Ovine Strains Research Articles

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.

Simulated Effects of Acute Left Ventricular Myocardial Infarction on Mitral Regurgitation in an Ovine Model.

Ischemic mitral regurgitation (IMR) occurs from incomplete coaptation of the mitral valve (MV) after myocardial infarction (MI), typically worsened by continued remodeling of the left ventricular (LV). The importance of LV remodeling is clear as IMR is induced by the post-MI dual mechanisms of mitral annular dilation and leaflet tethering from papillary muscle (PM) distension via the MV chordae tendineae (MVCT). However, the detailed etiology of IMR remains poorly understood, in large part due to the complex interactions of the MV and the post-MI LV remodeling processes. Given the patient-specific anatomical complexities of the IMR disease processes, simulation-based approaches represent an ideal approach to improve our understanding of this deadly disease. However, development of patient-specific models of left ventricle-mitral valve (LV-MV) interactions in IMR are complicated by the substantial variability and complexity of the MR etiology itself, making it difficult to extract underlying mechanisms from clinical data alone. To address these shortcomings, we developed a detailed ovine LV-MV finite element (FE) model based on extant comprehensive ovine experimental data. First, an extant ovine LV FE model (Sci. Rep. 2021 Jun 29;11(1):13466) was extended to incorporate the MV using a high fidelity ovine in vivo derived MV leaflet geometry. As it is not currently possible to image the MVCT in vivo, a functionally equivalent MVCT network was developed to create the final LV-MV model. Interestingly, in pilot studies, the MV leaflet strains did not agree well with known in vivo MV leaflet strain fields. We then incorporated previously reported MV leaflet prestrains (J. Biomech. Eng. 2023 Nov 1;145(11):111002) in the simulations. The resulting LV-MV model produced excellent agreement with the known in vivo ovine MV leaflet strains and deformed shapes in the normal state. We then simulated the effects of regional acute infarctions of varying sizes and anatomical locations by shutting down the local myocardial contractility. The remaining healthy (noninfarcted) myocardium mechanical behaviors were maintained, but allowed to adjust their active contractile patterns to maintain the prescribed pressure-volume loop behaviors in the acute post-MI state. For all cases studied, the LV-MV simulation demonstrated excellent agreement with known LV and MV in vivo strains and MV regurgitation orifice areas. Infarct location was shown to play a critical role in resultant MV leaflet strain fields. Specifically, extensional deformations of the posterior leaflets occurred in the posterobasal and laterobasal infarcts, while compressive deformations of the anterior leaflet were observed in the anterobasal infarct. Moreover, the simulated posterobasal infarct induced the largest MV regurgitation orifice area, consistent with experimental observations. The present study is the first detailed LV-MV simulation that reveals the important role of MV leaflet prestrain and functionally equivalent MVCT for accurate predictions of LV-MV interactions. Importantly, the current study further underscored simulation-based methods in understanding MV function as an integral part of the LV.

In Vitro Interaction between Mycoplasma agalactiae and Small Ruminants' Endogenous Bacterial Strains of Enterococcus spp. and Coagulase-Negative Staphylococcus.

Recently, an antimicrobial effect on Mycoplasma agalactiae (Ma), the main etiological agent of contagious agalactia (CA), was reported in vitro with strains of Enterococcus spp. from ovine and caprine milk. The aim of this work was to evaluate the interaction of Ma with the same Enterococcus spp. isolated from other anatomical locations (vagina) and other bacterial populations present in milk, such as coagulase-negative staphylococci (CNS). The vaginal Enterococcus strains and the raw milk CNS were isolated from sheep and goats. Experimental in vitro conditions were prepared to assess the growth of Ma with and without the presence of these strains. The selected vaginal strains were identified as Enterococcus (E.) hirae and E. mundtii, and the strains of CNS were identified as Staphylococcus petrasii. Different interactions of Ma with ovine and caprine wild vaginal strains of Enterococcus and dairy strains of CNS are described for the first time: Ma can grow exponentially during 15 h with the selected strains, although with certain strains, its optimal growth can be negatively affected (p < 0.05). The colonization and/or excretion of Ma could, therefore, be influenced by certain endogenous bacterial strains. Our results increase the knowledge about possible bacterial ecology dynamics surrounding CA.

