (59.8%) to other people. These results suggest that bovine brucellosis is endemic in Haryana, where Brucella-contaminated milk is likely being regularly sold. Brucellosis control efforts in Haryana should include education programs to raise awareness of the disease and means to control it in cattle and to prevent zoonotic transmission.

Multiple antigenic peptide-based flow through dot-blot assay for simultaneous antibody detection of infectious bronchitis virus and Newcastle disease virus.

The present study describes the development of a novel affordable and rapid visual dot-blot assay using synthetic multiple antigenic peptides (MAP) for simultaneous detection of antibodies to infectious bronchitis virus (IBV) and Newcastle disease virus (NDV). Antibody detection efficiencies of MAP peptides namely, NP1 MAP (Nucleoprotein IBV) and HN MAP (Haemagglutinin-neuraminidase NDV) were studied in solid-phase indirect peptide ELISA. In comparison with the commercial kit, the NP1 MAP showed 89.20% diagnostic sensitivity (DSn) and 85.90% diagnostic specificity (DSp) at 19.45% ROC cut-off. Similarly, HN MAP was evaluated and showed 89.70% DSn and 92.90% DSp at 19.90 % ROC cut-off. The peptides after evaluating their ELISA performance were further used to device a flow-through dot-blot assay (FT-DBA) for simultaneous detection of IBV and NDV antibodies. The kappa value for IBV by FT-DBA in comparison to commercial ELISA was 0.64 whereas for NDV, FT-DBA gave a kappa value of 0.68 in comparison to commercial ELISA indicating substantial agreement between the assays. In essence, the divergent MAP based diagnostic design could provide an alternative for antibody detection of IBV and NDV. Further, the FT-DBA approach could be used for low cost, rapid and pen-side detection of IBV and NDV antibodies simultaneously.

A bio-safe multiple antigenic peptide (MAP) enzyme-linked immunoassay for the detection of antibodies to infectious bronchitis virus in chickens.

The objective of the study was to develop a bio-safe synthetic peptide ELISA for the detection of antibodies against the infectious bronchitis virus (IBV) using a novel multiple antigenic peptide approach (MAP). After initial ELISA optimization, diagnostic sensitivity (DSn) and specificity (DSp) for the linear peptides were determined using receiver operator curve (ROC) analysis. The peptide IBVP1 showed 90.44% DSn and 88.64% DSp at ROC cut off 22.8% while IBVP2 showed 88.24% DSn and 85.23% DSp at ROC cut off 23.05%. The multimerization of linear peptides to MAP design resulted in the improvement of the diagnostic efficiency up to 94.85% DSn and 92.05% DSp for IBVM1 with 19.95% cut off. A similar improvement in the performance was also observed with 92.65% DSn and 90.91% DSp for IBVM2 at 20.72% cut off. All the peptides were tested for diagnostic specificity and did not show the cross-reactivity with Newcastle disease virus and infectious bursal disease virus positive serum samples. In addition, repeatability testing for all linear and multimeric peptide showed that the coefficient of variation for intra-assay was within the expected limits, ranging from 2.4 to 10.4% and inter-assay coefficient of variation was ranging from 5.56 to 14.3%. In a nutshell, the present study used predicted B cell epitope, the synthetic peptide in linear and multimeric design for IBV antibody detection. The study also highlights peptide antigen with modified scaffold design could be a safe alternative to whole virion-based ELISA for IBV antibody detection.

Carriage of Class 1 integrons and molecular characterization of intI1 gene in multidrug-resistant Salmonella spp. isolates from broilers.

AIM: The present study was conducted with the following aims: (i) To screen the Salmonella spp. isolates recovered from suspected cases of fowl typhoid for carriage of Class 1 integrons and analyze their association with antimicrobial resistance and (ii) to carry out molecular characterization and phylogenetic analysis of Class 1 integron-integrase (intI1) gene. MATERIALS AND METHODS: A total of 43 Salmonella isolates were subjected to polymerase chain reaction (PCR) assay to determine the presence of Class1 intI1. Differences between different serotypes in relation to their carriage of integrons and the differences between strains containing or not containing an integron and being resistant to different antimicrobials were analyzed by Fisher exact test using STATA™ (StataCorp, College Station, TX). Phylogenetic analysis was carried out using MEGA6 software. RESULTS: Out of 43 isolates, 40 (93.02%) were found positive for Class 1 integrons. 35/40 (87.5%) intI1-positive isolates were multidrug resistance (MDR) (resistant to ≥4 antibiotics), which support the hypothesis of an association between the presence of Class 1 integrons and emerging MDR in Salmonella. There was no significant difference among isolates resistant to different antimicrobials in Class 1 integron carrying isolates and the Class 1 integron negative isolates (p<0.05). Further, there was no significant difference among different serotypes in respect of their carriage of Class 1 integrons. CONCLUSION: It can be concluded that the high prevalence of Class 1 integrons indicates a high potential of Salmonella isolates for horizontal transmission of antimicrobial genes, especially among Gram-negative organisms.

