Ovis Strains Research Articles

Pangenomic and biochemical analyses of Helcococcus ovis reveal widespread tetracycline resistance and a novel bacterial species, Helcococcus bovis.

Helcococcus ovis (H. ovis) is an opportunistic bacterial pathogen of a wide range of animal hosts including domestic ruminants, swine, avians, and humans. In this study, we sequenced the genomes of 35 Helcococcus sp. clinical isolates from the uterus of dairy cows and explored their antimicrobial resistance and biochemical phenotypes in vitro. Phylogenetic and average nucleotide identity analyses classified four Helcococcus isolates within a cryptic clade representing an undescribed species, for which we propose the name Helcococcus bovis sp. nov. By establishing this new species clade, we also resolve the longstanding question of the classification of the Tongji strain responsible for a confirmed human conjunctival infection. This strain did not neatly fit into H. ovis and is instead a member of H. bovis. We applied whole genome comparative analyses to explore the pangenome, resistome, virulome, and taxonomic diversity of the remaining 31 H. ovis isolates. An overwhelming 97% of H. ovis strains (30 out of 31) harbor mobile tetracycline resistance genes and displayed significantly increased minimum inhibitory concentrations of tetracyclines in vitro. The high prevalence of mobile tetracycline resistance genes makes H. ovis a significant antimicrobial resistance gene reservoir in our food chain. Finally, the phylogenetic distribution of co-occurring high-virulence determinant genes of H. ovis across unlinked and distant loci highlights an instance of convergent gene loss in the species. In summary, this study showed that mobile genetic element-mediated tetracycline resistance is widespread in H. ovis, and that there is evidence of co-occurring virulence factors across clades suggesting convergent gene loss in the species. Finally, we introduced a novel Helcococcus species closely related to H. ovis, called H. bovis sp. nov., which has been reported to cause infection in humans.

Molecular detection and characterization of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in sheep and goats in Luxor, Egypt

BackgroundTick-borne diseases cause economically significant losses to animal production globally, and anaplasmosis and theileriosis are associated with the greatest losses. However, the spread of the relevant pathogens in flocks of domesticated animals in southern Egypt is little understood. Accordingly, in this study, we aimed to determine the prevalences of Anaplasma ovis, Theileria ovis, and Theileria lestoquardi in southern Egyptian sheep and goats through blood tests, and to make a molecular characterization of the A. ovis detected in sheep targeting a specific gene.ResultsWe collected blood samples collected from 300 sheep and goats (n=150 /species) in Luxor Province in southern Egypt, and analyzed them for the presence of A. ovis, T. ovis and T. lestoquardi with screening by conventional and nested PCR targeting the msp4 and msp5, 18S rRNA, and merozoite surface protein genes. For A. ovis 140/300 samples (46.66%) were positive overall, with 90/150 (60%) and 50/150 (33.33%) positive samples in sheep and goats, respectively. Two major surface protein genes of A. ovis, msp4 and msp5, were sequenced using DNA extracted from sheep and goat blood samples, for phylogenetic analysis and genotyping. The msp4 gene sequence revealed no significant genetic diversity, to contrast to data on A. ovis strains from other countries. For T. lestoquardi, 8/150 (5.33%) samples were positive in sheep, but no samples were positive in goats (0%). For T. ovis, 32/150 (21.33%) samples were positive in sheep, but no samples were positive in goats (0%). Sequencing targeting the merozoite surface protein gene for T. lestoquardi and the small subunit ribosomal RNA gene for T. ovis revealed no significant genetic diversity in the study, another contrast to data on A. ovis strains from other countries.ConclusionThis study provides valuable data on phylogenetic and molecular classifications of A. ovis, T. ovis and T. lestoquardi found in southern Egyptian sheep and goats. It also represents the first report on detection and molecular characterization of T. lestoquardi in southern Egyptian sheep based on the specific merozoite surface protein gene, thus providing valuable data for molecular characterization of this pathogen in southern Egypt.

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.

