Mycobacterium Avium Subspecies Paratuberculosis Research Articles

Lack of Exposure to Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis in Chilean Cervids, and Evidence of a New Mycobacterium-Like Sequence.

Screening of serum and fecal samples from huemul (Hippocamelus bisulcus) and pudu (Pudu puda) from southern Chile for Mycobacterium bovis and Mycobacterium avium paratuberculosis (MAP) found all but four samples Mycobacterium-negative. The positive sequences showed only 92-93% similarity with MAP and were from remote Isla Riesco populations.

Identification of Mycobacterium avium subspecies paratuberculosis in sheep farms in Bayannaoer, Inner Mongolia, China (short communication)

BackgroundParatuberculosis is a widespread chronic infection of Mycobacterium avium subspecies paratuberculosis (MAP) that causes significant economic losses to the sheep industry. The current study investigated this disease, which causes diarrhea in sheep, particularly, in Bayannaoer, Inner Mongolia, China. Diagnosis was based on clinical symptoms, pathological autopsy, histopathological inspection, and serological and molecular methods.ResultsMAP was confirmed using polymerase chain reaction using DNA extracted from tissue and fecal samples. Serum samples from 472 individual sheep were obtained to detect antibodies against MAP using an enzyme-linked immunosorbent assay. MAP antibodies were separately detected in 17.86% (35/196) and 18.48% (51/276) of sheep herds at approximately 6 months and ≥ 1 year of age, respectively. The tissue lesion and pathological section results were consistent with paratuberculosis infection.ConclusionsTo our knowledge, this is the first report of Mycobacterium avium subspecies paratuberculosis seroprevalence in Bayannaoer sheep in Inner Mongolia. Our findings show that MAP is not only prevalent, but also a potential threat to this region. Further investigations, including long-term epidemiological surveillance and isolation are needed for the awareness and effective treatment of paratuberculosis in sheep of Inner Mongolia.

Analysis of Colostrum and Udder Skin Swabs from a Dairy Goat Herd in Germany regarding the Occurrence of Mycobacterium avium Subsp. paratuberculosis.

Simple SummaryThe analysis of transmission routes for paratuberculosis as well as their prevention are critical for effective disease control. The aim of the present study was to assess the importance of colostrum and the udder skin as routes for transmission of Mycobacterium avium subspecies paratuberculosis (MAP) within a dairy goat herd. Swabs of the udder skin and colostrum samples were collected from goats of a paratuberculosis-infected herd during lambing season and analyzed for MAP DNA. Additionally, cultivation of the swabs was performed. MAP could not be detected in the colostrum. A low number of udder skin swabs was positive for MAP DNA but no bacteria could be cultured. Because the detection methods are imperfect, the occurrence of MAP in colostrum or on the udder skin can never be completely excluded. Therefore, present recommendations concerning colostrum and youngstock management should still be re-evaluated, but applied in goat herds within a paratuberculosis control program until the role of colostrum and udder skin for within-herd transmission of MAP is further investigated.Oral intake of Mycobacterium avium subspecies paratuberculosis (MAP) in first days of life is considered to be the main route of infection for paratuberculosis. This can be related to a direct contact to contaminated feces or feeding of MAP containing colostrum. Colostrum is believed to become contaminated either by lactogenic shedding or introduction of MAP from environmental sources. In this pilot study, the presence of MAP in individual and bulk colostrum samples from a paratuberculosis-infected, vaccinated dairy goat herd in Germany and the effect of udder skin disinfection on the MAP load of colostrum were examined. In order to distinguish between lactogenic shedding and fecal contamination, 49 udder skin swabs were cultivated on solid medium whereas 29 swabs were additionally analyzed by qPCR. qPCR was applied on 110 individual colostrum samples collected from 55 goats, one before and one after disinfection with a mycobactericidal disinfectant, and 14 bulk colostrum samples. MAP DNA was detected in 10.3% (3/29) of the swab samples, but no viable MAP was cultivated from any sample. These results indicate a low-level MAP contamination of the udder skin and colostrum of milking goats suggesting a low risk of MAP transmission via these routes.

