Assessment of the frequency of Mycobacterium bovis shedding in the faeces of naturally and experimentally TB infected cattle.

AIMS: To assess the prevalence of Mycobacterium bovis bacilli in faecal samples of tuberculous cattle, and to better understand the risk of environmental dissemination of bovine tuberculosis (TB) through the spreading of manure or slurry. METHODS AND RESULTS: Faecal samples were collected from 72 naturally infected cattle with visible lesions of TB that had reacted to the tuberculin skin test and 12 cattle experimentally infected with M. bovis. These were examined by microbial culture and PCR to assess the presence of M. bovis bacilli. There were no positive cultures from any naturally infected test reactor animal. A single M. bovis colony was cultured from a faecal sample from one of the experimentally infected animals. A single PCR positive result was obtained from the faecal sample of one naturally infected test reactor. CONCLUSIONS: The prevalence of M. bovis in the faecal samples of TB-infected cattle was extremely low. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the risk of spreading TB through the use of slurry or manure as an agricultural fertilizer is lower than that suggested in some historical literature. The results could inform a reconsideration of current risk assessments and guidelines on the disposal of manure and slurry from TB-infected herds.

A molecularly defined skin test reagent for the diagnosis of bovine tuberculosis compatible with vaccination against Johne's Disease.

Tuberculin Purified Protein Derivatives (PPDs) exhibit multiple limitations: they are crude extracts from mycobacterial cultures with largely unknown active components; their production depends on culture of mycobacteria requiring expensive BCL3 production facilities; and their potency depends on the technically demanding guinea pig assay. To overcome these limitations, we developed a molecularly defined tuberculin (MDT) by adding further antigens to our prototype reagent composed of ESAT-6, CFP-10 and Rv3615c (DIVA skin test, DST). In vitro screening using PBMC from infected and uninfected cattle shortlisted four antigens from a literature-based list of 18 to formulate the MDT. These four antigens plus the previously identified Rv3020c protein, produced as recombinant proteins or overlapping synthetic peptides, were formulated together with the three DST antigens into the MDT to test cattle experimentally and naturally infected with M. bovis, uninfected cattle and MAP vaccinated calves. We demonstrated significant increases in MDT-induced skin responses compared to DST in infected animals, whilst maintaining high specificity in unvaccinated or MAP vaccinated calves. Further, MDT can also be applied in in vitro blood-based interferon-gamma release assays. Thus, MDT promises to be a robust diagnostic skin and blood test reagent overcoming some of the limitations of PPDs and warrants full validation.

Flock health survey of Irish Texel society breeders and larynx examination in Texel sheep.

BACKGROUND: Laryngeal chondritis is a disease of undetermined aetiology, characterised by oedema, ulceration, abscessation and necrosis of the laryngeal mucosa and cartilage. The initial aim of the study was to document flock health issues identified by Irish pedigree Texel breeders using a questionnaire survey. Additionally, given the reports of breed predisposition for laryngeal chondritis in Texels, a further aim was to identify if laryngeal problems were perceived as an issue. Work was then conducted to identify if pre-clinical laryngeal mucosal pathology was identifiable in Texel sheep showing no overt clinical signs of respiratory disease and if associations existed between laryngeal measurements and laryngeal pathology.Thirty one larynges were collected from a Texel flock that previously had laryngeal chondritis diagnosed in fallen stock. Gross visual inspection was performed to identify and grade (0-5) laryngeal pathology. A series of measurements were then performed on larynges that had been formalin fixed. Associations between independent variables (larynx measurements) and the dependent variable (laryngeal pathology score) were examined. RESULTS: Respiratory disease was the most frequently identified health issue. Farmer-diagnosed 'throat problems' were reported by over 80% of respondents.Laryngeal pathology was noted in Texels showing no overt clinical signs of respiratory disease. Associations between laryngeal measurements and laryngeal pathology were identified relating to the angle between the cranial point of the cricoid cartilage and the vocal process of the arytenoid cartilage. CONCLUSIONS: Mild laryngeal pathology was noted in animals with no overt clinical signs of respiratory disease. Future research should examine whether significant associations between laryngeal measurements and laryngeal pathology identified in the current study can be measured ante mortem, and whether such ante mortem measurements will allow early identification of sheep at risk of developing laryngeal chondritis.

