Samples from the mesenteric lymph nodes (MS LNs) and ileocecal valves (ICV) of 105 goats, comprising 61 non-vaccinated and 44 vaccinated against Mycobacterium avium subspecies paratuberculosis (MAP), were collected at slaughter from a farm with a confirmed history of paratuberculosis (PTB). These goats had subclinical infections. PTB-compatible lesions in the MS LNs, ICV lamina propria (LP), and Peyer\'s patches (PPs) were graded separately. Furthermore, the load of acid-fast bacilli was quantified using Ziehl-Neelsen staining (ZN), MAP antigens by immunohistochemistry (IHC), and MAP DNA by PCR targeting the IS900 sequence. Gross PTB-compatible lesions were found in 39% of the goats, with 31.72% vaccinated (V) and 68.29% non-vaccinated (nV). Histopathological lesions induced MAP were observed in 58% of the animals, with 36.07% vaccinated and 63.93% non-vaccinated. The inclusion of histopathology as a diagnostic tool led to a 28% increase in diagnosed cases in MS LNs and 86.05% in ICV. Grade IV granulomas with central mineralization and necrosis were the most common lesions in MS LNs. In the ICV, mild granulomatous enteritis with multifocal foci of epithelioid macrophages was predominant, occurring more frequently in the PPs than in the LP. Furthermore, statistical differences in the presence of histopathological lesions between vaccinated and non-vaccinated goats were noted in MS LNs, ICV LPs, and ICV PPs. Non-vaccinated animals showed higher positivity rates in ZN, IHC, and PCR tests, underscoring the benefits of anti-MAP vaccination in reducing PTB lesions and bacterial load in target organs. Our findings emphasize the necessity of integrating gross and histopathological assessments with various laboratory techniques for accurate morphological and etiological diagnosis of PTB in both vaccinated and non-vaccinated goats with subclinical disease. However, further studies are required to refine sampling protocols for subclinical PTB in goats to enhance the consistency of diagnostic tools.

Johne\'s disease (JD), a chronic, infectious enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), affects wild and domestic ruminants. There is no cure or effective prevention, and current vaccines have substantial limitations, leaving this disease widespread in all substantial dairy industries causing economic, and animal welfare implications. Mycobacteriophages (MPs) have been gaining interest in recent years and are proposed as a promising solution to curtailing MAP infection. Using a well-validated infection model, we have demonstrated the preventative potential of MPs to protect dairy calves against MAP infection. Calves were supplemented daily with a phage cocktail from birth till weaning at 2 m of age and inoculated with MAP at 2 wk of age. Infection status was measured for 4.5 mo through blood, fecal, and postmortem tissue samples. Our findings highlight the remarkable efficacy of orally administered MPs. Notably, fecal shedding of MAP was entirely eliminated within 10 wk, in contrast to the infected control group where shedding continued for the entirety of the trial period. Postmortem tissue culture analysis further supported the effectiveness of MPs, with only 1 out of 6 animals in the phage-treated group testing positive for MAP colonized tissues compared to 6 out of 6 animals in the infected control group. Additionally, plaque assay results demonstrated the ability of phages to persist within the intestinal tract. Collectively, these results underscore the potential of orally administered MP cocktails as a highly effective intervention strategy to combat JD in dairy calves and by extension in the dairy industry.