Sequencing and characterization of Helcococcus ovis: a comprehensive comparative genomic analysis of virulence

BackgroundHelcococcus ovis (H. ovis) is an emerging bacterial pathogen that commonly causes opportunistic respiratory, mammary, and uterine infections across mammalian hosts. This study applied long- and short-read whole genome sequencing technologies to identify virulence factors in five H. ovis isolates with low, medium, and high virulence phenotypes.ResultsThe resulting assemblies contained one circular chromosome ranging from 1,744,566 to 1,850,083 bp in length and had a mean GC content of 27.6%. Phylogenetic and nucleotide identity analyses found low virulence strain KG38 to be part of a clade that forms an outgroup apart from the rest of the H. ovis taxon. Assembling the first complete genomes of the species revealed major genomic rearrangements in KG38. One to six prophage regions were identified in each genome. A novel pathogenicity island was found exclusively in the two high virulence strains (KG37 and KG104), along with two hypothetical transmembrane proteins designated as putative VFs. Finally, three zinc ABC transporters and three Type-II/IV secretion systems were identified as possible virulence determinants in this species. The low virulence strain KG38 has fewer intact paralogs of these operons in its genome compared to the higher virulence isolates, which strongly suggests a role in virulence. This strain is also missing four putative virulence factors (VFs) found in other isolates associated with adherence (collagen adhesin precursor), immune evasion (choline-binding protein A and a PspA-like hypothetical protein) and cell wall synthesis (glycerol-3-phosphate cytidylyltransferase).ConclusionsIn this study, we assembled reference-quality complete genomes for five H. ovis strains to identify putative virulence factors. Phylogenetic analyses of H. ovis isolates revealed the presence of a clade representing a potentially novel species within the genus Helcococcus. A novel pathogenicity island and two hypothetical transmembrane proteins were found exclusively in high-virulence strains. The identification of Zinc ABC transporters and Type-II/IV secretion systems as possible virulence determinants, along with the differences in operon content between the low and high virulence isolates, strongly suggests they also play a role in the bacterium’s pathogenicity. Taken together, these findings are a valuable first step toward deciphering the pathogenesis of H. ovis infections.

Molecular Prevalence and Genetic Diversity Based on Msp1a Gene of Anaplasma ovis in Goats from Türkiye.

Anaplasma ovis is a tick-borne obligated intraerythrocytic bacterium that infects domestic sheep, goats, and wild ruminants. Recently, several studies have been carried out using 16S rRNA and msp4 genes to identify the genetic diversity of A. ovis. Instead of these genes, which are known to be highly stable among heterologous strains, Msp1a, which is accepted as a stable molecular marker to classify A. marginale strains, was used in A. ovis genetic diversity studies. The genetic diversity of A. ovis strains according to the Msp1a gene has not been extensively reported. Therefore, the purpose of this study was to examine the genetic diversity of A. ovis in goats specifically using analysis of the Msp1a gene. Blood samples were taken from the vena jugularis to the EDTA tubes from 293 randomly selected goats (apparently healthy) in the Antalya and Mersin provinces of Mediterranean region of Türkiye. The Msp1a gene of A. ovis was amplified in all DNA samples through the use of PCR, using a specific set of primers named AoMsp1aF and AoMsp1aR. Among the amplified products, well-defined bands with different band sizes were subjected to sequence analysis. The obtained sequence data were converted into amino acid sequences using an online bioinformatics program and the tandem regions were examined. The Msp1a gene of A. ovis was amplified in 46.1% (135 out of 293) of the goats. Through tandem analysis, five distinct tandems (Ao8, Ao18, Tr15-16-17) were identified, and it was found that three of these tandems (Tr15-16-17) were previously unknown and were therefore defined as new tandems. The study also involved examination of ticks from goats. It was observed that the goats in the area were infested with several tick species, including Rhipicephalus bursa (888/1091, 81.4%), R. turanicus (96/1091, 8.8%), Dermacentor raskemensis (92/1091, 8.4%), Hyalomma marginatum (9/1091, 0.8%), and R. sanguineus s.l. (6/1091, 0.5%). This study provides important data for understanding the genetic diversity and evolution of A. ovis based on tandem repeats in the Msp1a protein.

