Biovar Ovis Research Articles

Isolation and Molecular Characterization of Corynebacterium pseudotuberculosis from Goats in Andaman and Nicobar Islands, India

Caseous lymphadenitis (CLA), caused by the bacteria Corynebacterium pseudotuberculosis, is a highly contagious disease of small ruminants, especially of goats and sheep. Here, we report an outbreak of the disease in goats for the first time from the Andaman and Nicobar Islands along with isolation and molecular characterization of the pathogen. A total of 22 goats were affected, with an attack rate of 12.02%, and six isolates were identified from the clinical samples. Molecular characterization of the pathogen was carried out based on the sequence information of 16S rRNA and RNA polymerase β subunit (rpoB) gene fragments. rpoB-based phylogenetic analysis indicated that the isolates belonged to Corynebacterium pseudotuberculosis biovar ovis. The antimicrobial resistance study revealed that the isolates were 100% resistant against erythromycin and rifampicin. Fifty percent resistance was found against amoxicillin/clavulanic acid, ciprofloxacin, penicillin, and vancomycin. All the isolates were sensitive to tetracycline, chloramphenicol, cotrimoxazole, sulphafurazole, ampicillin/cloxacillin, and oxytetracycline. In conclusion, the present study reports the occurrence of CLA in goats for the first time from an isolated archipelago of India and unveils the molecular signature and antibiotic resistance patten of the pathogen. The findings of this study will be helpful to control or eradicate the disease from the Andaman and Nicobar Islands.

Corynebacterium pseudotuberculosis: Whole genome sequencing reveals unforeseen and relevant genetic diversity in this pathogen.

Corynebacterium pseudotuberculosis (CPS) is an important bacterial animal pathogen. CPS causes chronic, debilitating and currently incurable infectious diseases affecting a wide range of livestock and wild herbivores including camelids worldwide. Belonging to the Corynebacterium diphtheriae complex, this pathogen can also infect humans. The classical characterization of CPS is typically based on the testing of nitrate reductase activity, separating the two biovars Equi and Ovis. However, more refined resolutions are required to unravel routes of infection. This was realized in our study by generating and analyzing whole genome sequencing (WGS) data. Using newly created core genome multilocus sequence typing (cgMLST) profiles we were the first to discover isolates grouping in a cluster adjacent to clusters formed by CPS biovar Equi isolates. This novel cluster includes CPS isolates from alpacas, llamas, camels and dromedaries, which are characterized by a lack of nitrate reductase activity as encountered in biovar Ovis. This is of special interest for molecular epidemiology. Nevertheless, these isolates bear the genes of the nitrate locus, which are characteristic of biovar Equi isolates. However, sequence analysis of the genes narG and narH of the nitrate locus revealed indels leading to frameshifts and inactivity of the enzymes involved in nitrate reduction. Interestingly, one CPS isolate originating from another lama with an insertion in the MFS transporter (narT) is adjacent to a cluster formed by ovine CPS isolates biovar Equi. Based on this knowledge, the combination of biochemical and PCR based molecular biological nitrate reductase detection can be used for a fast and uncomplicated classification of isolates in routine diagnostics in order to check the origin of camelid CPS isolates. Further analysis revealed that partial sequencing of the ABC transporter substrate binding protein (CP258_RS07935) is a powerful tool to assign the biovars and the novel genomovar.

Ovine and Caprine Strains of Corynebacterium pseudotuberculosis on Czech Farms-A Comparative Study.