Antibacterial potential of commercial and wild lactic acid bacteria strains isolated from ovine and caprine raw milk against Mycoplasma agalactiae.

The complexity of fighting contagious agalactia (CA) has raised the necessity of alternative antimicrobial therapies, such as probiotics. Lactic acid bacteria (LAB) are present in the mammary gland of small ruminants and their antimicrobial effect have been previously described against species like Mycoplasma bovis but never against Mycoplasma agalactiae (Ma). This in vitro study aims to evaluate the antimicrobial activity against Ma of ovine and caprine LAB strains and a human commercial probiotic (L2) of Lactobacillus spp. A total of 63 possible LAB strains were isolated from nine ovine and caprine farms in Spain, three isolates (33B, 248D, and 120B) from the 63 strains were selected, based on their capacity to grow in a specific medium in vitro, for an in vitro experiment to assess their antimicrobial activity against Ma in Ultra High Temperature (UHT) processed goat milk (GM). A women commercial vaginal probiotic was also included in the study. The inoculum of L2 was prepared at a concentration of 3.24 × 108 CFU/mL and the average concentration of the inoculum of the wild LAB varied from 7.9 × 107 to 8.4 × 108 CFU/mL. The commercial probiotic L2 significantly reduced the concentration of Ma to 0.000 log CFU/mL (p < 0.001), strain 33B reduced it from 7.185 to 1.279 log CFU/mL (p < 0.001), and 120B from 6.825 to 6.466 log CFU/mL (p < 0.05). Strain 248D presented a bacteriostatic effect in GM. Moreover, the three wild strains and the commercial probiotic produced a significative reduction of the pH (p < 0.001). This is the first in vivo report of the antimicrobial potential of LAB strains against Ma and its interaction. Our results support possible future alternative strategies to antibiotic therapy, previously not contemplated, to fight CA in small ruminants. Further studies are necessary to elucidate the action mechanisms through which these LAB are able to inhibit Ma and to assess the safety of using these strains in possible in vivo studies.

One clone to rule them all: Culture-independent genomics of Chlamydia psittaci from equine and avian hosts in Australia.

Chlamydia psittaci is an avian pathogen with zoonotic potential. In Australia, C. psittaci has been well reported as a cause of reproductive loss in mares which subsequently have been the source of infection and illness in some in-contact humans. To date, molecular typing studies describe the predominant and clonal C. psittaci sequence type (ST)24 strains in horse, psittacine, and human infections. We sought to assess the clonality between ST24 strains and the emergence of equine ST24 with a comprehensive genomics approach. We used culture-independent probe-based and metagenomic whole-genome sequencing to investigate 13 C . psittaci genomes from horses, psittacines, and a pigeon from Australia. Published genomes of 36 C . psittaci strains were also used to contextualise our Australian dataset and investigate lineage diversity. We utilised a single-nucleotide polymorphism (SNP) based clustering and multi-locus sequence typing (MLST) approach. C. psittaci has four major phylogenetic groups (PG1-4) based on core-genome SNP-based phylogeny. PG1 contained clonal global and Australian equine, psittacine, and human ST24 genomes, with a median pairwise SNP distance of 68 SNPs. PG2, PG3, and PG4 had greater genomic diversity, including diverse STs collected from birds, livestock, human, and horse hosts from Europe and North America and a racing pigeon from Australia. We show that the clustering of C. psittaci by MLST was congruent with SNP-based phylogeny. The monophyletic ST24 clade has four major sub-lineages. The genomes of 17 Australian human, equine, and psittacine strains collected between 2008 and 2021 formed the predominant ST24 sub-lineage 1 (emerged circa 1979). Despite a temporal distribution of 13 years, the genomes within sub-lineage 1 had a median pairwise SNP distance of 32 SNPs, suggesting a recent population expansion or potential cross-host transmission. However, two C. psittaci genomes collected in 2015 from Victorian parrots clustered into distinct ST24 sub-lineage 4 (emerged circa 1965) with ovine strain C19/98 from Germany. This work describes a comprehensive phylogenomic characterisation of ST24 and identifies a timeline of potential bird-to-equine spillover events.