Pathology, bacteriology and molecular studies on caseous lymphadenitis in Camelus dromedarius in the Emirate of Abu Dhabi, UAE, 2015-2020.

Caseous lymphadenitis (CLA) or pseudotuberculosis is a chronic zoonotic bacterial disease caused by Corynebacterium pseudotuberculosis, which affects livestock and humans. This study aimed to describe the pathology, bacteriology and confirm the identity of the pathogen by 16S rRNA gene sequencing in Camelus dromedarius. A total of 12 camels with suspected CLA in three regions of Abu Dhabi Emirate (Abu Dhabi, Al Ain and Al Dhafra), United Arab Emirate (UAE) were subjected to clinical and postmortem examinations from January 2015 to December 2020. Clinically, camels were emaciated and showed the presence of external caseous abscesses suggestive of CLA. Postmortem examination showed multiple abscesses of variable sizes with caseous material encapsulated by fibrous tissue in the liver, lungs, muscle, and lymph nodes. Following clinical and postmortem examination, blood, pus and different tissue samples were collected for subsequent analysis. Histopathological examination of all organs stained with Hematoxylin and Eosin (H&E) indicated a central caseo-necrotic core that was admixed with bacterial colonies and infiltration of chronic inflammatory cells, surrounded by a pyogenic membrane, and an outer fibrous connective tissue capsule. Bacterial culture identified the isolates of Corynebacterium pseudotuberculosis biotype ovis strain, and these isolates were shown to be sensitive to all antibiotics tested (penicillin, ampicillin, Co-trimoxazole, enrofloxacin and tetracycline). Moreover, the identity of the isolates was confirmed by partial sequencing of the 16S rRNA gene which showed a 100% identity to Corynebacterium pseudotuberculosis. Phylogenetic analysis based on 16S rRNA gene sequence clearly differentiates Corynebacterium pseudotuberculosis from other species of Corynebacterium. Briefly, this study provided the basic information for infection of Corynebacterium pseudotuberculosis in Camels and will help in controlling of this pathogen in the region.

Study of biological properties of Brucella ovis strains isolated on the territory of Ukraine during 1973–2019

The article presents data on the biological properties of Brucella strains, which were kept in the collection of microorganisms of NSC ‘IECVM’ previously and determined as Brucella ovis by biochemical tests. It was found that during long-term storage strains did not lose their properties according to passports. Four strains 78/3131, 157/4151, 169/87, and 68/Ж grew not only in the presence of thionine, which is characteristic of R-forms, but also in the media with fuchsin (1:50,000 and 1:100,000). When studying the antigenic properties, it was found that these strains have Brucella S-antigen and the absence of R-antigen. Additionally, molecular genetic typing revealed that four strains belonged to other species of Brucella

Doramectin efficacy against Psoroptes ovis in sheep: Evaluation of pharmacological strategies

The aims of this study were to evaluate the efficacy of two injectable formulations of doramectin (DRM) against Psoroptes ovis in sheep infested under controlled experimental conditions and to characterize the DRM plasma disposition kinetics in the infested animals.To this end, sheep were experimentally infested with a P. ovis strain from a farm with a history of treatment failure, and then treated either with DRM 1% (traditional preparation) on days 0 and 7 or with DRM 3.15% (long-acting formulation) on day 0. The efficacy of each treatment was calculated by counting live mites in skin scrapings. Plasma samples were obtained from each animal and DRM concentrations were measured by HPLC. After the two doses of DRM 1%, the maximum efficacy (98.8%) was reached on day 28, whereas after the single dose of DRM 3.15%, the maximum efficacy (100%) was reached on day 35 and ratified on day 42. The long-acting formulation allowed obtaining higher exposure and more sustained concentrations of DRM than the traditional preparation. Although both DRM formulations studied were effective according to international protocols, they did not reach 100% effectiveness in the time required for approved pharmaceutical products against sheep scab, according to Argentine regulations.

