Veterinaria Italiana (Journal)
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Epizootic situation on horse helminthiases in the northern region of Kazakhstan
Get PDFThe research relevance is predefined by the widespread distribution of parasitosis, including helminthiases, alongside constant depasture on the same areas of pastures, which annually causes massive re‑infection of horses. In this regard, the research aims to identify the age, seasonal, and territorial dynamics of infection of horses with helminthiases in the context of the districts of the northern region of Kazakhstan. The primary research method was an experimental analysis that allowing to consider the dynamics and causes of horse helminthiases, associated with the prepatent period, the resistance of the organism, and the system of keeping and feeding horses. The study presents the results of studies of horse helminthiases on the territory of the Northern region of Kazakhstan, covering 4 regions that currently exist in 61 agricultural formations. Faecal samples of 4,395 horses were examined by scatological methods and 126 samples – by incomplete helminthological autopsy. As a result of coprolarvoscopic and coprological studies, it was found that in horses, the dominant representatives among the invasive larvae of strongylates are small and large palisade worms. In scatological studies, strongylatosis of the gastrointestinal tract was recorded in 63.9% of cases, oxyurosis in 40.8%, and parascariasis in 25.9%. The study also presents the results of post‑mortem diagnostics, which was carried out during the slaughter or due to the death of the horse
Biological and molecular genetic properties of the epizootic culture of Streptococcus equi isolated from pathological material of a foal
Get PDFStrangles is one of the main constraining factors in the development of horse breeding. However, the veterinary practice still does not have effective drugs designed for the prevention and treatment of this disease. To date, the development of new effective methods and means for specific prevention and therapy of strangles is of scientific interest and meet the needs of the present day. In this regard, in the summer of 2020, the staff of the microbiology laboratory of the Research Institute of Biological Safety Problems brought samples (pus, exudate of submandibular lymph nodes, a smear of the oral and nasal cavities, a smear of the mucous membrane of the eye) from a foal sick with strangles from the village of Akterek, Jambyl district of Almaty region, and the strain was isolated. The aim of this article is to study molecular biological and genetic properties of Streptococcus equi isolated from pathological material of a foal. Streptococci were isolated by the inoculation of samples of pathological material on GRM-broth (nutrient medium for the cultivation of microorganisms), blood and GRM agar with the addition of 1% glucose and 10% sterile horse serum (pH 7.4-7.6). The results of bacteriological and molecular genetic studies confirmed that the isolate belongs to the species Streptococcus equi
Isolation and identification of Clostridium perfringens and its toxins from mutton in Lahore City, Punjab, Pakistan
Get PDFThe present study was aimed to detect C. perfringens and identify its toxins in mutton samples collected from Lahore City in the Punjab Province of Pakistan. A total of 40 samples of minced and non‑minced mutton were collected from local butcher and retail shops representing four areas of the city. The samples were subjected to ELISA for the detection of C. perfringens alpha, beta and epsilon toxins. The samples were simultaneously processed for bacterial isolation. The isolates were confirmed both by biochemical testing and a multiplex PCR targeting alpha, beta and epsilon toxin genes of C. perfringens. While 10% (4/40) of the samples were positive for C. perfringens alpha toxins, 17.5% (7/40) of the samples were positive for the alpha toxin gene. The present study indicated that the samples collected from the local butcher shops were contaminated with C. perfringens and its toxins. Interestingly, no such contamination was detected in any of the samples collected from retail meat shops. In conclusion, improper hygienic conditions at butcher shops could lead to the contamination of mutton with C. perfringens and its toxins
O5-6 Identification by Transposon-sequencing of essential genes for chronic infection by Brucella in mice
No full textBecause of the importance for public health and damages induced by Brucella (B.) infection in livestock farming, scientists and field workers try to control and eradicate the disease in animals. However, existing vaccines do not meet all the safety criteria for animals. Currently, the only vaccines able to induce a protection to natural domestic animal hosts against brucellosis are live attenuated vaccines (LAVs), as B.abortus S19 and B. melitensis Rev.1. Unfortunately, they have some risks of human contamination, interfere with diagnostic tests and importantly, they can induce abortions in animals. Developing safer vaccines for animals would help to reduce the impact of brucellosis. First generation LAVs relied on empirical and somewhat unpredictable attenuation. Recent advances in genetics make it possible to envisage the rational construction of LAVs. Our objective is to achieve by Transposon Sequencing (Tn-seq) a functional map of the Brucella melitensis genome in order to construct a candidate LAV capable of persisting long enough to induce protective immunity but incapable of persisting in tissues or invading the placenta in pregnant animals. For this purpose, Tn-seq analyses were performed under different conditions in the mouse model. To generate the Tn-seq, we have generated a library of about 106 transpositional mutants. We have identified in mice genes required for the persistence of B. melitensis in the lung after an intranasal inoculation and in the spleen after an intraperitoneal injection. By using these protocols, we avoided the bottleneck which is a limit of the method. Our results show that essential genes for the persistence ofBrucella vary according to the analyzed tissues. Many of these genes are involved in bacterial metabolism, suggesting that Brucella has different nutritional requirements depending on the colonized tissues. Tn-seq predictions were validated by constructing markerless deletion mutants and testing them in mice. On this basis, several candidate vaccines have been selected and are in the validation phase in mice
