Molecular detection and genotype characterization of Streptococcus dysgalactiae from sheep flocks with outbreaks of infectious arthritis.

Outbreaks of infectious arthritis in young lambs are a growing concern for the Norwegian sheep industry. In other countries, Streptococcus dysgalactiae subspecies dysgalactiae (SDSD) is a frequent cause of such outbreaks. The objectives of this study were to investigate the causes of outbreaks of infections arthritis in Norwegian sheep flocks, and describe the sources, colonization patterns and genetic diversity of SDSD in affected and healthy sheep flocks. Almost 2000 samples from joints, animal body sites and the indoor environment were analysed by qPCR and culturing for SDSD, which was detected in 27 of 30 flocks. The proportion of positive samples was greater in outbreak flocks compared to healthy flocks. Altogether, SDSD was detected in 48 % of the samples from lambs, 27 % of the samples from ewes and 48 % of environmental samples. A relatively high proportion (67 %) of ear tag wounds were SDSD positive. These wounds may provide a port of entry for SDSD. Whole genome sequencing revealed a clonal distribution of SDSD-isolates, and identified four different multi locus sequence types (STs), among which two STs, ST454 and ST531, dominated. These STs were found in geographically distant flocks. ST454 was almost exclusively found in outbreak flocks. The current study points to skin, wounds and mucous membranes of animals as the main reservoir of SDSD in sheep flocks. However, a significantly higher proportion of SDSD-positive environmental samples in outbreak flocks compared to healthy flocks suggests that also indirect transmission may play a role.

Flock-level risk factors for outbreaks of infectious arthritis in lambs, Norway 2018.

BACKGROUND: Outbreaks of infectious arthritis in young lambs associated with Streptococcus dysgalactiae subspecies dysgalactiae (SDSD) lead to reduced animal welfare, increased use of antibiotics and economic losses for sheep farmers. Understanding risk factors is essential when developing strategies to prevent such outbreaks. This questionnaire-based cross-sectional study classified sheep flocks of respondents as cases or controls. Flock-level risk factors for outbreaks of infectious arthritis were assessed using a multivariable logistic regression model. RESULTS: Eighty-four of 1498 respondents (5.6%) experienced an outbreak of infectious arthritis in their flock in 2018, the year of study. Factors associated with a higher risk of outbreak were larger flock size (OR 1.3, 95% CI 1.1-1.4, per 100 lambs), plastic mesh flooring in the lambing pen (OR 3.0, 95% CI 1.7-5.3) and a lambing percentage greater than 200 (OR 2.0, 95% CI 1.1-3.5). Flocks where farmers observed infections around the ear tags of lambs also had an increased risk of outbreak (OR 2.6, 95% CI 1.6-4.3). CONCLUSIONS: The risk factors identified in this study are characteristic of modern and intensively managed sheep farms in Norway. A distinguishing feature of Norwegian sheep farming is winter housing and indoor lambing. One might expect that this in itself is a risk factor because of high stocking densities during lambing. However, outbreaks of infectious arthritis in young lambs are reported by the industry to be a more recent phenomenon. The current study indicates that intensification of indoor management systems with larger flocks and higher production per ewe may predispose to outbreaks. The results provide a basis for further studies on transmission dynamics of SDSD in sheep flocks with indoor lambing.

Assessing the clinical and bacteriological outcomes of vaccination with recombinant Asp14 and OmpA against A. phagocytophilum in sheep.