Descriptive analysis of ovine mortality in sentinel sheep flocks in Ireland.

BACKGROUND: Studies of sheep mortality or cause-specific mortality, in Ireland or internationally, are relatively scarce but are important in presenting baseline levels and changing trends of endemic disease. This study assessed sheep mortality and cause-specific mortality in 33 sentinel sheep flocks in Ireland. METHODS: Sentinel flocks were requested to submit carcases of all sheep that died to the regional veterinary laboratories (RVLs) of the Department of Agriculture, Food and Marine during a calendar year (2016). Postmortem examinations were performed on 1247 submissions to Athlone, Kilkenny and Sligo RVLs. RESULTS: The median overall submission rate was 13.8 per cent (range 2.5 per cent-35.8 per cent) per adult female sheep in the flock in January 2016. The median fetal, perinatal, lamb and adult submissions per adult female sheep in the flock in January 2016 were 2.1 per cent (0.0 per cent-15.2 per cent), 3.5 per cent (0.0 per cent-20.0 per cent), 3.0 per cent (0.0 per cent-12.4 per cent) and 2.8 per cent (0.8 per cent-7.1 per cent), respectively. The frequency of detection of categories of postmortem diagnoses in fetuses, perinates, lambs and adults are presented. CONCLUSIONS: Comparisons with existing passive surveillance findings reflect some differences in the relative frequency of detection of certain categories of disease suggesting that sentinel flock surveillance could usefully supplement existing passive animal disease surveillance activities for ovine disease.

The CD4(+) T cell methylome contributes to a distinct CD4(+) T cell transcriptional signature in Mycobacterium bovis-infected cattle.

We hypothesised that epigenetic regulation of CD4(+) T lymphocytes contributes to a shift toward a dysfunctional T cell phenotype which may impact on their ability to clear mycobacterial infection. Combined RNA-seq transcriptomic profiling and Reduced Representation Bisulfite Sequencing identified 193 significantly differentially expressed genes and 760 differentially methylated regions (DMRs), between CD4(+) T cells from M. bovis infected and healthy cattle. 196 DMRs were located within 10 kb of annotated genes, including GATA3 and RORC, both of which encode transcription factors that promote TH2 and TH17 T helper cell subsets respectively. Gene-specific DNA methylation and gene expression levels for the TNFRSF4 and Interferon-γ genes were significantly negatively correlated suggesting a regulatory relationship. Pathway analysis of DMRs identified enrichment of genes involved in the anti-proliferative TGF-ß signaling pathway and TGFB1 expression was significantly increased in peripheral blood leukocytes from TB-infected cattle. This first analysis of the bovine CD4(+) T cell methylome suggests that DNA methylation directly contributes to a distinct gene expression signature in CD4(+) T cells from cattle infected with M. bovis. Specific methylation changes proximal to key inflammatory gene loci may be critical to the emergence of a non-protective CD4(+) T cell response during mycobacterial infection in cattle.

Application of quantitative real-time polymerase chain reaction for the diagnosis of toxoplasmosis and enzootic abortion of ewes.