Fetuin as a potential serum biomarker to detect subclinical shedder of bovine paratuberculosis

Paratuberculosis (PTB) is a chronic contagious granulomatous enteritis of wild and domestic ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). PTB causes considerable economic losses to the dairy industry through decreased milk production and premature culling. PTB-affected cattle undergo a subclinical stage without clinical signs and initiate fecal shedding of MAP into the environment. Current diagnostic tools have low sensitivity for the detection of subclinical PTB infection. Therefore, alternative diagnostic tools are required to improve the diagnostic sensitivity of subclinical PTB infection. In this study, we performed ELISA for three previously identified host biomarkers (fetuin, alpha-1-acid glycoprotein, and apolipoprotein) and analyzed their diagnostic performance with conventional PTB diagnostic methods. We observed that serum fetuin levels were significantly lowered in the subclinical shedder and clinical shedder groups than in the healthy control group, indicating its potential utility as a diagnostic biomarker for bovine PTB. Also, fetuin showed an excellent discriminatory power with an AUC = 0.949, a sensitivity of 92.6%, and a specificity of 94.4% for the detection of subclinical MAP infection. In conclusion, our results demonstrated that fetuin could be used as a diagnostic biomarker for enhancing the diagnostic sensitivity for the detection of subclinical MAP infections that are difficult to detect based on current diagnostic methods.

Bayesian estimation of sensitivity and specificity of fecal culture, fecal PCR and serum ELISA for diagnosis of Mycobacterium avium subsp. paratuberculosis infections in sheep

The objective of the present study was to evaluate the diagnostic accuracy of the individual fecal culture (IFC), fecal PCR (FPCR), and serum ELISA for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infections in sheep from four governorates in Egypt, using a latent class model (LCM) fitted within a Bayesian framework. Furthermore, the within-governorate prevalence of MAP infection in sheep was estimated as a secondary objective. Fecal and blood samples were collected from 370 sheep in four Egyptian governorates. Fecal samples were analyzed by IFC and RT-PCR based on ISMav2 gene, while ELISA was performed on serum samples. The sensitivity (Se) and specificity (Sp) of the three diagnostic tests were estimated using a three-tests-four-populations Bayesian LCM to obtain posterior estimates [medians and 95% Bayesian credible intervals (95% BCI)] for each parameter. The median Se estimates (95% BCI) for IFC, FPCR, and serum ELISA were 31.8% (22.8–41.4), 49.7% (31.8–79.9), and 61.2% (39.8–81.4), respectively. The median Sp estimates (95% BCI) for IFC, FPCR, and serum ELISA were 97.7% (96.1–98.9), 97.7% (95.6–99.5), and 98.4% (96.9–99.3), respectively. The median within-governorate paratuberculosis prevalence (95% BCI) was 5.2% (1.1–13.6), 8.4% (2.9–17.7), 9.4% (3.0–20.7), and 18.2% (10.5–29.5) for the Gharbia, Menoufia, Qalyubia, and Kafr El-Sheikh governorates, respectively. In conclusion, at a ratio of the optical density (OD) sample/OD positive control threshold of > 45%, ELISA showed the highest Se among the three tests and comparable Sp to IFC and FPCR. The test ELISA evaluated in this study is an interesting alternative for detecting MAP in sheep due to its higher Se, lower cost, and shorter turnaround laboratory time compared to IFC and FPCR.

The Development of 3D Bovine Intestinal Organoid Derived Models to Investigate Mycobacterium Avium ssp Paratuberculosis Pathogenesis.