Caseous lymphadenitis (CLA) is a worldwide disease of small ruminants caused by Corynebacterium pseudotuberculosis, a facultative intracellular pathogen that is able to survive and multiply in certain white blood cells of the host. In this study, 33 strains of C. pseudotuberculosis were isolated from sheep and goats suffering from CLA on nine farms in the Czech Republic. All these strains were tested for their antibiotic susceptibility, ability to form a biofilm and resistance to the effects of commonly used disinfectant agents. To better understand the virulence of C. pseudotuberculosis, the genomes of strains were sequenced and comparative genomic analysis was performed with another 123 genomes of the same species, including ovis and equi biovars, downloaded from the NCBI. The genetic determinants for the virulence factors responsible for adherence and virulence factors specialized for iron uptake and exotoxin phospholipase D were revealed in every analyzed genome. Carbohydrate-Active Enzymes were compared, revealing the presence of genetic determinants encoding exo-α-sialidase (GH33) and the CP40 protein in most of the analyzed genomes. Thirty-three Czech strains of C. pseudotuberculosis were identified as the biovar ovis on the basis of comparative genome analysis. All the compared genomes of the biovar ovis strains were highly similar regardless of their country of origin or host, reflecting their clonal behavior.

Corynebacterium pseudotuberculosis biovar ovis strains isolated from small ruminants herds from the Brazilian Amazon present clonal genomic profile

This study tested the hypothesis that Corynebacterium pseudotuberulosis strains isolated from small herds of ruminants in the state of Pará have a clonal genomic profile. Serum samples were collected from 169 animals and used in an indirect ELISA that detected specific antibodies against C. pseudotuberculsis in 24% of the animals monitored in the field. Among sheep and goats, 20 animals had a confirmed symptomatic diagnosis of Caseous Lymphadenitis Disease (CL) and caseous material from the abscesses was sampled. From these samples, seven bacterial strains were isolated and identified as C. pseudotuberculosis biovar ovis. Bacterial genomic DNA was extracted and subjected to fingerprint analysis (BOX-PCR and ERIC-PCR). The fingerprint analysis revealed the existence of two main groups of isolated strains, with one of these groups necessarily formed by strains A27 and A31 (at least 62% similarity), and the other group formed by the remaining isolated strains. The genome outline of the A27 and A31 strains was sequenced using Illumina NovaSeq 6000 technology. The preliminary genomes A27 and A31 grouped with other genomes of C. pseudotuberculosis biovar ovis when compared to 46 genomes of C. pseudotuberculosis belonging to both biovars (ovis and equi) isolated worldwide. More than 99% of similarity was observed when comparing the draft genomes A27 and A31 with other genomes of C. pseudotuberculosis biovar ovis strains isolated from the Brazilian Amazon. The mapping readouts generated with preliminary A27 and A31 genomes using the reference genome of C. pseudotuberculosis 42/02-A showed more than 99% coverage. The central genome consisted of 78% of the orthogroups in the pangenome analysis. The specific genes that encode unique proteins in the A27 and A31 genomes yield molecular information about how each species adapts to its ecological niches. This study shows that strains of C. pseudotuberculosis biovar ovis isolated from small ruminant herds in the Brazilian Amazon showed a clonal genomic profile.

Bioinformatic Approach of B and T Cell Epitopes of PLD and CP40 Proteins of Corynebacterium pseudotuberculosis ovis Mexican Isolate 2J-L towards a Peptide-Based Vaccine.

Mapping B and T cell epitopes constitutes an important action for peptide vaccine design. PLD and CP40 virulence factors of Corynebacterium pseudotuberculosis biovar ovis, a causal agent of Caseous Lymphadenitis, have been evaluated in a murine model as good candidates for vaccine development. Therefore, the goal of this work was to in silico analyze B and T cell epitopes of the PLD and CP40 proteins of a Mexican isolate of Corynebacterium pseudotuberculosis ovis. The Immune Epitope Data Base and Resource website was employed to predict the linear and conformational B-cell, T CD4+, and T CD8+ epitopes of PLD and CP40 proteins of Corynebacterium pseudotuberculosis ovis Mexican strain 2J-L. Fifty B cell epitopes for PLD 2J-L and forty-seven for CP40 2J-L were estimated. In addition, T CD4+ and CD8+ cell epitopes were predicted for PLD 2J-L (MHC I:16 epitopes, MHC II:10 epitopes) and CP40 2J-L (MHC I: 15 epitopes, MHC II: 13 epitopes). This study provides epitopes, paying particular attention to sequences selected by different predictor programs and overlap sequences as B and T cell epitopes. PLD 2J-L and CP40 2J-L protein epitopes may aid in the design of a promising peptide-based vaccine against Caseous Lymphadenitis in Mexico.