Detection and characterization of bopiviruses in domestic and wild ruminants.

Highly divergent picornaviruses (PVs) classified in the genus Bopivirus have been recently discovered on faecal samples from sheep and goats in Hungary and from fallow and red deer in Australia. In this study, we investigated the epidemiology of these novel viruses in domestic and wild ruminants from Northwestern Italian Alps by testing archival faecal samples collected from 128 sheep, 167 goats, 61 red deer (Cervus elaphus), 77 roe deer (Capreolus capreolus), 43 chamois (Rupicapra rupicapra) and 32 Alpine ibex (Capra ibex). Bopivirus RNA was detected in a total of 19 animals, including 14 sheep (10.9%), 2 red deer (3.3%), 1 roe deer (1.3%), 1 chamois (2.3 %) and 1 Alpine ibex (3.3 %), but not in goats. Upon sequence analysis of the 3DRdRp region, the sequences generated from chamois, roe deer, Alpine ibex and ovine faecal samples showed the highest nucleotide identity (96.8-100%) to bopiviruses detected in goats and sheep from Hungarian farms, whereas strains found in red deer displayed the closest relatedness (90.8%-91.2%) to bopiviruses identified in fallow and red deer in Australia. The nearly complete genome sequence of strains 12/2020/ITA (ON497046) and 14-73/2020/ITA (ON497047) detected in an Alpine ibex and in a sheep, respectively, was determined by combining a modified 3'-RACE protocol with Oxford Nanopore Technologies sequencing platform. On phylogenetic analysis based on the complete polyprotein, both strains segregated into the candidate species Bopivirus B along with ovine and caprine strains detected in Hungary (90.0-94.6% nucleotide and 94.6-98.0% amino acid identities). The findings of this study expand the host range of these novel viruses and hint to a possible virus circulation between domestic ruminants and wild animals.

Molecular Characterization of Small Ruminant Lentiviruses in Polish Mixed Flocks Supports Evidence of Cross Species Transmission, Dual Infection, a Recombination Event, and Reveals the Existence of New Subtypes within Group A.

Small ruminant lentiviruses (SRLVs) are a group of highly divergent viruses responsible for global infection in sheep and goats. In a previous study we showed that SRLV strains found in mixed flocks in Poland belonged to subtype A13 and A18, but this study was restricted only to the few flocks from Małopolska region. The present work aimed at extending earlier findings with the analysis of SRLVs in mixed flocks including larger numbers of animals and flocks from different part of Poland. On the basis of gag and env sequences, Polish SRLVs were assigned to the subtypes B2, A5, A12, and A17. Furthermore, the existence of a new subtypes, tentatively designed as A23 and A24, were described for the first time. Subtypes A5 and A17 were only found in goats, subtype A24 has been detected only in sheep while subtypes A12, A23, and B2 have been found in both sheep and goats. Co-infection with strains belonging to different subtypes was evidenced in three sheep and two goats originating from two flocks. Furthermore, three putative recombination events were identified within gag and env SRLVs sequences derived from three sheep. Amino acid (aa) sequences of immunodominant epitopes in CA protein were well conserved while Major Homology Region (MHR) had more alteration showing unique mutations in sequences of subtypes A5 and A17. In contrast, aa sequences of surface glycoprotein exhibited higher variability confirming type-specific variation in the SU5 epitope. The number of potential N-linked glycosylation sites (PNGS) ranged from 3 to 6 in respective sequences and were located in different positions. The analysis of LTR sequences revealed that sequences corresponding to the TATA box, AP-4, AML-vis, and polyadenylation signal (poly A) were quite conserved, while considerable alteration was observed in AP-1 sites. Interestingly, our results revealed that all sequences belonging to subtype A17 had unique substitution T to A in the fifth position of TATA box and did not have a 11 nt deletion in the R region which was noted in other sequences from Poland. These data revealed a complex picture of SRLVs population with ovine and caprine strains belonging to group A and B. We present strong and multiple evidence of dually infected sheep and goats in mixed flocks and present evidence that these viruses can recombine in vivo.