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne's Disease, a chronic enteritis of ruminants prevalent across the world. It is estimated that approximately 50% of UK dairy herds are infected with MAP, but this is likely an underestimate of the true prevalence. Infection can result in reduced milk yield, infertility and premature culling of the animal, leading to significant losses to the farming economy and negatively affecting animal welfare. Understanding the initial interaction between MAP and the host is critical to develop improved diagnostic tools and novel vaccines. Here we describe the characterisation of three different multicellular in vitro models derived from bovine intestinal tissue, and their use for the study of cellular interactions with MAP. In addition to the previously described basal-out 3D bovine enteroids, we have established viable 2D monolayers and 3D apical-out organoids. The apical-out enteroids differ from previously described bovine enteroids as the apical surface is exposed on the exterior surface of the 3D structure, enabling study of host-pathogen interactions at the epithelial surface without the need for microinjection. We have characterised the cell types present in each model system using RT-qPCR to detect predicted cell type-specific gene expression, and confocal microscopy for cell type-specific protein expression. Each model contained the cells present in the original bovine intestinal tissue, confirming they were representative of the bovine gut. Exposure of the three model systems to the K10 reference strain of MAP K10, and a recent Scottish isolate referred to as C49, led to the observation of intracellular bacteria by confocal microscopy. Enumeration of the bacteria by quantification of genome copy number, indicated that K10 was less invasive than C49 at early time points in infection in all model systems. This study shows that bovine enteroid-based models are permissive to infection with MAP and that these models may be useful in investigating early stages of MAP pathogenesis in a physiologically relevant in vitro system, whilst reducing the use of animals in scientific research.Bos taurus: urn:lsid:zoobank.org:act:4C90C4FA-6296-4972-BE6A-5EF578677D64

Prevalencia de Mycobacterium avium subsp. paratuberculosis y factores de riesgo asociados en lecherías bajo sistemas de sala de ordeño mecánico en Antioquia, Colombia

The present study aimed to determine Mycobacterium avium subsp. paratuberculosis (MAP) prevalence according to environmental samples and to explore the herd-level risk factors associated to MAP infection in dairy herds under mechanical milking parlor and pasture grazing-based systems. The study herds (n= 94) were located in 60 districts from five municipalities in the Northern region of the province of Antioquia, Colombia. Herds were visited once in 2016 to collect two composite environmental samples and to complete a risk assessment questionnaire. MAP identification was carried out using a quantitative real-time PCR method based on the IS900 sequence. A herd was considered as MAP-positive if one or both of the environmental samples were found positive by the molecular technique. The information on risk factors was analyzed using a multivariable logistic regression model. The apparent herd-level prevalence found was 14.9 % (14/94; 95 % CI: 7.7-22.1), ranging from 0 to 33.3 % at municipality-level. Herds where other than Pure-Holstein breeds were predominant (i.e. Jersey, Jersey crossbreeds) were more likely to be MAP-qPCR positively infected than those on which where pure-Holstein cattle was predominant (OR=3.7; 95 % CI: 1.1-15.2). The present study reports MAP prevalence in dairy herds in the province of Antioquia (Colombia), and the association between MAP environmental positivity with the predominant breed in the herd.

Modelling transmission of Mycobacterium avium subspecies paratuberculosis between Irish dairy cattle herds

Bovine paratuberculosis is an endemic disease caused by Mycobacterium avium subspecies paratuberculosis (Map). Map is mainly transmitted between herds through movement of infected but undetected animals. Our objective was to investigate the effect of observed herd characteristics on Map spread on a national scale in Ireland. Herd characteristics included herd size, number of breeding bulls introduced, number of animals purchased and sold, and number of herds the focal herd purchases from and sells to. We used these characteristics to classify herds in accordance with their probability of becoming infected and of spreading infection to other herds. A stochastic individual-based model was used to represent herd demography and Map infection dynamics of each dairy cattle herd in Ireland. Data on herd size and composition, as well as birth, death, and culling events were used to characterize herd demography. Herds were connected with each other through observed animal trade movements. Data consisted of 13 353 herds, with 4 494 768 dairy female animals, and 72 991 breeding bulls. We showed that the probability of an infected animal being introduced into the herd increases both with an increasing number of animals that enter a herd via trade and number of herds from which animals are sourced. Herds that both buy and sell a lot of animals pose the highest infection risk to other herds and could therefore play an important role in Map spread between herds.