Anaplasma phagocytophilum is a tick borne bacterium, causing disease in sheep and other mammals, including humans. The bacterium has great economic and animal welfare implications for sheep husbandry in Northern Europe. With the prospect of a warmer and more humid climate, the vector availability will likely increase, resulting in a higher prevalence of A. phagocytophilum. The current preventive measures, as pyrethroids acting on ticks or long acting antibiotics controlling bacterial infection, are suboptimal for prevention of the disease in sheep. Recently, the increased awareness on antibiotic- and pyrethorid resistance, is driving the search for a new prophylactic approach in sheep against A. phagocytophilum. Previous studies have used an attenuated vaccine, which gave insufficient protection from challenge with live bacteria. Other studies have focused on bacterial membrane surface proteins like Asp14 and OmpA. An animal study using homologous proteins to Asp14 and OmpA of A. marginale, showed no protective effect in heifers. In the current study, recombinant proteins of Asp14 (rAsp14) and OmpA (rOmpA) of A. phagocytophilum were produced and prepared as a vaccine for sheep. Ten lambs were vaccinated twice with an adjuvant emulsified with rAsp14 or rOmpA, three weeks apart and challenged with a live strain of A. phagocytophilum (GenBank acc.nr M73220) on day 42. The control group consisted of five lambs injected twice with PBS and adjuvant. Hematology, real time qPCR, immunodiagnostics and flow cytometric analyses of peripheral blood mononuclear cells were performed. Vaccinated lambs responded with clinical signs of A.phagocytophilum infection after challenge and bacterial load in the vaccinated group was not reduced compared to the control group. rAsp14 vaccinated lambs generated an antibody response against the vaccine, but a clear specificity for rAsp14 could not be established. rOmpA-vaccinated lambs developed a strong specific antibody response on days 28 after vaccination and 14 days post-challenge. Immunofluorescent staining and flow cytometric analysis of peripheral blood mononuclear monocytes revealed no difference between the three groups, but the percentage of CD4+, CD8+, γδ TcR+, λ-Light chain+, CD11b+, CD14+ and MHC II+ cells, within the groups changed during the study, most likely due to the adjuvant or challenge with the bacterium. Although an antigen specific antibody response could be detected against rOmpA and possibly rAsp14, the vaccines seemed to be ineffective in reducing clinical signs and bacterial load caused by A. phagocytophilum. This is the first animal study with recombinant Asp14 and OmpA aimed at obtaining clinical protection against A. phagocytophilum in sheep.

Molecular genetic typing reveals further insights into the diversity of animal-associated Staphylococcus aureus.

Staphylococcus aureus is an important pathogen of man, but is also able to colonize and cause disease in a wide variety of mammals and birds. An extended multilocus sequencing approach, involving multilocus sequence typing (MLST), sas typing, spa typing and agr typing, was used to examine the molecular diversity of 118 S. aureus isolates recovered from a range of host species and to compare these data with the known diversity of human-derived isolates. MLST revealed that the commonest animal-associated MLST types were ST133, ST5, ST71, ST97, ST126 and ST151. ST133 appears to be an ungulate-animal-specific genotype, as no evidence of ST133 associating with humans has yet been found in the literature. Novel and unique sas alleles were identified in the animal-associated strains that may represent animal-associated sas alleles. However, sas typing exhibited a lower typeability than MLST for the animal strains (91.3 %). Phylogenetic analyses using neighbour-joining and maximum-parsimony trees localized ruminant-associated MLST lineages to both previously identified S. aureus subspecies aureus subgroups, thus explaining the finding of all four agr types within the ruminant-associated strains. S. aureus isolates recovered from chickens and rabbits were genotypically more similar to known human genotypes than the ruminant-associated lineages.

Clinical mastitis in ewes; bacteriology, epidemiology and clinical features.

BACKGROUND: Clinical mastitis is an important disease in sheep. The objective of this work was to identify causal bacteria and study certain epidemiological and clinical features of clinical mastitis in ewes kept for meat and wool production. METHODS: The study included 509 ewes with clinical mastitis from 353 flocks located in 14 of the 19 counties in Norway. Clinical examination and collection of udder secretions were carried out by veterinarians. Pulsed-field gel electrophoresis (PFGE) was performed on 92 Staphylococcus aureus isolates from 64 ewes. RESULTS AND CONCLUSION: S. aureus was recovered from 65.3% of 547 clinically affected mammary glands, coagulase-negative staphylococci from 2.9%, enterobacteria, mainly Escherichia coli, from 7.3%, Streptococcus spp. from 4.6%, Mannheimia haemolytica from 1.8% and various other bacteria from 4.9%, while no bacteria were cultured from 13.2% of the samples. Forty percent of the ewes with unilateral clinical S. aureus mastitis also had a subclinical S. aureus infection in the other mammary gland. Twenty-four of 28 (86%) pairs of S. aureus isolates obtained from clinically and subclinically affected mammary glands of the same ewe were indistinguishable by PFGE. The number of identical pairs was significantly greater than expected, based on the distribution of different S. aureus types within the flocks. One-third of the cases occurred during the first week after lambing, while a second peak was observed in the third week of lactation. Gangrene was present in 8.8% of the clinically affected glands; S. aureus was recovered from 72.9%, Clostridium perfringens from 6.3% and E. coli from 6.3% of the secretions from such glands. This study shows that S. aureus predominates as a cause of clinical ovine mastitis in Norway, also in very severe cases. Results also indicate that S. aureus is frequently spread between udder halves of infected ewes.