P8-04 Molecular phylogenetic correlations of Brucella abortus strains isolated from specific regions in South Korea
No full textBovine brucellosis is a re-emerging disease in South Korea with a recent increase in incidence on cattle farms in specific regions. Given this, a molecular phylogenetic investigation is a practical approach to tracking the infection source to reveal the reliable etiological evidence of the incidence. We analyzed phylogenetic characteristics using multi-locus sequence type analysis (MLSA) to prove the genetic correlation ofBrucella abortus strains isolated from two specific regions, Jeonnam (JN) and Gyeongnam (GN). We analyzed 81 B. abortus strains from domestic cattle in nine provinces within these two regions in the last three years using MLSA with 18 specific single nucleotide polymorphisms (SNPs) which could display phylogenetic characteristics from genome sequences verified by draft whole genome sequencing (WGS). Fifty-eight strains isolated from eight JN provinces were divided into two sequence types (STs), either ST3 or ST5. All 23 strains from the one province in the GN region belonged in only ST3. In comparison with 172 internal reference strains isolated from other regions, 49 strains from four (4/8) provinces of JN region and all strains from the GN province were classified as ST3, which accounts for 31.2% of the internal reference strains. Formation of this ST3 cluster in five specific provinces over the three year period indicated a considerable molecular epidemiological relationship among these areas. Accordingly, we found B. abortus strains having the same genotypes distributed in some specific regions that could circulate across certain areas by indigenous strains. Furthermore, we propose that this investigation will be an integral part of providing reliable genetic evidence to support an effective action plan to control disseminating the disease. Work supportedby APQA B-1543081-2021-22-02
O5-3 Screening for vaccinal candidates in Brucella canis: A genomic based strategy for selection of potential DNA target regions.
No full textBrucella canis is transmitted among dogs and from dogs to humans mainly through contact with vaginal secretions, fetus and placenta from infected animals. In Costa Rica, a diversity of outbreaks related to the disease have occurred in commercial kennels, causing significant economic losses as well as awareness from health and animal authorities. To date, no commercial vaccines are available against canine brucellosis. In this work, we used successive passages in different in vitro culture media as a strategy to induce spontaneous mutations in a virulent strain of B.canis. Subsequently, a genomic-based approach was performed to screen for DNA targets that could be potentially selected to assess attenuation. Distinct strains with different number of passages in in vitro culture media were selected and genetically characterized through whole genome sequencing. Genomic analysis revealed no major genetic or structural rearrangements among passages in comparison to the original strain. In addition, we found 8 different Single Nucleotide Polymorphisms (SNPs) between the original strain and strains with different number of passages as potential targets, located in different genes and in intergenic regions. Non reverting deletion mutants of selected target SNPs were derived from virulent B.canis strain and were tested for attenuation in a murine model. Significant differences in virulence profiles were observed between the deletion mutants and the virulent B.canis parental strain. These results contribute on the searching for potential DNA targets for the development of vaccine candidates for canine brucellosis
O2-4 Targeted methodology for the identification of protein-peptide vaccine candidates
No full textEffective vaccines for infectious animal diseases, including zoonotic animal diseases of livestock and poultry, including brucellosis of ruminants, are often not available or not efficacious. This work presents a targeted approach and methodology to identify potential protein and peptide vaccine candidates. This methodology applies to all microbial organisms for which procedures are developed to study membrane proteins in particular. Certain microbial membrane proteins are important receptors of signal proteins of the host’s immune response system for the initiation of a protective immune response or alternatively, some membrane proteins are activated to allow the organism to escape the host’s immune surveillance system. Here we present an approach to directly target the membrane proteins which might afford effective protection. A membrane protein extraction procedure based on previously published procedures by investigators separates three fraction of membrane proteins using TX-100 differential protein extraction procedures. This method results in three types of protein fractions, detergent-soluble proteins, aqueous soluble proteins (including periplasmic proteins), and detergent-insoluble proteins. One and two-dimensional electrophoresis separates (and purifies) these proteins. Western Blots with antibodies from infected and naïve animals as controls are compared and appropriate protein bands or spots are excised, digested with a variety of proteolytic enzymes, and submitted for MALDI-TOF spectrometry. Analysis of the data with SwissProt database programs provides the protein ID and sequence. The identified sequences are then further analyzed by three-dimensional modeling using MolBio and protein external protein loops are identified. Sequences are further studied for loop/sequence homologies and function by the publicly available Resource Center of the Zhang Laboratory which identifies additional potential functionalities of the identified protein(s), providing a better understanding of the immune response regulation. Examples of proteins and peptides identified by this approach are presented