Mycobacterium avium subsp. paratuberculosis exploits miRNA expression to modulate lipid metabolism and macrophage polarisation pathways during infection

Pathogenic mycobacteria including Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne’s disease, manipulate host macrophages to persist and cause disease. In mycobacterial infection, highly plastic macrophages, shift between inflammatory M1 and permissive M2 phenotypes which alter the disease outcome and allow bacteria to survive intracellularly. Here we examine the impact of MAP infection on polarised macrophages and how increased lipid availability alters macrophage phenotype and bacterial persistence. Further, we assess if host microRNA (miRNA) are sensitive to macrophage polarisation state and how MAP can drive their expression to overcome innate responses. Using in vitro MAP infection, we find that increasing lipid availability through supplementing culture media with exogenous lipid increases cellular nitric oxide production. Lipid-associated miRs -19a, -129, -24, and -24-3p are differentially expressed following macrophage polarisation and lipid supplementation and are further regulated during MAP infection. Collectively, our results highlight the importance of host lipid metabolism in MAP infection and demonstrate control of miRNA expression by MAP to favour intracellular persistence.

Immunological and Hemato-biochemical alterations in diarrhoeic buffaloes screened for Mycobacterium avium subspecies paratuberculosis infection using ‘indigenous ELISA kit’

Paratuberculosis (ParaTB) in ruminants, commonly known as Johne’s disease (JD)is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Characteristic hallmark of JD is chronic inflammation of intestines. Similar infection of MAP in human population is called Crohn's disease (CD). In sub-tropical countries like India, domestic buffaloes are important source of milk and meat and vital for thenutritional security of human beings. Diarrhoea leading to weight loss and weakness invariably leads to reduced productivity, therefore huge financial losses to the livestock farmers. Evaluation of immuno-biochemical alterations and their management through appropriate therapeutic interventions would aid in preventing production losses caused by diarrhoea following MAP infection.Most of the studies were performed to understand immune-biochemical alterations in serum samples of cattle following experimental infections. But studies are limited in clinical cases of buffaloes suffering from diarrhoea and were suspected for MAP infection.We categorized diarrhoeic buffaloes brought at Veterinary Clinical Complex of Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, for therapeutic intervention; As MAP negatives / suspected, low positive, positive and strong positive based on indigenous ELISA kit results. We further compared immuno hemato-biochemical alterations in MAP negative/suspected low positive, positive and strong positive animals.Our major findings were; significant differences observed in creatinine, globulin, total leucocytic, neutrophils and in lymphocytes numbers. Decreasing trend in leucocytes was seen from suspected to strong positive animals, which indicated the progression of disease towards chronic paratuberculosis. However, differences were not observed in the Th1 cytokine levels.

Multiplex qPCR for differentiation of Mycobacterium avium subspecies paratuberculosis in active and passive infection of goats.

