Mycobacterium avium subspecies paratuberculosis (Map) is the cause of Johne’s disease, a chronic enteritis of cattle. A significant knowledge gap is how persistence of Map within the intestinal tract after infection contributes to progression of disease. To address this, we exposed calves to Map by direct ileocecal Peyer’s patch injection. Our objective was to characterize the persistence of Map in tissues, associated intestinal lesions, fecal Map shedding, and serum antibody responses, through the first 28-weeks post-inoculation (wpi). Previous work using this model showed 100% rate of Map infection in intestine and lymph node by 12 wpi. We hypothesized that direct inoculation of Map into the distal small intestine would induce intestinal Map infection with local persistence and progression towards clinical disease. However, our data show decreased persistence of Map in the distal small intestine and draining lymph nodes. We identified Map in multiple sections of distal ileum and draining lymph node of all calves at 4 and 12 wpi, but then we observed reduced Map in distal ileum at 20 wpi, and by 28 wpi we found that 50% of animals had no detectable Map in intestine or the lymph node. This provides evidence of resilience to Map infection following direct intestinal Map inoculation. Further work examining the immune responses and host–pathogen interactions associated with this infection model are needed to help elicit the mechanisms underlying resilience to Map infection.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the nasal mucosa of sheep and goats and is associated with enzootic nasal tumour virus (ENTV). As ENA is a common disease in North America and there are no vaccines against ENTV-1, diagnostic tests that can identify infected animals and assist with eradication and disease surveillance efforts are greatly needed. In this study, we endeavoured to develop a novel, non-invasive diagnostic tool that could be used not only to validate clinical signs of ENA but also to detect ENTV-1 infection prior to the onset of disease signs (i.e. pre-clinical diagnosis). Cytology, serology and reverse transcription (RT)-PCR-based diagnostic methods were investigated. Although the cytology-based assay was able to detect ENTV-1 infection in some animals, it had poor sensitivity and specificity and thus was not developed further as an ante-mortem diagnostic method. Three different assays, including ELISA, Western blotting and virus neutralization, were developed to detect the presence of ENTV-1-specific antibodies in sheep serum. Whilst a surprisingly large number of sheep mounted an antibody-mediated immune response against ENTV-1, and in some cases neutralizing, correlation with disease status was poor. In contrast, RT-PCR on RNA extracted from nasal swabs reliably detected exogenous ENTV-1 sequences, did not amplify endogenous ovine betaretroviral sequences, demonstrated high concordance with immunohistochemical staining for ENTV-1 envelope protein, and had perfect sensitivity and specificity. This report describes a practical and highly specific RT-PCR technique for the detection of clinical and pre-clinical ENA that may prove beneficial in future control or eradication programmes.

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BackgroundEnzootic nasal tumor virus (ENTV-1) is an exogenous betaretrovirus of sheep that transforms epithelial cells lining the ethmoid turbinates leading to a disease called enzootic nasal adenocarcinoma (ENA). A unique feature of ENA is the apparent absence of a specific humoral immune response to the virus, despite the highly productive infection in nasal tumors. The sheep genome contains approximately 27 copies of endogenous ovine betaretroviral sequences (enJSRVs) and expression of enJSRVs in the ovine placenta and uterine endometrium throughout gestation is thought to induce immunological tolerance to exogenous ovine betaretroviruses, a factor that may influence the likelihood of exogenous ENTV infection and disease outcome. Nevertheless, we recently demonstrated the presence of neutralizing antibodies directed against the ENTV-1 envelope glycoprotein in sheep naturally exposed to ENTV-1.FindingsHere, we employed an ENTV-1 envelope glycoprotein surface subunit specific ELISA and a virus neutralization assay to monitor serum antibody responses to ENTV-1 in a group of lambs experimentally infected with ENTV-1 virus containing filtered ENA tumor homogenate. Seroconversion and development of neutralizing antibodies was detected in one of six experimentally infected lambs.ConclusionsOur results demonstrate that sheep can respond immunologically and seroconvert following ENTV-1 infection suggesting that anti-viral immune responses may play a role in the development of ENA.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease, an enteric infection of ruminants that causes significant economic burden for dairy and beef producers. Efforts to control MAP in endemic herds typically focus on herd management practices such as limiting exposure or early culling of infected animals and, occasionally, vaccination. The ionophore monensin sodium may have protective effects against MAP both in vivo and in vitro; however, this has not been thoroughly evaluated experimentally. Using a direct intestinal MAP challenge model, we have observed similarities regarding persistence of MAP in tissues and apparent resilience to infection compared with experimental oral infection or natural disease.Here we sought to investigate the effects of oral monensin supplementation in experimentally MAP-infected calves. We examined the persistence of MAP in the intestinal tissues, MAP-induced intestinal inflammation, fecal MAP shedding, and seroconversion using a commercial serologic assay. Monensin-supplemented MAP-infected calves demonstrated evidence for resilience to MAP infection earlier in this study compared with monensin-free MAP-infected calves. However, statistical modeling did not identify a significant effect of monensin on outcomes of infection, and more work is required to understand how monensin affects early tissue colonization of MAP in calves.