Identification of a novel astrovirus in goats in China.

In this study, a total of 143 fecal samples (107 diarrheic and 36 non-diarrheic) were collected from 11 goat farms in southwest China, and 3.7% of diarrheic and 8.3% of non-diarrheic samples were detected as astrovirus-positive by RT-PCR. A nearly complete astrovirus genomic sequence (SWUN/F4/2019) of 6278 nucleotides (nt), which contained a 6186bp open reading frame, was successfully obtained. The genome of strain SWUN/F4/2019 shared the highest nt identity (77.0%) and the closest genetic relationship with CapAstV-G5.1. It is worth noting that in the nonstructural protein 1ab, strain SWUN/F4/2019 shared the highest amino acid (aa) identity (93.8%) with strain CapAstV-G5.1; however, its capsid protein shared the highest aa identity (72.7%) with the Sichuan takin astrovirus strain LLT03 and only shared 23.1-64.8% aa identities with all available ovine and caprine astrovirus strains. Interestingly, a region recombination event was predicted in the ORF2 gene of strain SWUN/F4/2019, with CapAstV-G5.1 as the putative major parental strain and CcAstV/roe_deer/SLO/D5-14/2014 as the possible minor parental strain. According to the species classification criteria of the International Committee on Taxonomy of Viruses (ICTV), SWUN/F4/2019 may represent a novel astrovirus in goats. To our knowledge, this is the first detection of astrovirus in goats in China and a novel astrovirus strain was identified in goats. These findings increase the understanding of the epidemic and the genetic evolution of astroviruses.

Diversity and characterization of temperate bacteriophages induced in Pasteurella multocida from different host species

BackgroundBacteriophages play important roles in the evolution of bacteria and in the emergence of new pathogenic strains by mediating the horizontal transfer of virulence genes. Pasteurella multocida is responsible for different disease syndromes in a wide range of domesticated animal species. However, very little is known about the influence of bacteriophages on disease pathogenesis in this species.ResultsTemperate bacteriophage diversity was assessed in 47 P. multocida isolates of avian (9), bovine (8), ovine (10) and porcine (20) origin. Induction of phage particles with mitomycin C identified a diverse range of morphological types representing both Siphoviridae and Myoviridae family-types in 29 isolates. Phage of both morphological types were identified in three isolates indicating that a single bacterial host may harbour multiple prophages. DNA was isolated from bacteriophages recovered from 18 P. multocida isolates and its characterization by restriction endonuclease (RE) analysis identified 10 different RE types. Phage of identical RE types were identified in certain closely-related strains but phage having different RE types were present in other closely-related isolates suggesting possible recent acquisition. The host range of the induced phage particles was explored using plaque assay but only 11 (38%) phage lysates produced signs of infection in a panel of indicator strains comprising all 47 isolates. Notably, the majority (9/11) of phage lysates which caused infection originated from two groups of phylogenetically unrelated ovine and porcine strains that uniquely possessed the toxA gene.ConclusionsPasteurella multocida possesses a wide range of Siphoviridae- and Myoviridae-type bacteriophages which likely play key roles in the evolution and virulence of this pathogen.

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.

