Veterinaria Italiana (Journal)
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Evidence of Salmonella enterica in Pakistani backyard poultry breeds: isolation, molecular characterization, and pathology
Get PDFThe present study evaluated the presence of Salmonella enterica in Pakistani backyard poultry. A total 48 chickens from 4 backyard poultry breeds with the clinical presentation of S. enterica infection were randomly selected from villages in the Punjab Province. Cloacal swabs from live poultry and liver samples from the dead birds were collected for bacterial culture and biochemical identification. Liver and spleen samples from dead birds were evaluated for gross and histopathological changes. Bacterial isolates were subjected to PCR and sequencing of ratA gene. Biochemical identification revealed 5/48 (10.42%) chickens positive for S. enterica. Gross pathology included enlarged, discoloured and congested liver and congested spleen. Histopathology demonstrated congestion of sinusoidal capillaries, cellular swelling and cellular/ballooning degeneration, congestion of central hepatic vein, granular hepatocytic cytoplasm and the presence of variable-sized vacuoles in hepatocytes. The PCR yielded a S. enterica specific amplicon (1047 bp). All liver samples that were positive for S. enterica by biochemical tests, were also positive by PCR. The ratA gene sequencing revealed a close resemblance with S. enteritidis isolates from humans. The present study highlights zoonotic risk from backyard poultry and suggests that PCR can be used as an alternate method for rapid detection of Salmonella serovars
Post-harvest biocontrol of Salmonella Enteritidis on Chicken breast meat and Shell eggs using multiphage cocktail
Get PDFThe study aimed to evaluate the efficacy of a phage cocktail to reduce Salmonella Enteritidis contamination on perishable food items viz. chicken breast meat and shell eggs using different concentrations. Initially, four bacteriophages €P54, €P59, €P66, and €P72 were isolated from sewage water using Salmonella Enteritidis as a target strain. €P54 and €P66 were found to be Myoviruses while €P59 and €P72 belonged to the Siphoviridae family. A phage cocktail was applied at a concentration of 100 and 10,000 multiplicity of infection (MOI) after artificially contaminating both food items with Salmonella Enteritidis. Results showed that, phage cocktail significantly (p ≤ 0.05) reduced Salmonella Enteritidis count at both concentrations. However, the increased reduction was witnessed at 10,000 MOI. In comparison to untreated control, on chicken breast meat bacterial count was reduced to 1.94 and 3.17 Log10 cfu/g at 100 and 10,000 MOI respectively at 4oC. Similarly, on shell eggs, the bacterial count was reduced to 3.09 and 2.81 Log10 cfu/mL at 10,000 MOI at 4°C and 25°C respectively, while at 100 MOI there was less drop in bacterial count at both 4°C and 25°C. The results showed a better reduction at 4°C as compared to 25°C. Our data showed that the phage cocktail is an effective alternative and additional measure compared to conventional bacterial control methods for meat and eggs
Canine Distemper Virus (CDV) in Grey Wolf (Canis lupus) in Fars Province of Iran
Get PDFEmerging and re-emerging viral diseases shared between wildlife and domestic animals are continually spreading to new geographic locations, influenced by human activities and environmental change. Canine distemper (CD) is probably one of the best examples of a disease that has been proved to be capable of compromising the conservation of several wild carnivore species. In this article, we describe a case report of CD in a grey wolf (Canis lupus) in Iran. A grey wolf was found in Fars Province close to Bamou national park. Clinical signs were characterized by neurologic signs, muscle twitching, hyperkeratosis of the footpads and nose and keratoconjunctivitis sicca. After the death of the animal, samples were taken from different organs and sent to collaborator laboratory of Fars Provincial Office of Veterinary Organization. RT-PCR assays confirmed canine distemper virus in the grey wolf. This is the first documented report of canine distemper virus in wild species from Fars Province of Iran
O2-1 Brucella Databases at BioCyc.org: Pathways, Omics Tools, and Enhanced Genome Annotation