Mycobacterium avium subspecies paratuberculosis (MAP) infection leads to chronic, persistent granulomatous enteritis, causing prolonged diarrhoea and emaciation. The disease is managed using medications such as antibiotics, live vaccines, mycobacteriophage therapies and other treatments; however, a notable proportion of affected animals do not show improvement with this approach. We hypothesise that immunoinhibitory receptors TIM-3 (T cell immunoglobulin mucin protein-3) and PD-1 (Programmed death receptor 1) may be upregulated on Peripheral blood mononuclear cells (PBMCs) of MAP-seropositive bovines, potentially contributing to immune exhaustion. Samples (blood and faeces) were collected from 32 diarrhoeic bovines suspected of MAP infection; eight apparently healthy buffaloes from the dairy farm at Hisar, Haryana and from 14 cows (suffering from chronic diarrhoea, weakness and emaciation) housed in stray cattle shed. MAP infection was estimated using indigenous ELISA (i-ELISA), faecal IS900 PCR, culture and acid-fast staining. TIM-3 and PD-1 gene expression on PBMCs were determined using qRT-PCR. TIM3 expression was relatively higher (~400-fold, 330-fold, 112-fold, 65-fold and 16-fold) in 5 chronically diarrhoeic PBMCs samples (MAP-seropositive), and higher PD-1 expression (around ~7-fold, 1.75-fold, 2.5-fold, 7.6-fold) was recorded in 4 diarrhoeic MAP-seropositive animals, compared to apparently healthy and other MAP-seronegative diarrhoeic animals. High co-expression of TIM-3 and PD-1 levels was also recorded in chronically diarrhoeic, emaciated stray cattle. Understanding immune responses in field conditions might aid in the therapeutic management of paratuberculosis.

The considerable epidemiological and economic implications of paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), have placed importance on control efforts aimed at preventing MAP transmission. In this context, Italy issued national guidelines for the control and status certification of MAP in dairy cattle in 2013.
We assessed the long-term outcomes of the Italian MAP control programme for 14 dairy farms located in northern Italy by retrospectively reviewing the results of yearly serological tests, presence of clinical cases, MAP faecal shedding in serologically positive animals, farm management and health ranking as indicators of herd health between 2014 and 2021.
A significantly higher number of serologically positive animals were observed between 2014 and 2016 than between 2017 and 2021, as well as an improving trend in the paratuberculosis health ranking for nine of the 14 farms. No clinical cases were reported. MAP shedding was detected in 9.4% of serologically positive animals. Discarding colostrum and prioritised culling of seropositive animals assisted by adoption of standardised serological testing were presumed to have a key role in MAP control, despite the reluctance of some farmers to address hygienic issues and improve the separation of calves from adult animals.
The small number of farms included in this study and the fact that these were not randomly selected may limit the generalisability of the findings.
The Italian paratuberculosis control plan has provided measures to limit the uncontrolled spread of MAP infection within and between herds by promoting animal trading between farms certified as negative or low risk.

OBJECTIVE: Ante-mortem diagnosis of Johne\'s disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is normally achieved through faecal culture, PCR, or serological tests, but agreement as to which samples are positive for Johne\'s disease is often poor and sensitivities are low, particularly in early-stage infections. The potential solution: Mycobacterial cells contain very complex characteristic mixtures of mycolic acid derivatives that elicit antibodies during infection; this has been used to detect infections in humans. Here, we explore its application in providing an assay differentiating infected from vaccinated animals (DIVA assay) for Johne\'s disease in cattle.
METHODS: Antibody responses to different classes of mycolic acid derivatives were measured using ELISA for serum from cattle positive for MAP by both faecal PCR and commercial serum ELISA, or just by PCR, and from animals from herds with no history of Johne\'s disease, bovine tuberculosis reactors, BCG-vaccinated, BCG-vaccinated and M. bovis-infected, and Gudair-vaccinated animals.
RESULTS: The best-performing antigens, ZAM295 and ST123-the latter a molecule present in the cells of MAP but not of Mycobacterium bovis-achieved a sensitivity of 75% and 62.5%, respectively, for serum from animals positive by both faecal PCR and a commercial MAP serum ELISA, at a specificity of 94% compared to 80 no-history negatives. Combining the results of separate assays with two antigens (ST123 and JRRR121) increased the sensitivity/specificity to 75/97.5%. At the same cut-offs, animals vaccinated with Gudair or BCG vaccines and bTB reactors showed a similar specificity. The specificity in BCG-vaccinated but M. bovis-infected animals dropped to 85%. Combining the results of two antigens gave a sensitivity/specificity of 37.5/97.5% for the full set of 80 PCR-positive samples, detecting 30 positives compared 16 for IDEXX.
CONCLUSIONS: Serum ELISA using synthetic lipids distinguishes effectively between MAP-negative cattle samples and those positive by both PCR and a commercial MAP serodiagnostic, without interference by Gudair or BCG vaccination. It identified almost twice as many PCR positives as the commercial serodiagnostic, offering the possibility of earlier detection of infection.