Mycobacterium avium paratuberculosis (MAP) is causative agent of Johne's disease (JD) in domestic animals and has broad host range. JD infected animals shed viable MAP in their milk, feces, blood, and tissues which get transmitted to human beings directly or indirectly by consumption of animal products, through contact, animal handling and through contaminated environment, aerosols. In this current study, we developed hydrolysis probe based TaqMan® real-time PCR assay where samples were investigated by targeting IS900 mRNA and ModD gene to differentiate live MAP shedders from inactive/dead MAP bacilli shedding animals. The IS900 mRNA and ModD gene primers were designed using discontiguous unique conserved sequences of IS900 more towards the 3' end and fibronectin attachment protein (FAP) genes, respectively. Two different reporter dyes Cy5 and TexasRed, with compatible quenchers BHQ-1 and BHQ-2, respectively, were used for probe designing of IS900 and ModD genes. Triplex PCR assay was developed by using serially diluted positive MAP culture in log10 dilution and probe and template titration. TaqMan® probe real-time PCR targeting IS900 mRNA and ModD gene detects the MAP infection at early stage with high sensitivity and specificity. The specificity of developed TaqMan probe real-time PCR was found to be high while validated by using Escherichia coli and Staphylococcus aureus in addition to the MAP culture as there is no non-specific signal from other microbes. The sensitivity of developed TaqMan® probe real-time PCR was computed based on copy numbers ranged from 4.14 × 1011 to 4.14 × 104 for IS900 (FAM), 1.27 × 1011 to 1.27 × 104 for IS900 mRNA (Cy5), and 3.68 × 1010 to 3.68 × 104 for ModD (TexasRed), and lowest limit to detect MAP was 4.14 × 104, 1.27 × 104, and 3.68 × 104 copies for respective genes. This assay would be of great aid to contain the MAP infection in the large herd, where silent shedders spread active infection can be differentiated from passive shedding by non-infected animals. This test would also be equivalent to culture test in terms of specificity and hence can be able to be undertaken in molecular epidemiological studies to represent the actual disease prevalence in the future. KEY POINTS: • Multiplex mRNA-based qPCR was developed to identify the actively infective MAP bacilli from passive ones. • ModD and IS900 used as targets to assess active MAP bacilli in fecal samples of suspected animals. • The LOD was computed using copy numbers with 4.14 × 104 and 3.68 × 104 copies for IS900 and ModD, respectively.

Optimization of the composition of a solid culture medium for Mycobacterium avium subsp. paratuberculosis using factorial design and response surface methodology.

AimTo develop an optimized solid culture medium for improved growth of Mycobacterium avium subsp. paratuberculosis (MAP).Methods and resultsSeven medium constituents (factors) were assessed at various concentrations for their ability to positively affect MAP growth. The factors tested were Tween 80, egg yolk, casitone, taurocholic acid, Mycobactin J, agar and either OADC or ADC supplement. After an initial screening of individual factors, a fractional factorial design and a response surface methodology (RSM) central composite design were used to assess the effects of multiple factors simultaneously and design a new solid culture medium. MAP growth became visible on streak plates of the optimized solid medium 2 weeks earlier than on Herrold's egg yolk medium (HEYM).ConclusionsMAP grew faster on the optimized solid medium than on HEYM. It consisted of Middlebrook 7H9 broth with 1.0% Tween 80, 0.019% casitone, 1.4% bacteriological agar, 10% egg yolk, 10% ADC and 1.65 μg ml−1 Mycobactin J.Significance and impact of the studyThis is the first study to use an RSM approach to optimize the composition of a solid medium for MAP culture. The new medium could improve MAP culture in future by reducing incubation times and increasing MAP colony numbers.

Detection of Low MAP Shedder Prevalence in Large Free-Stall Dairy Herds by Repeated Testing of Environmental Samples and Pooled Milk Samples.

Simple SummaryParatuberculosis is a disease which affects ruminants worldwide. Many countries have implemented certification and monitoring systems to control the disease, particularly in dairy herds. Monitoring herds certified as paratuberculosis non-suspect is an important component of paratuberculosis herd certification programs. The challenge is to detect the introduction or reintroduction of the infectious agent as early as possible with reasonable efforts but high certainty. In our study, we evaluated different low-cost testing schemes in herds where the share of infected animals was low, resulting in a low within-herd prevalence of animals shedding the bacteria that causes paratuberculosis in their feces. The test methods used were repeated pooled milk samples and fecal samples from the barn environment. Our study showed that numerous repetitions of different samples are necessary to monitor such herds with sufficiently high certainty. In the case of herds with a very low prevalence, our study showed that a combination of different sampling approaches is required.An easy-to-use and affordable surveillance system is crucial for paratuberculosis control. The use of environmental samples and milk pools has been proven to be effective for the detection of Mycobacterium avium subsp. paratuberculosis (MAP)-infected herds, but not for monitoring dairy herds certified as MAP non-suspect. We aimed to evaluate methods for the repeated testing of large dairy herds with a very low prevalence of MAP shedders, using different sets of environmental samples or pooled milk samples, collected monthly over a period of one year in 36 herds with known MAP shedder prevalence. Environmental samples were analyzed by bacterial culture and fecal PCR, and pools of 25 and 50 individual milk samples were analyzed by ELISA for MAP-specific antibodies. We estimated the cumulative sensitivity and specificity for up to twelve sampling events by adapting a Bayesian latent class model and taking into account the between- and within-test correlation. Our study revealed that at least seven repeated samplings of feces from the barn environment are necessary to achieve a sensitivity of 95% in herds with a within-herd shedder prevalence of at least 2%. The detection of herds with a prevalence of less than 2% is more challenging and, in addition to numerous repetitions, requires a combination of different samples.