O3-4 A Brucella effector that modulates host retrograde transport to promote intravacuolar replication
No full textBrucella spp. remodel host cellular membrane transport pathways via delivery of Type IV secretion (T4SS) effector proteins to generate a replication-permissive vacuole (rBCV) that supports their intracellular proliferation. Whether Type IV effectors also mediates intracellular growth within the replication vacuole is mostly unknown. Here we show that the T4SS effector BspF is not required for rBCV biogenesis but specifically promotes Brucella replication within rBCVs. Ectopically expressed and bacterially delivered BspF interfered with vesicular transport between the trans-Golgi network (TGN) and recycling endocytic compartment. BspF targeted the recycling endosome, inhibited retrograde traffic to the TGN and interacted with the Arf6 GTPase-activating Protein (GAP) ACAP1 to dysregulate Arf6-/Rab8a-dependent transport within the recycling endosome, which resulted in accretion of TGN-associated vesicles by rBCVs and enhanced bacterial growth. The predicted structure of BspF and interaction with ACAP1 identified residues within its Gcn5-related acetyltransferase (GNAT) domain required for BspF’s modulation of Arf6 activity through ACAP1, the resulting interference with retrograde transport and BspF’s role in bacterial intracellular growth. Altogether, these findings provide mechanistic insight into Brucella modulation of membrane transport that promotes their own proliferation within intracellular vacuole
O1-2 East African Brucella phylogenomics: introducing OrthoPhylo, A turn-key tool for generating ortholog based bacterial whole genome phylogenetic trees
No full textThe number of publicly available bacterial genome sequences is staggering (1.1milion assemblies in NCBI alone) and the rate of deposition is only increasing, with Brucella sequences being no exception. This wealth of data is juxtaposed with the lack of phylogenetic methods to robustly place these sequences within an evolutionary context. Not only does a phylogenetic placement aid in taxonomic classification, but also informs the evolution of novel phenotypes (host switching), targets of selection (host immune evasion mechanisms), and horizontal gene transfer (AMR/virulence genes). Methods fo reconstructing trees include comparing 16S ribosomal or other single loci, multi locus, whole genome alignments and Kmer based SNP analysis. All of these methods suffer from narrow taxonomic resolution, with 16S working well for higher taxon divisions and whole genome alignments working well for closely related samples. Here I present OrthoPhylo, a phylogenetic pipeline that takes bacterial genomes, annotates them and identifies orthologs, converts protein to nucleotide alignments, then builds species trees with both concatenated alignments and gene tree to species tree methods. The workflow has been designed to accept large numbers of input genomes (>1000) by identifying samples that represent the diversity of the whole dataset, and using these genomes to build models and identify orthologs. This strategy allows the generation of trees for ~1000 bacterial assemblies in ~30hrs using 30 cpus, with the majority of this time being taken up by ML based tree generation. This pipeline is designed to be an easy to install, turn-key solution for generating high resolution bacterial trees from species that can differ by more than 30% nucleotide identity. Here I present findingson the state of publicly available Brucella assemblies and their phylogenetic placements with a focus on sequences from East Africa, an understudied region of endemic Brucella infections. This effort is a part of a recently initiated five-year cross-sectional survey to assess risk factors associated with brucellosis in the East African countries of Tanzania and Rwanda
O9-1 Isolation of Brucella suis biovar 2 in a male roe deer (capreolus caprolus), in central italy
No full textIn May 2022, a moribund male roe deer (Capreolus capreolus) was rescued near Spoleto (Umbria region, Italy) and admitted to a Wildlife Rescue Center. On clinical examination, the subject, in older age, showed severe cachexia, disorientation, motor difficulties and marked increase in volume of the scrotal sac. The animal died few hours after admission. Anatomopathological examination and subsequent diagnostic tests were performed by IZSUM. Post mortem external examination showed ticks infestation, poor body condition score and a dorsal abscess of approximately 10 cm diameter. The animal showed reddened lungs and severe renal congestion, suggesting a stress myopathy as cause of death. The left scrotum appeared significantly increased in volume (10×6 cm) and, on cut surface, it showed areas of fibrosis with caseous-purulent orchitis in a well- organized abscess. The right one was slightly smaller in size (6×4 cm) and showed purulent epididymitis and orchitis. Serum was subjected to Rose Bengal test (RBT) and complement fixation test (CFT) for Brucellosis according to WOAH procedure. Spleen, kidney, liver, testes and testicular lymph nodes were taken for molecular (Fast qPCR) and bacteriologica investigations, while histology was performed also on epididymis and ascellar lymph nodes. RBT and CFT were both positive. CFT title was ≥ 1702,4 IU/ml. PCR resulted positive for Brucella spp.. WOAH procedures for Brucella isolation were performed and after one week of incubation, Brucella spp. strain was isolated from testis samples. The National Reference Laboratory for Brucellosis, at the IZS in Teramo, confirmed the presence of Brucella spp. and typing the isolated strains as Brucella suis serovar 2. Brucella suis can infect domestic and wild species. Brucella suis biovar 2 is the main strain responsible for brucellosis in wild boars in Italy and in Europe. This is the first case of Brucella suis isolation from a roe deer in Italy. Brucellosis in this roe deer might have been a consequence of transmission between the wild boar population and wild ruminants. In-depth epidemiological investigations of the affected territory will be necessary to understand the dynamics of the infection in wildlife and its potential spread to extensively farmed domestic species