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.

Genetic diversity of groEL and msp4 sequences of Anaplasma ovis infecting camels from Tunisia

To date, no information is available regarding the infection of camels (Camelus dromedarius) by Anaplasma ovis in North African region. Several animal species can be infected by A. ovis which further complicates its natural infection cycle. In this paper, we investigated the occurrence and the genetic diversity of A. ovis in camels and ticks collected from them in Tunisia and the risk factor analysis. Camel blood samples (n = 412) and tick (n = 300) samples, identified as Hyalomma dromedarii (n = 149, 49.6%), H. impeltatum (n = 142, 47.3%) and H. excavatum (n = 9, 3%), were analyzed by conventional PCR followed by the sequencing of msp4 and groEL genes. A. ovis DNA was identified in five camels (1.2%), but not in infesting ticks (0%). The microscopic examination revealed the specific infection of camel erythrocytes by Anaplasma inclusions. The msp4 and groEL typing confirmed the natural infection of camels by A. ovis and revealed two different msp4 genotypes earlier detected in Tunisian small ruminants and their infested ticks, and five different and novel groEL genetic variants forming a separately sub-cluster within A. ovis cluster. The occurrence of different A. ovis strains specific to camels associated with a low prevalence of this Anaplasma species in camels may enrich knowledge regarding the distribution and the transmission cycle of this bacterium in arid and Saharan areas of Tunisia.

Draft Genome Sequences of Helcococcus ovis Strains Isolated at Time of Metritis Diagnosis from the Uterus of Holstein Dairy Cows.

Helcococcus ovis is an emerging pathogen implicated in the pathogenesis of metritis in dairy cows. Herein, we report the first draft genome sequences of four Helcococcus ovis isolates from the uterus of dairy cows with metritis. This information will enable a better understanding of the bacterium's pathogenicity and antimicrobial resistance.

Anaplasma ovis genetic diversity detected by major surface protein 1a and its prevalence in small ruminants

Anaplasma ovis is a widely distributed tick-borne rickettsial pathogen of sheep, goats, and wild ruminants. The aims of this study were to assess the prevalence, associations of Anaplasma ovis in sheep and goats, as well as its genetic diversity based on analysis of the msp1α gene. A total of 416 DNA samples from sheep (n = 236) and goats (n = 180) from four provinces in southeastern Turkey were analyzed by PCR. The overall A. ovis prevalence was 18% (CI 14.4–22.1). The infection rates of A. ovis varied from 15.9% to 21.8% in sampled provinces, and they were not significantly different. There was no difference between Anaplasma ovis infection in sheep (20.3%, CI 15.4–26.0) and goats (15.0%, CI 10.1–21.1) or in infection rate of animals <1 year (21.8%, CI 14.9-30.1) compared to >1 year (16.4%, CI 12.4–21.2). A significant association between A. ovis infection and the presence of Rhipicephalus bursa and Rhipicephalus turanicus was observed (P < 0.05). Prevalence of A. ovis-positive animals was higher in animals showing co-infection with Babesia and Theileria compared to those not co-infected (P < 0.05). The Msp1a amino acid repeats were identified and used for the characterization of A. ovis strains. Forty partial msp1a gene sequences containing the repeated sequences of A. ovis were obtained, and 14 previously undescribed tandem repeats with 33 to 43 amino acids were found. Thirteen A. ovis genotypes were identified based on the structure of Msp1a tandem repeats. The majority of A. ovis isolates exhibited one Msp1a tandem repeat, with a maximum of three. This study revealed the Msp1a could be used as a marker for genotyping A. ovis, and high genetic diversity of A. ovis were found in small ruminants in Turkey.