Quantitative Risk Assessment of Exposure to Mycobacterium avium subsp. paratuberculosis (MAP) via Different Types of Milk for the Slovenian Consumer.

This study aimed to assess the risk of exposure to Mycobacterium avium subsp. paratuberculosis (MAP) via milk for the Slovenian consumer. MAP is suspected to be associated with several diseases in humans, therefore the risk of exposure should be better understood. The primary source of MAP for humans is thought to be cattle, in which MAP causes paratuberculosis or Johne’s disease. We developed a stochastic quantitative risk assessment model using Monte Carlo simulations. Considering the assumptions and uncertainties, we estimated the overall risk of exposure to MAP via milk to be low. For people consuming raw milk from MAP positive farms, the risk was high. On-farm pasteurisation reduced the risk considerably, but not completely. The risk of exposure via pasteurised retail milk was most likely insignificant. However, with a higher paratuberculosis prevalence the risk would also increase. Given the popularity of raw milk vending machines and homemade dairy products, this risk should not be ignored. To reduce the risk, consumers should heat raw milk before consumption. To prevent a potential public health scare and safeguard farmers’ livelihoods, a reduction in paratuberculosis prevalence should be sought. Our results show that culling clinically infected cows was insufficient to reduce milk contamination with MAP.

Use of uncoated magnetic beads to capture Mycobacterium smegmatis and Mycobacterium avium paratuberculosis prior detection by mycobacteriophage D29 and real-time-PCR

Uncoated tosyl-activated magnetic beads were evaluated to capture Mycobacterium smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) from spiked feces, milk, and urine. Centrifugation and uncoated magnetic beads recovered more than 99% and 93%, respectively, of 1.68 × 107 CFU/mL, 1.68 × 106 CFU/mL and 1.68 × 105 CFU/mL M. smegmatis cells resuspended in phosphate buffer saline. The use of magnetic beads was more efficient to concentrate cells from 1.68 × 104 CFU/mL of M. smegmatis than centrifugation. Likewise, the F57-qPCR detection of MAP cells was different whether they were recovered by beads or centrifugation; cycle threshold (Ct) was lower (p < 0.05) for the detection of MAP cells recovered by beads than centrifugation, indicative of greater recovery. Magnetic separation of MAP cells from milk, urine, and feces specimens was demonstrated by detection of F57 and IS900 sequences. Beads captured no less than 109 CFU/mL from feces and no less than 104 CFU/mL from milk and urine suspensions. In another detection strategy, M. smegmatis coupled to magnetic beads were infected by mycobacteriophage D29. Plaque forming units were observed after 24 h of incubation from urine samples containing 2 × 105 and 2 × 103 CFU/mL M. smegmatis. The results of this study provide a promising tool for diagnosis of tuberculosis and Johne's disease.