No full textBioCyc.org is a web portal for >20,000 sequenced microbes, including 54 Brucella genomes. BioCyc couples high-quality curated data with a wide range of easy-to-use bioinformatics tools. Each of the Brucella databases in BioCyc was constructed using a similar methodology. A series of computational inferences were applied to the annotated genome from RefSeq, including prediction of metabolic reactions and metabolic pathways, transport reactions, operons, Pfam domains, and orthologs with other genomes in BioCyc. When available, additional data were imported from related databases, including protein features and Gene Ontology terms from UniProt, protein localization data from PSORTDB, and gene essentiality data from OGEE. Manual curation was performed on the databases for B. abortus 2308 and B. ovis ATCC 25840 to integrate information from the experimental literature. Mini-review summaries, literature references, and evidence codes were authored for selected proteins and pathways. The resulting B. abortus database contains 224 metabolic pathways and 3,874 protein features and the B. ovis database contains 236 pathways and 3,745 protein features. A particular focus was curation of virulence factors, such as thelipopolysaccharide and type IV secretion system. The BioCyc website provides extensive bioinformatics tools for searching and analyzing these databases and leveraging them for analysis of omics datasets. Genome-related tools include a genome browser, sequence search and alignment tools, and extraction of sequence regions. Pathway-related tools include pathway diagrams and navigation of zoomable organism-specific metabolic map diagrams. Operons, regulatory sites, and the full regulatory network can be displayed when such data are present. Comparative analysis tools enable comparisons of genome organization, of orthologs, and of pathway complements. Omics data analysis tools support enrichment analysis and painting of transcriptomics and metabolomics data onto individual pathways and the full metabolic mapdiagrams. The Omics Dashboard tool enables hierarchical exploration of omics datasets. A unique feature called SmartTables enables users to construct and store tables of genes, metabolites, or pathways, and to perform multiple analyses, such as converting a list of genes or metabolites into a list of all pathways in which those genes/metabolites participate
P2-01 Envelope stress response in Brucella abortus
No full textStudies performed with Escherichia coli indicated that following an envelope stress, a signalling sensor is activated and induces a series of regulatory cascades which modify the expression of target genes allowing an adaptative response. While envelope integrity is obviously crucial for survival and growth, the envelope stress response in B. abortus is under-investigated. We therefore undertook a Tn-seq analysis in the presence of deoxycholate (DOC) in rich medium, compared to a control without DOC. DOC is a soft anionic detergent, used here to stress the envelope. We found that a two-component system (TCS) CenK-CenR and a homologous system to Mla pathway are required to grow in the presence of DOC. According to a Tn-seq performed with B. abortus in RAW 264.7 macrophages, CEnR, CEnK and MLA Mutants are attenuated, suggesting that mutants failing to adapt to envelope stress are indeed impaired for their ability to survive and/or grow inside these cells. A homologous system of CenK-CenR was first identified in Caulobacter crescentus, the model for alpha-proteobacteria, where it plays an essential role in maintaining cell envelope integrity. In Brucella this TCS is poorly characterized and previous studies focus only on the response regulator, that seems to play a role in responses to osmotic stress and acidic pH. We generated markerless ∆cenR and ∆cenR∆cenK mutants, and we show that they have a growth defect on DOC, confirming the phenotype suggested by the Tn-seq analysis on DOC. These results indicate that cenK and cenR genes are indeed involved in envelope stress sensing. In E. coli the Mla pathway maintains the lipid asymmetry and membrane integrity. This pathway allows a mainly retrograde transport of phospholipids between inner and outermembranes. The mla mutants in E. coli have a disruption of outer membrane permeability and integrity. We identified homologues of the Mla proteins in B. abortus. We generated markerless deletions strains for mlaE, mlaF, mlaD, mlaA and mlaEFDA and we found that each deletion strain has a growth defect on DOC, confirming the phenotype suggested by Tn-seq indicating that this system is indeed involved in envelope integrity
P8-03 16S Microbiome Characterization of Brucella Positive and Negative Raw Milk Samples Collected from three regions in Tanzania.