Lymphocyte subpopulations and neutrophil function in calves during the first 6 months of life.

Fifteen clinically healthy calves were sampled every week during the first 5 weeks of life and thereafter every month until the age of 6 months. The percentages and absolute values of CD4+, CD8+ gammadelta TCR+ and WC1+ T cells, CD21+ B cells and NKp46+ NK cells were determined by flow cytometry, and the expression of the interleukin-2 receptor alpha chain (CD25) was measured to assess the level of activation of the lymphocyte subpopulations. Neutrophil phagocytosis, respiratory burst and bactericidal activity were measured in five different neutrophil function assays. Most of the parameters examined reached a stable level during the first 6 months of life. The proportions of CD4+ and CD8+ lymphocytes remained relatively stable during the study period, while there was a moderate decrease in the relative percentage of gammadelta T cells from birth to approximately 5 months of age. However, the absolute numbers of gammadelta T cells per millilitre of blood remained stable throughout the study period and did not display significant variation with age. The percentage of cells expressing the B-cell maturation marker CD21 increased significantly over the first 5 months of life. The proportion of NK cells showed substantial variation during the study. Marked differences in the relative proportions of the lymphocyte subpopulations were noted between the individual calves, and the individual ranking of the animals was largely maintained over time. CD25 expression was detected on a mean of 6.6% of the CD4+ cells, while a lower percentage of the other lymphocyte subpopulations expressed this receptor. Phagocytic activity was demonstrated in approximately 90% of the neutrophils, and this proportion remained stable during the entire study period, while respiratory burst activity showed a moderate decrease during the first 2 months of life. The present study shows that the T-cell subpopulations are present in peripheral blood of calves at levels comparable with adult values, while the B-cell population increases significantly with age. The decrease in the relative percentage of gammadelta T cells appears to be attributable to an increase in the absolute numbers of CD4+ and CD21+ cells, rather than a change in absolute gammadelta T-cell numbers. Furthermore, the results indicate that the neutrophilic granulocytes are functional and able to mount an effective response in young calves from the first week of life.

Staphylococcus aureus enterotoxin D is secreted in milk and stimulates specific antibody responses in cows in the course of experimental intramammary infection.

An enterotoxin D (SED)-producing strain of Staphylococcus aureus was used to infect one mammary gland of each of 17 lactating dairy cows. All glands became infected and shed bacteria over a sampling period of 3 weeks. Serum and milk antibodies specific for SED were monitored by an enzyme-linked immunosorbent assay for 12 weeks. Elevated anti-SED antibodies were detected in all cows after infection, and immunoglobulin of the G2 subclass comprised most of the specific serum response. SED was detected in mastitic milk samples from two cows at levels of 5 to 10 ng/ml. An in vitro lymphocyte proliferation assay showed that SED at levels below 10 pg/ml induced proliferation of bovine lymphocytes and that sheep antiserum specific for SED neutralized this proliferative response. Sera obtained from the cows pre- and postinfection inhibited lymphocyte proliferation at SED concentrations of 10 and 50 ng/ml, respectively. The addition of SED to whole blood or to isolated neutrophils had no significant effect on neutrophil function in vitro. The results show that SED is secreted during mammary gland infection, is mitogenic for bovine lymphocytes, and stimulates the production of specific antibodies.

Genetic diversity of Staphylococcus aureus isolated from ovine intramammary infections in Norway.