Toxoplasma gondii and Chlamydophila abortus are the 2 most common infectious causes of ovine abortion worldwide. These obligate intracellular pathogens are associated with severe placentitis leading to abortion or stillbirth in pregnant ewes, and resulting in significant economic losses. The objectives of the current study were the development, validation, and application of a duplex real-time polymerase chain reaction (PCR) assay capable of quantifying the burden of infection by T. gondii and C. abortus in material submitted for diagnostic purposes. The validation was carried out using samples from ewes experimentally infected with these organisms. Based on the numbers of genome copies detected, an arbitrary cutoff level was established to correlate with significant pathological changes sufficient to give rise to abortion. When the PCR assay was applied to samples from 66 Irish farms with naturally occurring outbreaks of ovine abortion, toxoplasmosis and enzootic abortion of ewes (EAE) accounted for 14% and 20% of the farms, respectively, while on 6% of the farms, there was evidence of dual infection. When standard diagnostic techniques including histopathological examination, serological analysis, chlamydial antigen detection, and bacteriological culture, were used on samples from the same farms, toxoplasmosis was diagnosed in 17% of farms, and EAE in 12%; dual infection was diagnosed on 3% of the farms. In general, good agreement was found between the PCR and the standard methods. The duplex real-time PCR assay developed in this study has proved to be a very sensitive and rapid tool that might provide a valuable addition to the methods currently available for routine diagnosis of ovine abortions.

Amniotic and allantoic fluids from experimentally infected sheep contain immunoglobulin specific for Chlamydophila abortus.

Chlamydophila abortus, the aetiological agent of enzootic abortion of ewes (EAE), replicates in trophoblast cells leading to their destruction and dissemination of the bacterium to foetal organs. To further understand the pathogenesis of EAE, amniotic and allantoic fluids were collected from experimentally infected pregnant ewes at 30 (7 samples from each fluid), 35 (8 samples from each fluid), 40 (10 samples from each fluid) and 43 (6 amniotic fluids and 7 allantoic fluids) days post-infection to determine pathogen numbers and other markers of infection. Whilst experimentally infected ewes had characteristic placental lesions, only two amniotic and seven allantoic fluid samples were positive for C. abortus by real-time PCR. In contrast, all amniotic and allantoic fluids were positive for immunoglobulin. Immunoglobulins were generally detected earlier in allantoic fluid than in amniotic fluid and the numbers of samples containing immunoglobulins increased as infection progressed. IgG in amniotic and allantoic fluids was shown to be specific for C. abortus, and reacted with the major outer membrane proteins, polymorphic outer membrane protein and macrophage infectivity potentiator protein. A comparison of two-dimensional immunoblots using purified IgG from the allantoic fluid, amniotic fluid, ewe serum and foetal serum of a C. abortus infected animal at 40 days post infection indicated a pattern of reactivity intermediate between that of the ewe serum and the foetal serum. Results suggest that a maternal source of immunoglobulin is predominant at 30 days post-infection but that foetal derived antibodies may be contributed at a later stage.

Monitoring clinical outcomes, pathological changes and shedding of Chlamydophila abortus following experimental challenge of periparturient ewes utilizing the natural route of infection.

Enzootic abortion of ewes (EAE) caused by Chlamydophila abortus is an important disease resulting in significant lamb loss in most sheep producing countries. Ewes are considered to be naturally infected with C. abortus via the oral-nasal route and may become persistent carriers, shedding during subsequent oestrous cycles and at lambing. The aim of this study was to monitor the clinical outcomes, pathological changes and shedding of C. abortus in 18 periparturient orally infected sheep for two breeding seasons. In the first season, C. abortus was detected by real-time PCR (rt-PCR) in 13/18 conjunctival swabs at oestrus. Three out of the 15 pregnant ewes gave birth to 1 live and 1 dead lamb, and 2 of them aborted. Following parturition/abortion, C. abortus was detected in 12/15 vaginal swabs and in all the collected foetal membranes. However, only those membranes containing high copy numbers of the bacterium displayed the EAE typical lesions. In the second season, none of the 13 pregnant ewes aborted, and 5 of them gave birth to dead or weak lambs. C. abortus was not detected in conjunctival or vaginal swabs at oestrus or parturition. The bacterium was detected at low levels in 36% of the foetal membranes, but with no evidence of histopathological lesions. These results indicate that C. abortus can be detected in a large proportion of animals during the first pregnancy after oral infection. However, this proportion is reduced at the subsequent breeding season, confirming the occurrence of a chronic low level persistent infection in post-abortion/lambing ewes.