Identification of Immunodominant Outer Membrane Proteins of Fusobacterium necrophorum from Severe Ovine Footrot By MALDI-TOF Mass Spectrometry

The aim of this study was to identify the immunodominant outer membrane proteins (OMPs) of Fusobacterium necrophorum from sheep affected with severe foot-rot. The OMP profile of ovine strains of F. necrophorum has not been well studied. We analyzed the OMP profile of the most frequent lktA variant JKS-F3 of F. necrophorum associated with severe ovine foot-rot with lesion score 4 in order to identify its major immunodominant OMPs. Electrophoretic separations of extracted OMPs showed a number of spots in two-dimensional electrophoretic gels. Two immunoreactive proteins of size around 43 kDa were identified through western blotting using hyperimmune sera raised in rabbits. These two immunogenic OMPs were analyzed by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF/MS) which revealed that these two OMPs of lktA variant JKS-F3 of F. necrophorum showed 46 and 42 percent protein sequence coverage and scores of 125 and 114, respectively, with the reported 43 kDa outer membrane protein of F. necrophorum strain H05, a putative porin having properties similar to pore-forming proteins of anaerobic Gram-negative bacteria. These identified immunogenic OMPs will contribute to our understanding of the pathogenic role played by this organism in ovine foot-rot and could be exploited to devise an effective control strategy through development of an OMP-based recombinant vaccine to mitigate foot-rot in sheep and goats.

Assessment of in vitro growth characteristics of Mycoplasma ovipneumoniae

Mycoplasma ovipneumoniae (Movp) is an emerging pathogen that causes respiratory disease in small ruminants worldwide. It is considered to be difficult and time consuming to grow, which complicates diagnostic and control measures including isolation (an essential step required prior to the characterisation of strains), antimicrobial susceptibility testing and the development of vaccines. The objectives of this study were to analyse in vitro growth patterns of Movp strains, and the effects of different media used to support their growth. The study was conducted on 20 ovine and caprine Movp strains, isolated using Thiaucourt’s medium. The rapid growth phase varied among the strains from 24 h to 72 h, although 60% of strains (12 of 20) reached a peak at 48 h. All strains were viable at 72 h after incubation, and declining viability was observed at 96 h (13 of 20 remained viable; 65%), 120 h (9 of 20; 45%) and 144 h (4 of 20; 20%). Growth was not detected at 168 h. All strains were able to grow in modified tryptone soy broth, while PH mycoplasma medium–Hayflick modified medium supported the growth of only two strains. Improved techniques of Movp cultivation require consideration of the growth variability among strains, the time of subculturing (during the first three days of incubation) and selection of appropriate media.

Non-structural Enterotoxin (NSP4) Gene based Molecular Characterization of Caprine and Ovine Rotavirus A, India

Rotavirus A (RVA) causes viral gastroenteritis in humans and animals, including calves, piglets, and foals. The current study reports the genetic characterization of the full-length enterotoxin gene, NSP4, from caprine and ovine species. Upon characterizing eight full-length NSP4 genes by sequencing, it was found that the four caprine and three ovine RVAs NSP4 genes are of E2 genotype and the sole ovine RVA isolate was found to be of E1 genotype. In the sequence and phyloanalysis of the NSP4 gene the seven E2 genotypes clustered with bovine, human, and caprine isolates from India and Bangladesh, respectively. The E1 genotype of ovine RVA was closer to human RVA isolate from India. The nucleotide per cent identity analysis revealed that all E2 genotype strains of caprine and ovine species ranged from 88.4% to 90.4% and it was found common to both the reference human RVA isolates DS-1 and AU-1. Whereas, the E1 genotype ovine strain clustered with human RVA isolates with 93.1% nucleotide per cent identity. The RVA strains circulating in caprine and ovine populations may share a common origin which is usually found in artiodactyl species because humans share a common dwelling with animals. Future studies are needed to confirm these findings of their relationship with humans and large animals.