SEROLOGICAL IDENTIFICATION OF OVINE PARATUBERCULOSIS IN VERACRUZ, MEXICO

This study aimed to determine the presence of Mycobacterium avium subspecies paratuberculosis (MAP) in three sheep production regions in the state of Veracruz, México. The study regions were the Totonaca, Capital, and Tuxtlas. The study included 414 animals distributed in 55 farms. Ewes and males used as rams over three months old were randomly selected to collect serum for its subsequent analysis by indirect ELISA standard kits. Overall seroprevalence was 0.96% (IC95% 0.02-1.90), 23.07% (IC95% 0.017-45.98) by municipality, and 5.45% (IC95% 0.00-11.46) by flock. This could be the first report of exposition to MAP in sheep from the state of Veracruz. Since it is the third ovine producer in Mexico, the undetected threat of paratuberculosis (PTB) could induce economic losses and commercial disadvantage to producers. Meanwhile, the subclinical animals deemed as seropositive in this study, could be use as breed. Therefore, subclinical animals might maintain and disseminate the pathogen within herds. Furthermore, exposure to MAP was identified in the three regions studied (which have different climate conditions). These results confirm the environmental resistance of MAP and imply a health risk to ruminants and other susceptible species. Besides, in extensive livestock production, local wildlife could acquire MAP and transmit to other flocks or regions. None of the studied variables were considered as risk factor associated with MAP. Results suggest the need to develop further research to establish the distribution and impact of MAP in Veracruz. This work also prompts to enhance biosecurity measures amongst ovine producers.

Ex vivo Platforms to Study the Primary and Recall Immune Responses to Intracellular Mycobacterial Pathogens and Peptide-Based Vaccines.

Progress in the study of the immune response to pathogens and candidate vaccines has been impeded by limitations in the methods to study the functional activity of T-cell subsets proliferating in response to antigens processed and presented by antigen presenting cells (APC). As described in this review, during our studies of the bovine immune response to a candidate peptide-based vaccine and candidate rel deletion mutants in Mycobacterium avium paratuberculosis (Map) and Mycbacterium bovis (BCG), we developed methods to study the primary and recall CD4 and CD8 T-cell responses using an ex vivo platform. An assay was developed to study intracellular killing of bacteria mediated by CD8 T cells using quantitative PCR to distinguish live bacteria from dead bacteria in a mixed population of live and dead bacteria. Through use of these assays, we were able to demonstrate vaccination with live rel Map and BCG deletion mutants and a Map peptide-based vaccine elicit development of CD8 cytotoxic T cells with the ability to kill intracellular bacteria using the perforin-granzyme B pathway. We also demonstrated tri-directional signaling between CD4 and CD8 T cells and antigen-primed APC is essential for eliciting CD8 cytotoxic T cells. Herein, we describe development of the assays and review progress made through their use in the study of the immune response to mycobacterial pathogens and candidate vaccines. The methods obviate some of the major difficulties encountered in characterizing the cell-mediated immune response to pathogens and development of attenuated and peptide-based vaccines.

Attenuation of Excess TNF-α Release in Crohn’s Disease by Silencing of iRHOMs 1/2 and the Restoration of TGF-β Mediated Immunosuppression Through Modulation of TACE Trafficking

TNFα converting enzyme (TACE) is a transmembrane metalloprotease that sheds an assortment of signaling receptors, cytokines, growth factors, and pro-inflammatory mediators. In Crohn’s disease (CD), TACE activity is upregulated, resulting in a marked increase of TNFα secretion and inflammation. Although treatment of CD with TNFα monoclonal antibodies is beneficial, many patients are at risk for acquiring opportunistic infections, and the treatment efficacy of TNFα monoclonal antibodies typically decreases over time. This study investigated an alternative approach for mitigating TNFα release by knocking down TACE membrane translocation in macrophages via inhibitory rhomboid proteins 1 and 2 (iRHOMs 1/2) siRNA treatment. First we measured TGFβRII shedding in ex vivo plasma samples collected from CD patients and healthy control subjects (N=40 per group). Then, we measured TGFβRII shedding and the expression and production of TGFβ ligand, TNFα, IL-6, IL-1β, IL-10, and total versus membranous TACE in vitro with THP-1 derived macrophage infected with Mycobacterium avium subspecies paratuberculosis (MAP), a highly studied CD-related pathogen. We determined that TGFβRII shedding was significantly higher in CD patients compared to healthy controls [515.52 ± 54.23 pg/mL vs 310.81 ± 43.16 pg/mL, respectively], and MAP-infected CD plasma samples had significantly more TGFβRII shedding (601.83 ± 49.56 pg/mL) than MAP-negative CD samples (430.37 ± 45.73 pg/mL). Moreover, we also determined that TACE production; TGFβ ligand expression and production; and TGFβRII shedding were also higher in MAP-infected THP-1 macrophages. Nevertheless, once we transfected the MAP infected macrophages with iRHOM siRNA, TACE production and membrane localization were significantly decreased, resulting in a significant decrease in TGFβRII shedding; an increase in Smad3 phosphorylation; a decrease in the expression and production of pro-inflammatory cytokines; and a decrease in the expression and production of stricture-associated factor, plasminogen activator inhibitor-1 (PAI-1). Our data clearly demonstrates that the regression of TACE trafficking, via iRHOM 1/2 silencing, significantly reduces the release of TNFα and restores the immunosuppressive capabilities of TGFβ signaling, which ultimately reverses inflammatory tissue damage. Accordingly, this study may provide a framework for the creation of newer, safer therapeutic options designed to treat inflammatory autoimmune diseases such as CD and rheumatoid arthritis.