No full textBrucellosis is endemic in many low- and middle-income countries, including Tanzania, where the disease is often transmitted via the consumption of raw milk or direct contact with infected animals/products. Brucella species can localize in the mammary lymph nodes and glands of infected dairy animals that may shed the pathogen in milk for extended periods of time and present a significant health risk to consumers of unpasteurized dairy products and individuals in direct contact with infected animals. Since the sale and consumption of raw and unprocessed milk is common amongst pastoral and agropastoral communities in Tanzania, large proportions of the population are susceptible to milk-borne and zoonotic diseases, including brucellosis. We have recently initiated a five-year cross-sectional survey to assess risk factors associated with brucellosis, including raw milk consumption in Tanzania. To determine the total microbial diversity present inraw milk and to identify potential associations with other pathogens or microbial signatures of milk from infected animals, characterization of Brucella-positive and negative samples using next-generation sequencing is being performed. In brief, we have collected 176 bovine milk samples from dairy cattle (76), and Boran crossbred cattle (100) from seven herds in three regions of Tanzania, Dodoma (Central), Morogoro (East), and Tanga (Northeast). Milk samples were collected aseptically, and Fluorescence Polarization Assay (FPA) was used to screen the milk samples as an initial screen to ascertain Brucella’s status. DNA extraction from the FPA+ and select FPA- milk samples were performed by applying a magnetic-based extraction method, followed by real-time PCR-based detection of the IS711 insertion sequence to confirm the Brucella status. Studies to assess 16S rRNA-based microbiome diversity will next be performed, and genomic DNA will undergo PCR with primers 515F-806R to amplify the V4 region of the 16S gene for microbiome profiling following the Earth Microbiome Project protocol Next-generation sequencing of the 16S RNA and Illumina short-read sequencing and are in progress
O3-3 Lipopolysaccharide synthesis and traffic in the envelope of the pathogen Brucella abortus
No full textThe lipopolysaccharide (LPS) is essential for most Gram-negative bacteria as it is a main component of the outer membrane (OM). In the pathogen Brucella abortus, smooth LPS (S-LPS) is crucial for virulence. Being part of the Rhizobiales, Brucella displays unipolar growth and its LPS was shown to be incorporated at the growth sites, i.e. the new pole and the constriction site. When synthesized, the rough LPS (R-LPS) is anchored to the cytoplasmic leaflet of the inner membrane (IM) and is then flipped to the periplasmic leaflet by MsbA, an essential ABC transporter. The O-antigen is independently flipped to the periplasmic leaflet of the IM and is then ligated by a periplasmic ligase to a fraction of the R-LPS, giving S-LPS. S-LPS and R- LPS are translocated by the essential LPS translocation machinery (Lpt). LptDE and LptB2CFG form the OM and IM complexes, respectively. Nothing is known about the localization of MsbA or the Lpt complex, and the O-antigen ligase remains unidentified in Brucella. Here, we showed that the IM complex (LptC and LptF) is localized at the growth sites, while LptD is dispersed all over the OM. We hypothesize that LPS could be translocated only when the entire complex is formed at the growth sites and that a fraction of LptD would be inactive. Surprisingly, MsbA was mainly localized at the old pole and time-lapse microscopy suggestedthat MsbA was able to move during growth. Based on this, we proposed that MsbA would be mobile in the IM, actively flipping LPS. Then, LPS would diffuse in the IM until it encounters the Lpt pathway. In addition, we identified WadA as the main O-antigen ligase in Brucella spp. WadA presents a N-terminal glycosyltransferase domain, that was shown to add the last glucose onto the R-LPS core, and a C-terminal predictedto be an O-antigen ligase domain. We showed that deleting the biggest periplasmic loop of the O-antigen ligase domain generates a rough phenotype, that could be restored to smooth by adding a copy of the O-antigen ligase domain only. This work highlights a new class of bifunctional O- antigen ligase
P2-02 Comparison of the Brucella abortus population structure based in genotyping methods of distinct levels of resolution
No full textNumerous techniques are currently available with different principles, costs, and levels of resolution to understand the transmission dynamics of brucellosis worldwide. We aimed to compare the B. abortus population structure of 53 Brazilian genomes using eight different genotyping methods [multiple-locus variable-number tandem-repeat analysis (MLVA8, MLVA11, MLVA16), multilocus sequence typing (MLST9, MLST21), core genome MLST (cgMLST) and two techniques based on single nucleotide polymorphisms (SNP) detection (parSNP and NASP)]. The strains were isolated from six different states between 1977 and 2008 and were previously analysed using MLVA8, MLVA11, and MLVA16. Then, their whole genomes were sequenced, assembled, and submitted to MSLT9 MLST21, cgMLST, and SNP analyses, using the mlst,chewbbaca, parSNP, and NASP programs, respectively. All the genotypes were compared in the R program (dendextend package). MLST9 and MLST21 were the two techniques with the lowest