Genetic Diversity Among Mycobacterium avium Subspecies Revealed by Analysis of Complete Genome Sequences.

Mycobacterium avium comprises four subspecies that contain both human and veterinary pathogens. At the inception of this study, twenty-eight M. avium genomes had been annotated as RefSeq genomes, facilitating direct comparisons. These genomes represent strains from around the world and provided a unique opportunity to examine genome dynamics in this species. Each genome was confirmed to be classified correctly based on SNP genotyping, nucleotide identity and presence/absence of repetitive elements or other typing methods. The Mycobacterium avium subspecies paratuberculosis (Map) genome size and organization was remarkably consistent, averaging 4.8 Mb with a variance of only 29.6 kb among the 13 strains. Comparing recombination events along with the larger genome size and variance observed among Mycobacterium avium subspecies avium (Maa) and Mycobacterium avium subspecies hominissuis (Mah) strains (collectively termed non-Map) suggests horizontal gene transfer occurs in non-Map, but not in Map strains. Overall, M. avium subspecies could be divided into two major sub-divisions, with the Map type II (bovine strains) clustering tightly on one end of a phylogenetic spectrum and Mah strains clustering more loosely together on the other end. The most evolutionarily distinct Map strain was an ovine strain, designated Telford, which had >1,000 SNPs and showed large rearrangements compared to the bovine type II strains. The Telford strain clustered with Maa strains as an intermediate between Map type II and Mah. SNP analysis and genome organization analyses repeatedly demonstrated the conserved nature of Map versus the mosaic nature of non-Map M. avium strains. Finally, core and pangenomes were developed for Map and non-Map strains. A total of 80% Map genes belonged to the Map core genome, while only 40% of non-Map genes belonged to the non-Map core genome. These genomes provide a more complete and detailed comparison of these subspecies strains as well as a blueprint for how genetic diversity originated.

Evidence for zoonotic transmission of species A rotavirus from goat and cattle in nomadic herds in Morocco, 2012-2014.

Species A rotaviruses (RVAs) are a leading cause of diarrhea in children and in the young of a large variety of mammalian and avian host species. The purpose of this study was to identify RVA in nomadic goats and calves during severe diarrhea outbreaks in 2012 and 2014 in Bouaarfa, Morocco, and to characterize the complete genomic constellation of two bovine and caprine strains (S18 and S19) and their genetic relatedness with the human strain ma31 detected in 2011 in Morocco. Partial nucleotide sequencing of VP4 and VP7 genes for the twenty-two positive samples revealed three circulating genotypes: G6P[14], G10P[14], and G10P[5] with predominance of G6P[14] genotype. Full-genome sequencing for both strains S18 and S19 presented, respectively, the following genomic constellations: G6-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3 and G10-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3. Phylogenetic analyses and the analysis of the VP8* antigenic epitopes for S18, S19 and ma31 revealed a shared similarity with bovine, caprine, ovine and human strains from Morocco and other countries. The VP2 and NSP1 genes of the S19 strain were closely related to those of the cognate genes of the human ma31 strain, while the VP4 gene of S18 strain was closely related to the cogent gene of the ma31 strain. Our findings revealed cases of zoonotic transmission and confirmed the risk of emergence of new genotypes in some environments such as nomadic regions, where close physical proximity between human and livestock is common. The present study is novel in reporting whole-genome analyses of RVA isolates obtained from nomadic livestock in Morocco.Electronic supplementary materialThe online version of this article (10.1007/s11262-020-01778-w) contains supplementary material, which is available to authorized users.

Experimental Ixodes ricinus-Sheep Cycle of Anaplasma phagocytophilum NV2Os Propagated in Tick Cell Cultures.