Concurrent infection of Bluetongue and Peste-des-petits-ruminants virus in small ruminants in Haryana State of India.

Bluetongue (BT) and peste-des-petits-ruminants (PPR) are major transboundary diseases of small ruminant, which are endemic in India. Testing of bluetongue virus (BTV) and peste-des-petits-ruminants virus (PPRV) from recent outbreaks (2015-2016) in different regions of Haryana State of India revealed that 27.5% of the samples showed the presence of dual infection of BTV and PPRV. Analysis of Seg-2 of BTV (the serotype-determining protein) showed the presence of BTV-12w in several isolates. However, analysis of N gene fragment amplicons showed that viruses belong to lineage IV were most closely related to a pathogenic strain of PPRV from Delhi. This is the first report of co-circulation of PPRV lineage IV and bluetongue virus serotype 12 in the state.

First Draft Genome Sequence of Salmonella enterica Serovar Gallinarum Strain VTCCBAA614, Isolated from Chicken in India.

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum causes fowl typhoid (FT), which results in huge economic losses to poultry farmers in India. We report the draft genome sequence of Salmonella biovar Gallinarum strain VTCCBAA614, isolated from a chicken in an FT affected broiler flock.

Prevalence and epidemiology of Salmonella enterica serovar Gallinarum from poultry in some parts of Haryana, India.

AIM: The present study was investigated to ascertain the epidemiological status of fowl typhoid (FT) in broilers in some parts of Haryana during January 2011 to December 2013. MATERIALS AND METHODS: To elucidate the epidemiological status of FT in broiler chickens for the 3 years (2011-2013) and to study the prevalence of various Salmonella serovars in poultry on the basis of culture characteristics, biochemical features, serotyping, and their antibiogram profile from some parts of Haryana (India). RESULTS: A total of 309 outbreaks of FT were recorded in chickens during this period. Overall percent morbidity, mortality, case-fatality rate (CFR) in broiler chicks due to FT during this period was 9.45, 6.77, and 71.55. The yearly observations were divided into quarters A (January-March), B (April-June), C (July-September) and D (October-December). Maximum number of outbreaks - 106 (34.3%) was recorded in quarter D followed by quarters B - 84 (27.3%), C - 64 (20.7%), and A - 55 (17.7%). Salmonella isolates (253) were recovered from disease outbreaks in broilers from different parts of Haryana. Typical morphology and colony characters on MacConkeys Lactose Agar and Brilliant Green agar, biochemical reactions, serotyping along with antibiogram profiles were able to group these isolates into 3 groups namely Salmonella Gallinarum (183), Salmonella Enteritidis (41) and Salmonella Typhimurium (29). The antibiogram pattern of 183 isolates of S. Gallinarum revealed that most of the isolates were sensitive to gentamicin (76%) followed by amikacin (72%), kanamycin (71%). CONCLUSION: FT is prevalent in commercial broiler flocks in different parts of Haryana and is responsible for considerably high morbidity and mortality in affected flocks. Isolation of S. Gallinarum (9, 12:183) from FT cases suggest it to be the primary pathogen, however, isolation of S. Typhimurium and S. Enteritidis from these cases is a major concern. The detection of S. Enteritidis and S. Typhimurium from FT cases assumes significance from public health point of view.

Fowl adenovirus (FAdV) in India: evidence for emerging role as primary respiratory pathogen in chickens.

Adenoviruses have been isolated from both clinically healthy and diseased birds worldwide. The pathogenic role of most of the FAdVs is still questionable. They can quickly take on the role of opportunistic pathogens when additional factors, particularly concurrent infections, adversely affect the health of the avian host. Immnosuppressing agents especially chicken infectious anemia and infectious bursal disease viruses are known to enhance the pathogenicity of FAdVs upon coinfection. The aim of the present study was to screen for the involvement of FAdV in poultry flocks affected with respiratory disease complex by RT-PCR. The samples were also screened by RT-PCR/PCR for other respiratory pathogens. Thirty two commercial poultry flocks with the history of respiratory disease complex from various parts of India. FAdV nucleic acid could be detected in tissue samples of 13 out of 34 farms investigated. Out of 13 FAdV positive farms, FAdV and CIAV were alone detected in 4/13 (31%) whereas, in other farms more than two respiratory pathogens were detected together. CIAV was detected in all the farms (34/34) investigated. Eosinophilic intranuclear inclusion bodies were noticed in FAdV infected laryngeal and tracheal epithelium under light microscopy. The findings of the study assert that FAdV can play the role of primary respiratory pathogen in immunocompromised birds and also in the presence of other respiratory pathogens.