The complete genome sequence of the most ancestral type SI strain of Mycobacterium avium subspecies paratuberculosis 6756, isolated from a sheep, was determined. The genome was sequenced using PacBio technology, yielding a genome size of 4,830,294 nucleotides with no identified plasmids.

Analysis of the primary and recall responses to a membrane molecule (MMP), encoded by MAP2121c demonstrated that tri-directional signaling between the antigen-presenting cell (APC), CD4 and CD8 is essential for eliciting a CD8 cytotoxic T cell (CTL) response against Mycobacterium avium subsp. paratuberculosis. As reported here, RNA-sequencing was used to initiate the characterization of the signaling pathways involved in eliciting the development of CD8 CTL, starting with the characterization of the activation status of genes in monocyte-derived macrophages (MoMΦ) following uptake and processing MMP for the presentation of antigenic epitopes to CD4 and CD8 T cells. Activation status was compared with the uptake and processing of LPS, a nonspecific stimulator of macrophages. 1609 genes were identified that were upregulated, and 1277 were downregulated three hours after uptake and processing MMP. No significant difference was observed in the cytokine genes selected for analysis of the signaling that must occur between APC, CD4, and CD8 for the development of CTL. The initial observations indicate screening of the transcriptome should include genes involved in signaling between APC and CD4, and CD8 regardless of their activation status. Four genes of interest in this study, IL12A, IL12B, IL15, and IL23A, were not significantly different from control values. The initial studies also indicate MoMΦ can be included with dendritic cells and monocyte-derived dendritic cells for further analysis of the tri-directional signaling required for the development of CTL.
A análise das respostas primárias e de recall a uma molécula de membrana (MMP), codificada por MAP2121c demonstrou que a sinalização tridirecional entre a célula apresentadora de antígeno (APC), CD4 e CD8 é essencial para provocar uma resposta de células T citotóxicas CD8 (CTL) contra Mycobacterium avium subsp. paratuberculose. Conforme relatado aqui, o sequenciamento de RNA foi usado para iniciar a caracterização das vias de sinalização envolvidas na indução do desenvolvimento de CTL CD8, começando com a caracterização do status de ativação de genes em macrófagos derivados de monócitos (MoMΦ) após captação e processamento de MMP para a apresentação de epítopos antigênicos às células T CD4 e CD8. O status de ativação foi comparado com a captação e processamento de LPS, um estimulador inespecífico de macrófagos. Foram identificados 1.609 genes que foram regulados positivamente e 1.277 foram regulados negativamente três horas após a captação e processamento de MMP. Nenhuma diferença significativa foi observada nos genes de citocinas selecionados para análise da sinalização que deve ocorrer entre APC, CD4 e CD8 para o desenvolvimento de CTL. As observações iniciais indicam que o rastreio do transcriptoma deve incluir genes envolvidos na sinalização entre APC e CD4 e CD8, independentemente do seu estado de activação. Quatro genes de interesse neste estudo, IL12A, IL12B, IL15 e IL23A, não foram significativamente diferentes dos valores de controle. Os estudos iniciais também indicam que o MoMΦ pode ser incluído com células dendríticas e células dendríticas derivadas de monócitos para análise adicional da sinalização tridirecional necessária para o desenvolvimento de CTL.