Pilot study on the distribution of caseous lymphadenitis in Korean native goats and the relationship between sex and age in disease occurrence

Caseous lymphadenitis (CLA) caused by Corynebacterium pseudotuberculosis is a chronic disease that affects goats. The Korean native goat (KNG) is the most popular goat breed raised in Korea. In this study, the distribution of CLA in the KNG population and the relevance of sex and age in disease development were determined. From March 2020 to February 2021, 1,177 KNGs from 110 farms were assessed. The distribution of CLA in animals was determined by a veterinary inspector who performed gross examinations of goat carcasses and confirmed diagnoses via polymerase chain reaction and bacteriological examination. The CLA detection rate in the KNG population was 19.80%, and more than half of the farms were affected by the disease (56.36%). A statistically significant difference was observed among the sex groups, with castrated males (13.98%) having the lowest detection rate, followed by intact males (22.48%) and females (24.09%), suggesting that castration has a positive effect on reducing the risk of CLA (p < 0.05). In terms of age groups, an increased detection rate of 28.16% was observed in the late adult (>2 years) group. Phylogenetic analysis indicated that the RNA polymerase beta subunit-encoding gene can effectively differentiate biovar ovis from biovar equi and can be used for further epidemiological studies of C. pseudotuberculosis. This is the first nationwide surveillance study of CLA distribution to confirm the continuous occurrence of CLA in Korean goat farms. Future studies should include risk factor analyses of CLA based on herd levels to prevent and control this disease in Korea.

Molecular Characterization and Antimicrobial Susceptibilities of Corynebacterium pseudotuberculosis Isolated from Caseous Lymphadenitis of Smallholder Sheep and Goats.

Caseous lymphadenitis (CLA) is a bacterial infection caused by Corynebacterium pseudotuberculosis (C. pseudotuberculosis) that affects sheep and goats, leading to abscess formation in their lymph nodes. The present study aimed to isolate and identify C. pseudotuberculosis from CLA in smallholder sheep and goats, and determine the resistance patterns, virulence, and resistance genes of the isolates. Additionally, genotypic and phylogenetic analysis of the isolates was conducted using ERIC-PCR and DNA sequencing techniques. A cross-sectional study examined 220 animals (130 sheep and 90 goats) from 39 smallholder flocks for clinical signs of CLA. Fifty-four (24.54%) animals showed CLA-compatible lesions, confirmed by C. pseudotuberculosis isolation and PCR identification. Sheep had a lower infection rate of CLA (18.46%) compared with goats (33.3%). Antimicrobial susceptibility testing of 54 C. pseudotuberculosis isolates to 24 antimicrobial drugs revealed that they were 100% resistant to bacitracin and florfenicol, while none of the isolates were resistant to norfloxacin. A high resistance rate was observed for penicillin and erythromycin (92.6% each). Interestingly, 16.7% of C. pseudotuberculosis isolates recovered from sheep showed vancomycin resistance. Molecular characterization of C. pseudotuberculosis isolates revealed that PLD, PIP, and FagA virulence genes were present in all examined isolates. However, the FagB, FagC, and FagD genes were detected in 24 (100%), 20 (83%), and 18 (75%) of the sheep isolates, and 26 (87%), 26 (87%), and 18 (60%) of the goat isolates, respectively. The β-lactam resistance gene was present in all isolates. Furthermore, 83% of the sheep isolates carried the aminoglycoside (aph(3″)-lb), chloramphenicol (cat1), and bacitracin (bcrA) resistance genes. Among the isolates recovered from goats, 73% were found to contain macrolides (ermX), sulfonamide (sul1), and bacitracin (bcrA) resistance genes. It is worrisome that the glycopeptide (vanA) resistance gene was detected in 8% of the sheep isolates as a first report. ERIC-PCR genotyping of 10 multi-drug-resistant C. pseudotuberculosis isolates showed a high similarity index of 83.6% between isolates from sheep and goats. Nucleotide sequence analysis of partial 16S rRNA sequences of C. pseudotuberculosis revealed 98.83% similarity with biovar Ovis of globally available reference sequences on the Genbank database. Overall, our findings might indicate that C. pseudotuberculosis infection in smallholders in Egypt might be underestimated despite the significant financial impact on animal husbandry and potential health hazards it poses. Moreover, this study highlights the importance of implementing a sustainable control strategy and increasing knowledge and awareness among smallholder breeders to mitigate the economic impact of CLA.