Immunogenicity of major cell surface protein(s) of Brucella melitensis Rev 1.

Brucella melitensis Rev 1 organisms were salt-extracted and the cell surface proteins (BCSPs) were found to be mainly 39-42 kDa (group 2 porin proteins) in addition to 31.6, 32.5, 58.5 and 14.7 kDa proteins. DEAE-Sephadex anion-exchange column chromatography of BCSPs yielded fraction 1, which contained one major protein (39.8-42.0 kDa) and a minor protein (31.6 kDa). All these proteins were found to be immunogenic by Western blotting. Fraction 1 along with monophosphoryl lipid A and trehalose dicorynomycolate adjuvants as well as BCSPs alone induced significant (p < or = 0.05) protection in BALB/c mice. Both these immunizing agents produced almost equivalent protection to live B. melitensis Rev 1 vaccine at 15 and 30 days post challenge. Lymphocyte stimulation test as well as delayed-type hypersensitivity reaction revealed that both these preparations induced cell-mediated immune response. These preparations also induced humoral immune response as indicated by indirect ELISA. Neither of the immune responses was significantly less (p < or = 0.05) than that with live B. melitensis Rev 1 vaccine, except that their duration was short.

Toxicity of aflatoxin B1 in broiler chicks and its reduction by activated charcoal.

A study was conducted on 60 broiler chicks of the effect of activated charcoal (200 ppm) on the toxicity of 0.5 ppm aflatoxin B1 (AFB1) in feed fed from day 1 to day 42. Activated charcoal was found to be moderately effective in reducing the harmful effects of AFB1 as assessed by growth response and various biochemical parameters. The feeding of activated charcoal along with AFB1 reduced the inhibitory effect of AFB1 on bodyweights and feed intake. There was also a significant improvement in the serum aspartate aminotransferase, alkaline phosphatase, total proteins, calcium and phosphorus levels. However, no significant improvement was observed in cholesterol levels.

Comparison of serological tests for Brucella melitensis infection in sheep.

A comparative study of the standard tube agglutination test (SAT), Rose Bengal plate agglutination test and counter immuno-electrophoresis (CIEP) was made on 647 sera from naturally aborting ewes, orchitic, in-contact and apparently healthy sheep with no history of vaccination against brucellosis. No individual test could detect all the 13 known positive reactors (the foetuses of which yielded Brucella melitensis) but by combination of two tests all 13 were positive. The SAT detected more reactors during the early stage of infection while CIEP performed better in later stages of infection. All these tests may be carried out in a field laboratory at very low cost.

Brucellosis--cause of abortion in sheep and its public health significance.

Present study was undertaken to determine the association of brucellosis with abortions occurring naturally in sheep at an organized local sheep breeding farm. A total of 15 strains of Brucella melitensis biovar I were isolated from the abortion material. Serologically the aborted ewes were positive for brucellosis by one or more tests. During acute infection (abortion), standard tube agglutination test (SAT) detected more positive reactors (70.7%) while counter immunoelectrophoresis (CIE) detected more positive reactors (33.9%) in chronic infection (in-contact and apparently healthy sheep). Personnel handling the abortion material at the farm were found positive clinically as well as serologically for brucellosis. These observations suggest the zoonotic importance of brucellosis.

An improved micro-scale liquid-chromatographic assay for piperacillin in plasma and urine.

An accurate, sensitive, and specific liquid-chromatographic method is described for measuring piperacillin in plasma and urine. Plasma samples deproteinized with two volumes of acetonitrile containing 1.2 mg of the internal standard, p-nitrobenzene sulfonamide, per liter are centrifuged. The clear supernate is evaporated under nitrogen, and the residue is reconstituted in 50 microL of the mobile phase (32/68 by vol acetonitrile/water, adjusted to pH 2.5 with 85% phosphoric acid), of which 10 microL is injected onto a reversed-phase (C-18) column. Urine samples are diluted 10-fold with distilled water, an equal volume of acetonitrile containing 3 mg of the internal standard per liter is added, and 20 microL is chromatographed. Stability studies indicate that storage conditions are critical for both plasma and urine. Piperacillin in plasma is stable at -70 degrees C for at least six weeks, but 100% of it is degraded during the same time at -20 degrees C. Piperacillin in urine is also stable at -70 degrees C for six weeks, but 20% is degraded during six weeks at -20 degrees C.