Modeling Paratuberculosis Transmission in a Small Dairy Herd Typical of Slovenia Suggests That Different Models Should Be Used to Study Disease Spread in Herds of Different Sizes.

Simple SummaryParatuberculosis is widespread in cattle throughout the world and is an economically important disease, especially in dairy cattle. It is caused by Mycobacterium avium subsp. paratuberculosis and is problematic due to complex transmission and unreliable diagnostic tests. The disease is present in Slovenia, where milk production is one of the most important sectors of agriculture. Our aim was to understand the disease spread in a typical Slovenian dairy herd of about 17 cows. To our knowledge, this is the first study specifically looking at the dynamics of disease spread in a small dairy herd. We relied on real data from the competent authorities on herd structure and data from the literature on disease spread, as well as expert opinion. The results showed that herd size has an influence on disease spread and that the prevalence in a small herd is probably lower than in larger herds because farmers tend to sell and buy animals frequently, which allows for the elimination of diseased animals from small herds. However, this can also reintroduce the disease into the herd. The results suggest that different assessments should be used to study the spread and control of this disease in herds of different sizes.This study aimed to investigate the possible dynamics of paratuberculosis or Johne’s disease in a typical Slovenian dairy herd of about 17 cows. Paratuberculosis is a worldwide endemic disease of cattle caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is associated with significant economic losses. We developed a stochastic compartmental model with two pathways of disease progression, infections of adult cows and infections of young animals through horizontal and vertical transmission, and transmission through animal movements. The average proportions of subclinically and clinically infected cows were 4% and 0.47%, respectively. The prevalence within the herd, which included latently infected animals, averaged 7.13% and ranged from 0% to 70.59%. Under the given circumstances, the results showed a relatively high rate of spontaneous elimination (0.22 per herd per year) of the disease and a high rate of reinfection (0.18 per herd per year) facilitated by active animal trade. To our knowledge, this stochastic compartmental model is the first to be developed specifically to represent a small dairy herd and could apply to other countries with a similar structure of dairy farms. The results suggest that different models should be used to study MAP spread in herds of various sizes.

First molecular investigation John's disease (paratuberculosis) in water buffalo in Iraq

This study was conducted to exam 100 fecal samples and 50 tissue samples from (100 ) buffaloes above one years at different villages and townships in Al-Najaf governorate from (January 2020 to January 2021) to detect of specific insertion Sequence IS900 in Mycobacterium paratuberculosis (John's disease) nested PCR. Out of 150 (100 fecal and 50 tissue) samples tested by Nested PCR, 14(9.3%) were positive for IS900 specific gen, Significant difference at P&lt;0.05 in molecular result were observed between tissues and fecal samples. The molecular results revealed that 6(6%) out of 100 fecal sample were positive, while 8(16%) of tissue sample were positive for IS900 specific gen . There was no significant difference at P&lt;0.05 in relationship between age gender and location with paratuberculosis prevalence.