Characterization of Anaplasma ovis strains using the major surface protein 1a repeat sequences

Anaplasma ovis is one of the tick-transmitted pathogens of small ruminants. It causes ovine anaplasmosis and widely distributed in the world. In contrast to extensive worldwide genetic diversity of A. marginale and A. phagocytophilum, there are few reports on the classification of A. ovis strains. This study was conducted to investigate the occurrence and characterize A. ovis strains from goats and sheep from 12 provinces in China. The occurrence of A. ovis DNA was tested in 552 goats and sheep, by PCR based on the msp4 gene. Positive samples were used for the amplification of the msp1a gene of A. ovis. The Msp1a amino acid repeats were further identified and used for the characterization of A. ovis strains. The results showed that 79 (14.3%) goats and sheep were positive for A. ovis. The infection rates of A. ovis among different study sites ranged from 0 to 100%, and were significantly higher in sheep (26.6%, 45/169) than in goats (8.9%, 34/383) (χ 2 = 21.403, df = 1, P < 0.001). The msp4 gene sequences of these isolates were 99.8–100% identical to each other, and they represented two sequence types. Forty-four partial msp1a gene sequences containing the repeat sequences were obtained from A. ovis-positive samples. After translation to amino acid sequences, 24 Msp1a repeats with 33 to 47 amino acids, which corresponded to 19 genotypes of A. ovis, were recognized in goats and sheep in China. Anaplasma ovis is widely distributed in the investigated geographical regions. The msp4 gene of A. ovis had high sequence identity and was unable to be used to discriminate different strains. The Msp1a could be used as a genetic marker for characterizing A. ovis, and 19 genotypes of A. ovis were recognized in domestic small ruminants in China. The present study revealed, for the first time, the genetic diversity of A. ovis based on the analyses of Msp1a amino acid repeats.

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.

Antigenic characterization of Moraxella bovis, Moraxella bovoculi and Moraxella ovis strains with potential use in vaccines

Moraxella bovis is historically known as the primary agent of infectious bovine keratoconjunctivitis (IBK). However, Moraxella bovoculi and Moraxella ovis are also reported to be involved in the pathogenesis of IBK, therefore, these three species should be included in the development of a new vaccine with a broad-spectrum protection against the disease natural challenge. In this study we investigated the antigenic properties of clinical isolates and reference strains of M. bovis, M. bovoculi and M. ovis using a novel in vitro approach for vaccine evaluation based on two techniques, flow cytometry and western blotting (WB). Here, we demonstrated that rabbit antisera produced against reference M. bovis strain and commercial bacterin showed low number of IgG with capacity to recognize a panel of heterologous strains composed by M. bovoculi and M. ovis. On the other hand, the antisera generated against two clinical isolates of M. ovis (Mov2 and Mov3) presented high cross-reactivity levels against all M. ovis and M. bovis strains evaluated. Similarly, the antisera against Mbv3 (clinical isolate of M. bovoculi) had high levels of IgG associated on the surface of all M. bovoculi strains and most of the M. ovis strains analyzed. The WB analysis demonstrated that Moraxella spp. has multiple immunogenic antigens and most of them are shared between the three species. Based on the cross-reactivity analysis and considering the relative number of IgGs associated on the bacterial surface, we suggest that a multivalent vaccine including Mbv3, Mov2 and Mov3 strains may provide a strong and broad protection against all strains involved in IBK outbreaks.

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.

Cultivation of Anaplasma ovis in the HL-60 human promyelocytic leukemia cell line