level of resolution, both presenting 4 genotypes. MLVA8, MLVA11, and MLVA16 showed a progressive level of resolution as more loci were analysed, with 6, 16, and 44 genotypes, respectively. The cgMLST showed the highest level of resolution, depicting 45 genotypes, followed by the SNPs methods, both with 44 genotypes. Hierarchical grouping by the mean distance among the correlation of the techniques showed clustering of MLST9 and MLST21, the second clustering of MLVA8, MLVA11, and MLVA 16 and a third clustering grouping parSNP, NASP, and cgMLST. In the assessed population, MLVA was more discriminatory than MLST and was more practical and cheaper to perform. Furthermore, SNP techniques and cgMLST provided the highest levels of resolution, showing high agreement with each other, which could be explained by the high clonality of B. abortus genomes (90% of its gene repertoire belongs to the core genome). In conclusion, it was observed that different techniques might be more indicated depending on available resources. It investigated epidemiological scenarios, with MLVA being a simpler and ideal technique for countries that are still in the diseasecontrol phase, and SNP and cgMLST techniques would be better choices for outbreak investigations and for surveillance in countries in the eradication or where brucellosis is already eradicated
P2-03 Study of outer membrane vesicles from brucella species
No full textGram-negative bacteria release nanovesicles from their outer membrane, named outer membrane vesicles. The purpose of these vesicles in bacteria physiology is still unknow, and some models have been proposed to explain their production. In last decades, proteomics becomes an important tool to determine the proteins carried in the vesicles. Vesicles contain proteins able to elicit an immune response, so they have been proposed as a model to develop acellular vaccines. Previously, vesicles from Brucella abortus and B. melitensis have been studied and demonstrating to be protective in mice after the challenge with a virulent Brucella strain. In this study, OMVs of Brucella suis, B. ovis, B.canis, and B. neotomae were purified and analyzed by SDS-PAGE, transmission electron microscopy and liquid chromatography coupled to mass spectrometry to determine the pan-proteome. Additionally, antigenic proteins were detected by western blot using rabbit anti-Brucella specific sera. Brucella suis ATCC 23444 (1330), B. ovis ATCC 25840 (63/290), B.canis ATCC 23365 (RM6/66) and B. neotomae ATCC 23459 (5K33) were used in this study. OMVs of B. melitensis, B. ovis, B.canis and B. neotomae showed a spherical shape and bilayer lipid membrane, and sizes between 30-80 nm. The pan-proteome showed many homologous proteins, such as Omp16, Omp25, Omp31, SodC, Omp2a, and BhuA. Proteins contained in the vesicles from different Brucella species were detected by anti-Brucella sera. Also, the proteins found in the Brucella species tested here were also identified within the OMVs of B. suis and B. abortus. The presence of these proteins shared in vesicles of different Brucella species made them candidates to be evaluated as an acellular brucellosis vaccine or in diagnostic tests
P5-02 Exploring new candidates to develop a B. ovis vaccine based on S-LPS devoid mutant H38ΔwbkF and core defective derivates
No full textBrucella ovis, though non-zoonotic, is a serious cause of reproductive failure in sheep. No country has been declared free from this infection and eradication by test-and-slaughter has been claimed very seldom. Rev.1, the only available vaccine for small ruminants, is effective against B. melitensis (zoonotic) and B. ovis. However, Rev.1 is banned in those regions where B. melitensis is eradicated to avoid interferences in B. melitensis serosurveillance. Consequently, B. ovis is re-emerging in B. melitensis-free countries and a B. ovis specific vaccine not interfering in the smooth lipopolysaccharide (LPS) based tests used to diagnose B. melitensis is needed. In a recent work1, we demonstrated that subcutaneous vaccination of rams with the rough (R) O-polysaccharide (O-PS) mutant H38ΔwbkF confers protection similar to Rev.1 against B. ovis, while not interfering in the Rose Bengal and Complement Fixation tests used for B. melitensis diagnosis. However, since H38ΔwbkF interferes in B. ovis serodiagnosis, we also tested a B. ovis mutant (Bov::CAΔwadB) defective in the LPS-core lateral branch. While Bov::CAΔwadB did not provide protection, it caused low if any interference in the B. ovis agar gel immunodiffusion (AGID) test recommended by WOAH, an observation related to its modified core epitopes. The aim of this work was to explore whether similar LPS-core defects in H38ΔwbkF reduces the interference in B. ovis diagnosis while maintaining its efficacy against B. ovis. We constructed two mutants (H38ΔwbkFΔwadB and H38ΔwbkFΔwadC) carrying the expected core and O-PS defects, as shown by SDS-PAGE and Western-blot. In mice, both mutants showed marked attenuation with respect to H38ΔwbkF and did not protect against B. ovis. Thus, both were discarded for further research in sheep. These results confirmed the important role of the core for Brucella virulence and that over attenuation leads to ineffective vaccines. Therefore, we are exploring the use of the conjunctival route with H38ΔwbkF as a strategy to reduce the persistence of vaccinal antibodies and assessing protective efficacy of H38ΔwbkF by this route in rams