Molecular characterization and antimicrobial susceptibility of Corynebacterium pseudotuberculosis isolated from skin abscesses of native Korean goats (Capra hircus coreanae).

This study aimed to investigate the molecular characterization and antimicrobial susceptibility of Corynebacterium pseudotuberculosis from skin abscesses of Korean native black goats (KNBG, Capra hircus coreanae) in South Korea. A total of 83 isolates were recovered from skin abscesses of KNBG. Of these isolates, 74 isolates were identified as C. pseudotuberculosis by phospholipase D (PLD) gene-based PCR assay. Each of the isolates possessed all 18 virulence genes (FagA, FagB, FagC, FagD, SigE, SpaC, SodC, PknG, NanH, OppA, OppB, OppC, OppD, OppF, CopC, NrdH and CpaE). The genetic diversity of C. pseudotuberculosis isolates was assessed by the phylogenetic analysis using the concatenated sequences (3073 bp) of five housekeeping genes (fusA, dnaK, infB, groeL1 and leuA) for investigating their genetic diversity. In the results, the isolates belonged to three groups: group 1 (67 isolates), group 2 (one isolate) and group 3 (six isolates) within biovar ovis. However, the groups exhibited low genetic diversity (0.20%-0.41%). In the antimicrobial susceptibility test, most isolates were susceptible to tetracycline, vancomycin, chloramphenicol, ciprofloxacin, erythromycin, enrofloxacin, cefoxitin, ampicillin, gentamycin, cephalothin and doxycycline, whereas they were not susceptible to cefotaxime, trimethoprim and streptomycin. This results suggest the involvement of relatively few clones of C. pseudotuberculosis in Korea. Further, present isolates can threaten public health due to potentially virulent strains with all 18 virulence genes and non-susceptible strains to clinically important antibiotics (CIA) and highly important antibiotics. This study is the first to investigate the genetic diversity and potential pathogenicity of C. pseudotuberculosis biovar ovis isolates from skin abscesses of KBNG in South Korea, and could provide useful information in controlling its infections.

Caracterização molecular de Corynebacterium pseudotuberculosis, C. silvaticum e C. auriscanis pelo ERIC-PCR

ABSTRACT: The aims of the present study were (i) to genotype Corynebacterium pseudotuberculosis, C. silvaticum, and C. auriscanis strains using enterobacterial repetitive intergenic consensus (ERIC-PCR), and (ii) to analyze the epidemiological relationships among isolates according to biovar (Equi and Ovis), species, host, and geographical origin of the C. pseudotuberculosis strains. Sixty-eight C. pseudotuberculosis, nine C. silvaticum, and one C. auriscanis, C. pseudotuberculosis ATCC® 19410™ strain and the attenuated C. pseudotuberculosis 1002 vaccinal strain were fingerprinted by ERIC 1+2-PCR. Field strains were isolated from various hosts (cattle, buffaloes, sheep, goats, horses, dogs, and pigs) in six countries (Mexico, Portugal, Brazil, Equatorial Guinea, Egypt, and Israel). High genetic diversity was found among the studied Corynebacterium spp. isolates, clustering in 24 genotypes with a Hunter & Gaston diversity index (HGDI) of 0.937. The minimal spanning tree of Corynebacterium spp. revealed three clonal complexes, each associated with one bacterial species. Twenty-two genotypes were observed among C. pseudotuberculosis isolates, with an HGDI of 0.934. Three major clonal complexes were formed at the minimal spanning tree, grouped around the geographic origin of C. pseudotuberculosis isolates. These results reinforce the high typeability, epidemiological concordance, and discriminatory power of ERIC-PCR as a consistent genotyping method for C. pseudotuberculosis, which could be useful as an epidemiological tool to control caseous lymphadenitis. Moreover, our results also indicate the potential of ERIC 1+2-PCR for the genotyping of other species of Corynebacterium other than C. pseudotuberculosis.