The tick-borne bacterium Anaplasma ovis is a widely distributed pathogen affecting sheep, goats and wild ruminants. Here, the HL-60 human promyelocytic leukemia cell line was used to isolate A. ovis from PCR-positive sheep and goats in Heilongjiang Province, China. Two weeks after inoculation, morulae were observed in cytoplasmic vacuoles in four different HL-60 cultures. Confocal microscopy using a Cy3-labeled A. ovis-specific probe confirmed that the HL-60 cells were infected with A. ovis. Cells from the 6th HL-60 subculture displayed positive fluorescence when incubated with A. ovis antiserum in the indirect fluorescent antibody assay. PCR amplification and sequencing of 16S rRNA, groEL, gltA, msp2 and msp4 Anaplasma genes revealed that the four A. ovis culture isolates were identical. Phylogenetic analysis showed that the sequences clustered with other A. ovis strains but could clearly be distinguished from other Anaplasma species. When the 18th subculture of infected HL-60 cells was examined by electron microscopy, lysosomes were often observed near the vacuoles. After the 24th subculture, Giemsa staining and PCR indicated that the HL-60 cells were negative for A. ovis. Although A. ovis can infect HL-60 cells for only four months, the ability of the organism to infect and multiply in HL-60 cells provides a tool to study intra-erythrocytic Anaplasma and host cell interactions.Emerging Microbes & Infections (2017) 6, e83; doi:10.1038/emi.2017.70; published online 20 September 2017

Life Span of a Parapoxvirus Ovis Vector and Local Immune Activation in a Non-Permissive Host

Objective: Promising vector vaccine candidates derive from parapoxvirus ovis (PPVO) strain D1701. Thorough analysis of their safety is therefore of great relevance. We investigated the safety of and the immune response induced by the reporter gene β-galactosidase-expressing vector PPVO D1701-VrV. It is important to define the life span of the vector in vaccinated hosts. We chose non-permissive mice for a first study of PPVO longevity and clearence. Methods: Mice were inoculated i.m. with a TCID50 of 106.5 PPVO D1701-VrV and with PBS into the quadriceps and contralateral quadriceps muscle, respectively. The muscles were analyzed for viral genome, infective virus particles and infiltrating leukocytes. Day 21 was fixed as a late vector virus clearance option. The immune cell content of the draining lymph nodes, IFN-γ production by in vitro restimulated splenocytes, and expression of β- galactosidase in myoblasts in vitro was characterized. Results: Upon i.m. application infective PPVO D1701-VrV particles were detectable at the inoculation site for at least seven days but cleared at the fixed late time point. Among the re-isolated vector virus aberrantly shaped PPVO particles were detected, but the reporter gene expression remained stable. The PPVO D1701-VrV-induced local immune activation was characterized by the presence of inflammatory infiltrates and, even more important for the priming capability of the viral vector, significantly enhanced numbers of CD11c+ B220- conventional DC, CD11c+ B220+ plasmacytoid DC, CD4+ T cells, CD8+ T cells, and B220+ B cells in the muscle-draining inguinal lymph nodes. Elevated interferon-gamma (IFN-γ) production by splenocytes of PPVO D1701-VrV-inoculated mice indicated a systemic immunostimulatory effect. The detection of β-galactosidase expression by inoculated myoblasts shows that PPVO D1701-VrV is able to enter murine myoblasts and express the foreign antigen. Conclusions: Our data support the safety of PPVO D1701 as a vector virus under non-permissive conditions.

Multilocus Sequencing of Corynebacterium pseudotuberculosis Biotype Ovis Strains.

Thirteen Corynebacterium pseudotuberculosis biotype ovis strains isolated from clinical cases of caseous lymphadenitis in Hungary were characterised using multilocus sequencing and their phylogenetic comparison was carried out on the basis of four housekeeping genes (groEL1, infB, dnaK, and leuA). The in silico analysis of the 16 frequently studied housekeeping genes showed that C. pseudotuberculosis strains could be readily distinguished from C. diphtheriae and C. ulcerans strains; however, sequences of the same genes in the two biotypes of the C. pseudotuberculosis were highly similar; the heterogeneity values were low. Genes dnaK, infB, groEL1, and leuA showed marked genetic variation within C. pseudotuberculosis, and strains of the two biotypes of C. pseudotuberculosis could be differentiated. Analysis of the individual genes showed a fairly conservative nature of C. pseudotuberculosis biotype ovis strains. The greatest genetic differentiation was seen in the dnaK and infB genes and concatenations of these two genes were very useful in the genetic separation of the studied strains.