UNASSIGNED: The etiology of Multiple sclerosis (MS) is complicated and can be affected by several environmental factors, such as Mycobacterium avium subspecies paratuberculosis (MAP) infection in genetically predisposed individuals. The link between MAP and MS depends on host genetic and epigenetic aspects and population-based features that require further investigation. We aimed to study the possible role of MAP in triggering MS using molecular and serological methods.
UNASSIGNED: This case-control study examined 200 blood samples (100 MS patients and 100 HCs) to search for the MAP-specific IS900 gene. In addition, ELISA was conducted to determine the humoral response against MAP_402718-32 and its human IRF5424-434 peptide homolog.
UNASSIGNED: The frequency of MAP detection based on the molecular method in MS patients and HCs was 48 % and 13 %, respectively (p < 0.0001). The presence of antibodies against MAP_402718-32 and IRF5424-434 was 55 % and 65 % in MS patients versus 9 % and 7 % in HCs, respectively (p < 0.0001). A good correlation was observed between MAP_4027 and IRF5 antibodies (r = 0.5782, p < 0.0001), indicating that the same antibodies recognized common peptide epitopes.
UNASSIGNED: Our research revealed a significant association between MAP and MS, highlighting the possible role of MAP as an important infection trigger factor of MS. It is hypothesized that cross-reactivity between MAP4027 and IRF5 may dysregulate immune homeostasis.

Johne\'s disease (JD) is a chronic disease of ruminants endemic in the UK and other countries and responsible for large economic losses for the dairy sector. JD is caused by Mycobacterium avium subspecies paratuberculosis (MAP), which typically infects calves that remain latently infected during a long period, making early detection of infection challenging. Cow to calf transmission can occur in-utero, via milk/colostrum or faecal-orally. Understanding of the different transmission routes to calves is important in informing control recommendations. Our aim in this longitudinal study was to measure the association between the transmission routes via the dam and the environment on a calf subsequently testing serologically positive for MAP. The study population comprised of 439 UK dairy calves from 6 herds enrolled between 2012 and 2013. These calves were followed up from birth until 2023. At birth individual calf data was captured. During follow-up, individuals entering the milking herd were quarterly tested for the presence of MAP antibodies using milk ELISA. Cox regression models were used to measure the association between exposure from the dam (in-utero and/or colostrum) or from the environment (long time in dirty yard) and time to first detection of MAP infection. An association between calves born to positive dams and probability of having a MAP positive test result remained after excluding potential MAP transmission via colostrum (Hazard ratio: 2.24; 95% CI: 1.14 - 4.41). Calves unlikely to be infected with MAP via the in-utero or colostrum route, had 3.68 (95% CI: 3.68 1.45-9.33) higher hazard of a positive test result when they stayed longer in a dirty calving area. The effect of the dam infection status on transmission to calves precedes the dam\'s seroconversion and persists after excluding the potential role of transmission via colostrum. The association between time spent in a dirty calving area and probability of a MAP positive test result highlights the role of environmental contamination as a source of infection in addition to the dam.

Paratuberculosis (PTB), caused by Mycobacterium avium subspecies paratuberculosis (MAP), causes significant financial losses in the ruminant industry. The aim of this study is to describe the concomitant pathological findings as well as PTB-induced lesions in 39 naturally infected goats (15 vaccinated and 24 non-vaccinated). All animals exhibited MAP-induced microscopic lesions affecting target organs, although only 62% of those were detected grossly. Mainly concomitant inflammatory pathologies were recognized affecting the hemolymphatic, respiratory and gastrointestinal systems. Non-vaccinated animals exhibited both moderate and marked granulomatous enteritis in contrast with vaccinated ones which presented mild intestinal affection. Our results demonstrate that non-vaccinated animals presented pneumonia in all age groups studied (from 12 up to >48 months old). A significantly higher prevalence of ileocecal valve PTB lesions was detected in non-vaccinated animals with pneumonic lesions (p = 0.027). Furthermore, a reduction of gastrointestinal non-PTB processes was described in vaccinated goats. In conclusion, a PTB infected goat herd can be affected by a wide range of concomitant pathologies, mostly inflammatory in origin. Anatomic pathology is of crucial importance for correct herd diagnosis and histopathology is an indispensable tool for lesion detection. Additionally, anti-MAP vaccination could have a beneficial effect on the reduction of respiratory and gastrointestinal non-PTB diseases.