Three hundred and eighty-four Staphylococcus aureus isolates obtained from mammary secretions from 332 ewes kept for meat production were typed by pulsed-field gel electrophoresis (PFGE). The ewes were from 242 flocks located in 13 counties distributed in four regions of Norway. In total, 64 different pulsotypes were identified, 31 of these were represented by a single isolate. Fifty-nine percent of the isolates belonged to one of five closely related pulsotypes. This group of pulsotypes occurred in all the counties. Although widely disseminated, the proportions of the prevalent and closely related pulsotypes differed between the regions. Nine pulsotypes were unique to single regions but the number of isolates belonging to each of these pulsotypes was low. Resistance to penicillin was found in only 3 of the 384 S. aureus isolates. These represented three different single banding patterns, not related to any of the prevalent pulsotypes found.

Staphylococcus aureus capsular polysaccharide types 5 and 8 reduce killing by bovine neutrophils in vitro.

Isogenic variants of Staphylococcus aureus strain Reynolds expressing either no capsule or capsular polysaccharide (CP) type 5 (CP5) or type 8 (CP8) were used to assess the effect of CP on bacterial killing and the respiratory burst of bovine neutrophils. The effects of antisera specific for CP5 and CP8 were also evaluated. The killing of live bacteria by isolated neutrophils was quantified in a bactericidal assay, while the respiratory burst after stimulation with live bacteria in whole blood was measured by flow cytometry. The expression of a CP5 or CP8 capsule protected the bacteria from being killed by bovine neutrophils in vitro (P <0.001), and the capsule-expressing variants did not stimulate respiratory burst activity in calf whole blood. The addition of serotype-specific antisera increased the killing of the capsule-expressing bacteria and enhanced their stimulating effect in the respiratory burst assay (P <0.01). When the S. aureus variants were grown under conditions known not to promote capsule expression, there were no significant differences between them. The present study demonstrates that the expression of S. aureus CP5 or CP8 confers resistance to opsonophagocytic killing and prevents the bacteria from inducing respiratory burst of bovine neutrophils in vitro and that these effects can be reversed by the addition of serotype-specific antisera.

Flow cytometric measurement of neutrophil respiratory burst in whole bovine blood using live Staphylococcus aureus.

A rapid and simple method for measurement of respiratory burst in neutrophil granulocytes in whole bovine blood is described. The respiratory burst was stimulated by live Staphylococcus aureus, and the production of reactive oxygen species quantified by the conversion of intracellular dihydrorhodamine 123 to the green fluorescent rhodamine 123, measured by flow cytometry. Assay conditions, including bacterial and dihydrorhodamine 123 concentrations and incubation time, were determined. Repeatability and precision of the method were assessed by testing parallel samples from clinically healthy dairy cows. In vitro and in vivo inhibition of respiratory burst was investigated, and labelling with a granulocyte marker antibody was performed. Stimulation with live S. aureus induced green fluorescence in the neutrophil granulocytes in a whole blood preparation. The fluorescence intensity increased with increasing bacterial concentration and increasing incubation time. Agreement analysis showed that the method gave repeatable results, and the intra-assay variability of the method was relatively low. The method is considered a useful technique for measurement of neutrophil respiratory burst in whole bovine blood.

Repeatability of flow cytometric and classical measurement of phagocytosis and respiratory burst in bovine polymorphonuclear leukocytes.

Five methods for measurement of phagocytosis and respiratory burst activity of bovine blood polymorphonuclear leukocytes (PMNs) were evaluated. Eight cows were repeatedly sampled over a two week period and parallel samples tested in all five assays to assess the repeatability and stability of the methods. In the flow cytometric phagocytosis assay, ingestion of fluorescein labeled bacteria was measured, and in the flow cytometric assay for respiratory burst, oxidation of a dye by reactive oxygen species was recorded. In the classical assays, bactericidal effect on opsonized, live bacteria was quantified by the conversion of an indicator substance, superoxide anion production was assayed by the reduction of cytochrome c, whereas myeloperoxidase activity was determined with a radioactive iodination assay. The results showed that the Phagotest, Bursttest, cytochrome c and iodination assays gave repeatable results when samples were run in the same setup on the same day. Although day-to-day variability was significant in all assays, the described methods comprise a panel of useful tests for the evaluation of phagocytosis and respiratory burst activity in bovine PMNs. The flow cytometric methods represent a convenient alternative to the classical methods for measurement of phagocytosis and respiratory burst in bovine blood PMNs.