The causative agent of tick-borne fever and human granulocytic anaplasmosis, Anaplasma phagocytophilum, is transmitted by Ixodes ricinus, and is currently considered an emerging disease throughout Europe. In this study, we established a model of A. phagocytophilum sheep infection and I. ricinus transmission using the European Norway variant 2 ovine strain (NV2Os) propagated in both IDE8 and ISE6 tick cells. Two sheep were inoculated with IDE8 tick cells infected with NV2Os. Both sheep developed A. phagocytophilum infection as determined by qPCR and PCR, the presence of fever 4 days post inoculation (dpi), the observation of morulae in granulocytes at 6 dpi, and the detection of A. phagocytophilum antibodies at 14 dpi. A. phagocytophilum was detected by PCR in skin, lung, small intestine, liver, spleen, uterus, bone marrow, and mesenteric lymph node from necropsies performed at 14 and 15 dpi. One sheep was infested during the acute phase of infection with I. ricinus nymphs from a pathogen-free colony. After molting, A. phagocytophilum transstadial transmission in ticks was validated with qPCR positive bacterial detection in 80% of salivary glands and 90% of midguts from female adults. Infected sheep blood collected at 14 dpi was demonstrated to be able to infect ISE6 tick cells, thus enabling the infection of two additional naive sheep, which then went on to develop similar clinical signs to the sheep infected previously. One of the sheep remained persistently infected until 115 dpi when it was euthanized, and transmitted bacteria to 70 and 2.7% of nymphs engorged as larvae during the acute and persistent infection stages, respectively. We then demonstrated that these infected nymphs were able to transmit the bacteria to one of two other naive infested sheep. As expected, when I. ricinus females were engorged during the acute phase of infection, no A. phagocytophilum transovarial transmission was detected. The development of this new experimental model will facilitate future research on this tick-borne bacterium of increasing importance, and enable the evaluation of any new tick/transmission control strategies.

Genomic characteristics of classical swine fever virus strains of bovine origin according to primary and secondary sequence-structure analysis.

Background:Classical swine fever virus (CSFV), species member of the family Flaviviridae, is generally considered restricted to domestic and wild suids. The circulation of CFSV has been detected in cattle herds in China and India. Natural infection appeared associated with clinical signs in some cases.Aim:The secondary structures of the internal ribosome entry site in the 5′ untranslated region (UTR) were used for the genomic characterization of bovine strains.Methods:Sequences have been compared to the representative CSFV strains isolated from pigs, vaccines, and contaminants from porcine cell lines and an ovine strain isolated in Spain.Results:The observed sequences from cattle showed a genetic relatedness with live-attenuated vaccine strains used in pigs. Sequence characteristics of the Chinese strain S171 are genetically distant from the previously reported CSFV genotypes, suggesting a new outgroup in the species, described for the first time, and named CSFV-d. Other Chinese strains were genetically closely related to CSFV genotype a2 (Alfort type) pig strains. Indian strains, reported from the states of Tamil Nadu and Meghalaya, were genetically closely related to CSFV genotype a1 (Brescia type) and a5 pig strains, respectively.Conclusion:These preliminary observations are new and relevant in countries, where CSFV control and eradication strategies are applied.

A51 Genetic variability of small ruminant lentiviruses in sheep and goats from single-species flocks from Poland