Molecular Characterization of Corynebacterium pseudotuberculosis Isolated over a 15-Year Period in Switzerland.

Corynebacterium pseudotuberculosis biovar Ovis is the etiological agent of the contagious and chronic disease caseous lymphadenitis (CLA) in sheep and goats. The economic impact of CLA in Switzerland remains largely unknown, and the transmission modalities, as well as the genetic diversity of circulating strains, are poorly understood. This work presents further characterization data for 215 C. pseudotuberculosis isolates from sheep, goats and a dromedary originating from Switzerland and the Principality of Liechtenstein, collected over a 15-year period. The isolates were classified into the two biovars Ovis and Equi, analyzed for the presence of the diphtheria-like toxin gene and characterized using MLSA. All sheep and goat isolates were classified as C. pseudotuberculosis biovar Ovis. The isolate from a dromedary was classified as biovar Equi. No isolates harboring the diphtheria-like toxin gene were detected. Phylogenetic analysis of the concatenated sequences of four genes revealed the existence of 24 clusters. There was no correlation between MLSA sequence types, year of isolation and the geographical origin of the isolates. These findings confirm the presence of several MLSA sequence types in the study area and over a 15-year period. Moreover, no sheep- and goat-specific MLSA sequence types were found.

Genetic Characterization of Corynebacterium pseudotuberculosis Isolates in Egypt

Corynebacterium pseudotuberculosis is a small Gram-positive bacillus containing mycolic acid in the structure of the cell wall. The bacterium is responsible for Caseous Lymphadenitis (CLA) in small ruminants (sheep and goats). The bacteria are also responsible for Ulcerative Lymphangitis in equines. The disease causes great economic loss in the animal industry. This work aimed to check the ability of Quadruplex PCR (Q-PCR) for genotyping and identification of Egyptian isolates of C. pseudotuberculosis and sequence analysis of phospholipase D (PLD) gene of local isolates. Four of C. pseudotuberculosis local isolates previously biochemically identified were tested for narG gene (nitrate reductase gene). Both nitrate negative biovar (ovis) and nitrate positive biovar (equi) showed a positive result for 16S rRNA, rpoB and PLD genes of C. pseudotuberculosis species. The sequence analysis of our local isolates' PLD gene revealed minor changes in PLD proteins between ovine and equine strains compared with other published PLD sequences in GenBank. It was concluded that the Q-PCR method is able to differentiate between C. pseudotuberculosis equi and ovis biovars. Also, the sequence of PLD gene of local isolates representing the two biovars revealed some variation, which leads to an accurate diagnosis of C. pseudotuberculosis biovars and generates a mapping of immerged local isolates. Further, the PCR and sequence of these isolates provide rapid and accurate genotyping, especially with hyperimmune serum's unavailability.

An Outbreak Of Caseous Lymphadenitis (Pseudotuberculosis) In Dromedary Camels At Qassim Region, Saudi Arabia

This study was carried out to investigate an outbreak of lymphadenitis in dromedary camels in private farm at Qassim region, Central of Saudi Arabia. Out of 220 camels included in this study, lymphadenitis was observed in 42 camels representing morbidity rate of 19.09%. The morbidity rate did not differ significantly among different age groups (p < 0.4 and Odds Ratio = 0.7) or between different sex (p < 0.1 and Odds Ratio = 0.5). Clinically, infected camels showed enlargement and abscessation of superficial lymph nodes, emaciation in some cases with normal body temperature. Corynebacterium pseudotuberculosis biovar ovis was the only microorganism isolated from the pus. Haematological examination revealed significant decrease in red blood cells and packed cell volume in addition to significant increase in the total white blood cells and neutrophils in lymphadenitis-infected camels compared to healthy ones. Penicillin therapy and surgical intervention in addition to some control measures as isolation of healthy camels away from infected herd and thorough disinfection of the contaminated environment were effective measures in the control of the outbreak.

In silico functional prediction of hypothetical proteins from the core genome of Corynebacterium pseudotuberculosis biovar ovis.

Corynebacterium pseudotuberculosis is a pathogen of veterinary relevance diseases, being divided into two biovars: equi and ovis; causing ulcerative lymphangitis and caseous lymphadenitis, respectively. The isolation and sequencing of C. pseudotuberculosis biovar ovis strains in the Northern and Northeastern regions of Brazil exhibited the emergence of this pathogen, which causes economic losses to small ruminant producers, and condemnation of carcasses and skins of animals. Through the pan-genomic approach, it is possible to determine and analyze genes that are shared by all strains of a species—the core genome. However, many of these genes do not have any predicted function, being characterized as hypothetical proteins (HP). In this study, we considered 32 C. pseudotuberculosis biovar ovis genomes for the pan-genomic analysis, where were identified 172 HP present in a core genome composed by 1255 genes. We are able to functionally annotate 80 sequences previously characterized as HP through the identification of structural features as conserved domains and families. Furthermore, we analyzed the physicochemical properties, subcellular localization and molecular function. Additionally, through RNA-seq data, we investigated the differential gene expression of the annotated HP. Genes inserted in pathogenicity islands had their virulence potential evaluated. Also, we have analyzed the existence of functional associations for their products based on protein–protein interaction networks, and perform the structural prediction of three targets. Due to the integration of different strategies, this study can underlie deeper in vitro researches in the characterization of these HP and the search for new solutions for combat this pathogen.

A comparative pan-genomic analysis of 53 C. pseudotuberculosis strains based on functional domains

Corynebacterium pseudotuberculosis is a pathogenic bacterium with great veterinary and economic importance. It is classified into two biovars: ovis, nitrate-negative, that causes lymphadenitis in small ruminants and equi, nitrate-positive, causing ulcerative lymphangitis in equines. With the explosive growth of available genomes of several strains, pan-genome analysis has opened new opportunities for understanding the dynamics and evolution of C. pseudotuberculosis. However, few pan-genomic studies have compared biovars equi and ovis. Such studies have considered a reduced number of strains and compared entire genomes. Here we conducted an original pan-genome analysis based on protein sequences and their functional domains. We considered 53 C. pseudotuberculosis strains from both biovars isolated from different hosts and countries. We have analysed conserved domains, common domains more frequently found in each biovar and biovar-specific (unique) domains. Our results demonstrated that biovar equi is more variable; there is a significant difference in the number of proteins per strains, probably indicating the occurrence of more gene loss/gain events. Moreover, strains of biovar equi presented a higher number of biovar-specific domains, 77 against only eight in biovar ovis, most of them are associated with virulence mechanisms. With this domain analysis, we have identified functional differences among strains of biovars ovis and equi that could be related to niche-adaptation and probably help to better understanding mechanisms of virulence and pathogenesis. The distribution patterns of functional domains identified in this work might have impacts on bacterial physiology and lifestyle, encouraging the development of new diagnoses, vaccines, and treatments for C. pseudotuberculosis diseases. Communicated by Ramaswamy H. Sarma

Corynebacterium pseudotuberculosis biovar ovis: evaluación de la sensibilidad antibiótica in vitro