Previous phylogenetic analyses of small ruminant lentivirus (SRLV) sequences found in Poland revealed the circulation of subtype A1 in both sheep and goats, subtypes B1 in goats, and subtypes B2, A12, and A13 in sheep only. This study aimed to analyze the genetic nature of SRLV circulating in sheep and goats from single-species flocks. In order to analyze the degree of genetic variability, the fragments of gag and env genes of 24 SRLV strains were amplified by PCR, cloned into plasmid vectors, sequenced, and consensus sequences were aligned to each other and to reference sequences available from GenBank. Phylogenetic analysis was performed using the Geneious tree-builder tool, and phylogenetic trees were constructed using Mr Bayes (using the general time reversible substitution model) within Geneious Pro 5.3. Pairwise genetic distances were calculated in MEGA 6. Phylogenetic analysis revealed that the strains were highly heterogeneous and represented ovine strains belonging to subtypes A12 and B2 and caprine strains grouped in subtypes B1, B2, A1, and A12. In addition, two novel subtypes, A16 and A17, were found in goats. The mean pairwise genetic distances of gag and env sequences of both clusters were above 15 per cent nucleotide divergence when compared to all other subtypes within group A, which is a criterion required to distinguish a new subtype. Additionally, the existence of two separated clusters was confirmed by high bootstrap values. Co-infections with strains belonging to different subtypes within A and B groups were detected in one sheep and four goats originating from four flocks. Since the co-infection with more than one lentivirus genotype offers an opportunity for viral recombination, the possible recombination events were tested based on RDP analysis. For all co-infected animals, no evidence of recombination was found within the gag gene; however, env sequences showed some recombination patterns in three samples. In conclusion, we have demonstrated extended genetic variability of SRLV in sheep and goats from Poland with the existence of co-infection and recombination events.

Archive Mining Brings to Light a 25-Year Old Astrovirus Encephalitis Case in a Sheep.

In mammals, the small, positive-sense single-stranded RNA astroviruses are known as being mostly enterotropic and host-specific. Over the past years, however, they were identified several times in central nervous system tissues of humans, minks, cattle, sheep, and pigs with nonsuppurative inflammatory disease of that organ system. We recently reported such neurotropic astroviruses, amongst which bovine astrovirus CH15 (BoAstV-CH15) in two cows, and ovine astrovirus CH16 (OvAstV-CH16) in a sheep, which were genetically almost identical to one another. In order to investigate the occurrence of this virus species in Switzerland over time, we selected formalin-fixed, paraffin-embedded (FFPE) brain tissues of small ruminants diagnosed with severe encephalitis between 1969 and 2012 and screened those by immunohistochemistry for the capsid protein of BoAstV-CH15/OvAstV-CH16. We found one sheep, which died in 1992, that displayed positive immunostaining in various brain regions, and observed that immunostained cells were generally co-localized with the strongest histopathological lesions. We confirmed the virus presence with a second immunohistochemical protocol and demonstrated its close genetic relationship to other BoAstV-CH15/ OvAstV-CH16 strains by next-generation sequencing of an RNA extract from FFPE brain material. Our findings demonstrate that astrovirus BoAstV-CH15/OvAstV-CH16 existed in Switzerland already more than 2 decades ago and underline again the close relationship of the bovine and ovine strains of this virus.

Insights into the genome sequence of ovine Pasteurella multocida type A strain associated with pneumonic pasteurellosis

Pasteurella multocida is a causative agent of pneumonic pasteurellosis in ruminants. Bovine and porcine strains of P. multocida have been sequenced and analyzed worldwide. However limited genomic information is available on ovine strains involved in causation of pneumonic pasteurellosis. Therefore, the present study focused on genome sequencing and analysis of a P. multocida type A isolate recovered from respiratory infection of sheep to unravel the genetic contents and its involvement in the disease.P. multocida type A strain NIVEDI/PMS-1 genome was 2,543,931bp in size, assembled into 67 contigs, consisted of 1879 protein-coding regions, 19 genomic islands (GIs) and 10 phage regions. The identified GIs and phage regions showed high sequence identity mostly with other Pasteurellaceae family respiratory pathogens indicating horizontal gene transfer in ovine respiratory niche. The phylogenetic analysis revealed strain NIVEDI/PMS-1 as closest neighbor to P. multocida P1933 strain reported from bovine. Comparative genomic analysis of strain NIVEDI/PMS-1 with virulent GX-Pm and avirulent Pm70 strains revealed presence of 45 virulence genes common with virulent strain. Further, various virulence factors and accessory genes needed for survival and pathogenesis were also identified. Dermonecrotic toxin (ToxA) like protein presence in the strain appears interesting and further study is needed to understand its role in ovine pasteurellosis.