The aims of this work were to study the antibiotic susceptibility in Corynebacterium pseudotuberculosis isolated from small ruminants and to determine the presence of integrons that contain resistance genes. Fifteen isolates of different sources were analysed using the diffusion and the dilution methods. When the diffusion method was performed, amoxicillin-clavulanic, ampicillin, cefotaxime, cefoxitin, ciprofloxacin, chloramphenicol, erythromycin, streptomycin, gentamicin, imipenem, kanamycin, norfloxacin, penicillin, rifampicin, tetracycline, trimethoprim-sulfamethoxazole and vancomycin were effective against the 100% of isolates, while amikacin showed variable results. The isolates that were able to grow with amikacin, were studied in relation to the presence of integron class1. The result was negative, suggesting the absence of integron. Using dilution method, the antibiotics belonging to the cephalosporin, glycopeptide, macrolide, quinolone, and tetracycline groups were the most active ones for the C.pseudotuberculosis biovar ovis isolates. Less activity of β-lactam and aminoglycosides were observed. There was no observation of variability in the antibiotic patterns in the strains coming from different sources.

Transcriptome analysis of Corynebacterium pseudotuberculosis biovar Equi in two conditions of the environmental stress

Corynebacterium pseudotuberculosis has been widely studied in an effort to understand its biological evolution. Transcriptomics has revealed possible candidates for virulence and pathogenicity factors of strain 1002 (biovar Ovis). Because C. pseudotuberculosis is classified into two biovars, Ovis and Equi, it was interesting to assess the transcriptional profile of biovar Equi strain 258, the causative agent of ulcerative lymphangitis. The genome of this strain was re-sequenced; the reassembly was completed using optical mapping technology, and the sequence was subsequently re-annotated. Two growth conditions that occur during the host infection process were simulated for the transcriptome: the osmotic and acid medium. Genes that may be associated with the microorganism's resilience under unfavorable conditions were identified through RNAseq, including genes present in pathogenicity islands. The RT-qPCR was performed to confirm the results in biological triplicate for each condition for some genes. The results extend our knowledge of the factors associated with the spread and persistence of C. pseudotuberculosis during the infection process and suggest possible avenues for studies related to the development of vaccines, diagnosis, and therapies that might help minimize damage to agribusinesses.

Investigation of an outbreak of caseous lymphadenitis in goats

Caseous lymphadenitis (CLA) is a chronic and sub-clinical disease of sheep and goats with high prevalence in domestic animals. It is caused by Corynebacterium pseudotuberculosis biovar ovis. An outbreak of CLA in superficial and visceral forms was observed in Barbari and Jakhrana goat herds maintained at ICAR-CIRG, Makhdoom (UP) in July, 2016. A total of 147 goats was affected by the infection of external lymph nodes and two cases in Barbari goats showed internal abscess on necropsy. Biopsy pus samples were collected and identified the organism after Gram's staining. All biopsy samples were inoculated to blood agar and subjected to biochemical (catalase, oxidase and nitrate) tests. Isolation of DNAfrom culture and 16S rRNAgene-based PCR for C. ovis was carried out for confirmatory diagnosis. Gross and histopathological studies were conducted in cases of internal abscess. Antibiotic sensitivity tests were conducted for all the isolates obtained during the study. External lymph nodes were predominantly affected involving pre-scapular and post-scapular (94), pre-femoral (44) and mandibular lymph nodes (9). After incising abscesses, white thick pus oozed out. In Grams ’stained smears, the organism was pleomorphic rods arranged in Chinese letters. On blood agar, round, raised, greyish colonies were observed and biochemical examination showed catalase positive and oxidase negative. The 16S rRNA genebased PCR confirmed the presence of C. ovis. In two cases, visceral abscess was observed with oozing pus from affected lungs. Histopathology of affected lung tissue showed infiltration of inflammatory cells including neutrophils, lymphocytes and macrophages within infected tissue and concentric fibrous layers separated by inspissated caseous exudate. Based on morphology, biochemical tests and PCR, causal organism was confirmed as C. ovis and based on 16 antibiotics tested for sensitivity, ciprofloxacin was the only antibiotic found to be effective against isolates.

Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi

BackgroundCorynebacterium pseudotuberculosis is classified into two biovars, nitrate-negative biovar Ovis which is the etiologic agent of caseous lymphadenitis in small ruminants and nitrate-positive biovar Equi, which causes abscesses and ulcerative lymphangitis in equines. The aim of this study was to develop a quadruplex PCR assay that would allow simultaneous detection and biovar-typing of C. pseudotuberculosis.MethodsIn the present study, genomes of C. pseudotuberculosis strains were used to identify the genes involved in the nitrate reduction pathway to improve a species identification three-primer multiplex PCR assay. The nitrate reductase gene (narG) was included in the PCR assay along with the 16S, rpoB and pld genes to enhance the diagnosis of the multiplex PCR at biovar level.ResultsA novel quadruplex PCR assay for C. pseudotuberculosis species and biovar identification was developed. The results of the quadruplex PCR of 348 strains, 346 previously well-characterized clinical isolates of C. pseudotuberculosis from different hosts (goats, sheep, horse, cattle, buffalo, llamas and humans), the vaccine strain 1002 and the type strain ATCC 19410T, were compared to the results of nitrate reductase identification by biochemical test. The McNemar’s Chi-squared test used to compare the two methods used for C. pseudotuberculosis biovar identification showed no significant difference (P = 0.75) [95% CI for odds ratio (0.16–6.14)] between the quadruplex PCR and the nitrate biochemical test. Concordant results were observed for 97.13% (338 / 348) of the tested strains and the kappa value was 0.94 [95% CI (0.90–0.98)].ConclusionsThe ability of the quadruplex assay to discriminate between C. pseudotuberculosis biovar Ovis and Equi strains enhances its usefulness in the clinical microbiology laboratory.

Label-free quantitative proteomics of Corynebacterium pseudotuberculosis isolates reveals differences between Biovars ovis and equi strains

Corynebacterium pseudotuberculosis is a pathogen classified into two biovars: C. pseudotuberculosis biovar ovis, the etiologic agent of caseous lymphadenitis and C. pseudotuberculosis biovar equi, which causes ulcerative lymphangitis. The available whole genome sequences of different C. pseudotuberculosis strains have enabled identify difference of genes related both virulence and physiology of each biovar. To evaluate be this difference could reflect at proteomic level and to better understand the shared factors and the exclusive ones of biovar ovis and biovar equi strains, we applied the label-free quantitative proteomic to characterize the proteome of the strains: 1002_ovis and 258_equi, isolated from goat (Brazil) and equine (Belgium), respectively. From this analysis, we characterized a total of 1230 proteins in 1002_ovis and 1220 in 258_equi with high confidence. Moreover, the core-proteome between 1002_ovis and 258_equi obtained here is composed of 1122 proteins involved in different cellular processes, which could be necessary for the free living of C. pseudotuberculosis. In addition, 120 proteins from this core-proteome presented change in abundant with statistically significant differences. Considering the exclusive proteome, we detected strain-specific proteins to each strain. When correlated, the exclusive proteome of each strain and proteome with change in abundant, the proteomic differences, between the 1002_ovis and 258_equi, this related to proteins involved in cellular metabolism, information storage and processing, cellular processes and signaling. This study reports the first comparative proteomic study of the biovars ovis and equi of C. pseudotuberculosis. The results generated in this study provide information about factors which can contribute to understanding both the physiology and the virulence of this pathogen.

Draft Genome Sequence of Corynebacterium pseudotuberculosis Strain PA07 Biovar ovis, Isolated from a Sheep Udder in Amazonia.

ABSTRACTIn this work, we present the draft genome sequence of Corynebacterium pseudotuberculosis strain PA07 biovar ovis, isolated from a caseous secretion from a sheep udder in Pará, Brazil. The genome contains 2,320,235 bp, 52.2% G+C content, 2,191 coding sequences (CDSs), five pseudogenes, 48 tRNAs, and three rRNAs.