A rapid phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis in milk.

Paratuberculosis is an incurable gastroenteritis among ruminants that is promoted by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast mycobacterium. To accelerate the detection of viable pathogen, a conventional (peptide mediated magnetic separation: PMS) and novel (phage-bead qPCR: PBQ) phage based assay was optimized. A superior limit of detection (LOD) of 10 MAP per 10 mL milk was suggested for PBQ compared to 100 cells/10 mL for PMS-phage assay. Via PBQ, viable MAP was found in 48.78% out 41 unpasteurized sheep and goat milk samples. Sheep milk samples (n = 29) that were tested by PMS-phage assay contained no viable MAP. The absence of viable MAP in milk collected from 21 of the recent sheep animals was also confirmed by PBQ after a 2-week gap. Although, the two phage assays comparably detected no viable MAP in the milk samples, MAP DNA and antibodies against MAP were recognized in milk and sera of some of these animals within two instances of sampling representing that some sheep animals were MAP shedders. In conclusion, PBQ and PMS-phage could be promising methods for the assessment of MAP viability in milk samples. However, PBQ was privileged over the PMS-phage assay due to the lower LOD, rapidity, higher sensitivity, lack of need to M. smegmatis and consequent virucidal treatment that are essential in PMS-phage assay for making lawn and inactivation of exogenous mycobacteriophages respectively.

Identification of loci associated with pathological outcomes in Holstein cattle infected with Mycobacterium avium subsp. paratuberculosis using whole-genome sequence data.

Bovine paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic granulomatous enteritis that affects cattle worldwide. According to their severity and extension, PTB-associated histological lesions have been classified into the following groups; focal, multifocal, and diffuse. It is unknown whether these lesions represent sequential stages or divergent outcomes. In the current study, the associations between host genetic and pathology were explored by genotyping 813 Spanish Holstein cows with no visible lesions (N = 373) and with focal (N = 371), multifocal (N = 33), and diffuse (N = 33) lesions in gut tissues and regional lymph nodes. DNA from peripheral blood samples of these animals was genotyped with the bovine EuroG MD Bead Chip, and the corresponding genotypes were imputed to whole-genome sequencing (WGS) data using the 1000 Bull genomes reference population. A genome-wide association study (GWAS) was performed using the WGS data and the presence or absence of each type of histological lesion in a case-control approach. A total of 192 and 92 single nucleotide polymorphisms (SNPs) defining 13 and 9 distinct quantitative trait loci (QTLs) were highly-associated (P ≤ 5 × 10-7) with the multifocal (heritability = 0.075) and the diffuse (heritability = 0.189) lesions, respectively. No overlap was seen in the SNPs controlling these distinct pathological outcomes. The identified QTLs overlapped with some QTLs previously associated with PTB susceptibility, bovine tuberculosis susceptibility, clinical mastitis, somatic cell score, bovine respiratory disease susceptibility, tick resistance, IgG level, and length of productive life. Pathway analysis with candidate genes overlapping the identified QTLs revealed a significant enrichment of the keratinization pathway and cholesterol metabolism in the animals with multifocal and diffuse lesions, respectively. To test whether the enrichment of SNP variants in candidate genes involved in the cholesterol metabolism was associated with the diffuse lesions; the levels of total cholesterol were measured in plasma samples of cattle with focal, multifocal, or diffuse lesions or with no visible lesions. Our results showed reduced levels of plasma cholesterol in cattle with diffuse lesions. Taken together, our findings suggested that the variation in MAP-associated pathological outcomes might be, in part, genetically determined and indicative of distinct host responses.

A Mycobacterium species for Crohn's disease?

In ruminants Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of a chronic granulomatous inflammatory bowel disease called Johne's disease (JD). Some researchers have hypothesised that MAP is also associated with Crohn's disease (CD), an inflammatory bowel disease in humans that shares some histological features of JD. Despite numerous attempts to demonstrate causality by researchers, direct microbiological evidence of MAP involvement in CD remains elusive. Importantly, it has not been possible to reliably and reproducibly demonstrate mycobacteria in the tissue of CD patients. Past attempts to visualise mycobacteria in tissue may have been hampered by the use of stains optimised for Mycobacterium tuberculosis complex (MTB) and the lack of reliable bacteriological culture media for both non-tuberculous mycobacteria (NTM) and cell-wall-deficient mycobacteria (CWDM). Here we describe a Ziehl-Neelsen (ZN) staining method for the demonstration of CWDM in resected tissue from patients with Crohn's disease, revealing the association of CWDM in situ with host tissue reactions, and posit this as a cause of the tissue inflammation. Using the ZN stain described we demonstrated the presence of CWDM in 18 out of 18 excised tissue samples from patients diagnosed as having Crohn's disease, and in zero samples out of 15 non-inflammatory bowel disease controls.

Prediction of Johne's disease state based on quantification of T cell markers and their interaction with macrophages in the bovine intestine.

Cell-mediated immune responses to Mycobacterium avium subsp. paratuberculosis (MAP) are regulated by various types of T lymphocytes. The aim of this study was to quantitate T cell subsets in the mid-ileum of cows naturally infected with MAP to identify differences during different stages of infection, and to determine whether these subsets could be used as predictors of disease state. Immunofluorescent labeling of T cell subsets and macrophages was performed on frozen mid-ileal tissue sections archived from naturally infected dairy cows in either subclinical or clinical disease status, and noninfected control cows. Comprehensive IF staining for CD4, CD8α, TcR1-N24 (gamma delta), FoxP3, CXCR3 and CCR9 served to define T cell subsets and was correlated with macrophages present. Clinically affected cows demonstrated significantly higher numbers of CXCR3+ (Th1-type) and CCR9+ (total small intestinal lymphocytes) cells at the site of infection compared to the subclinical cows and noninfected controls. Further, predictive modeling indicated a significant interaction between CXCR3+ and AM3K+ (macrophages) cells, suggesting that progression to clinical disease state aligns with increased numbers of these cell types at the site of infection. The ability to predict disease state with this model was improved from previous modeling using immunofluorescent macrophage data. Predictive modelling indicated an interaction between CXCR3+ and AM3K+ cells, which could more sensitively detect subclinical cows compared to clinical cows. It may be possible to use this knowledge to improve and develop an assay to detect subclinically infected animals with more confidence during the early stages of the disease.

Mycobacterium avium subspecies paratuberculosis and bovine leukemia virus seroprevalence and associated risk factors in dairy herds in Minas Gerais state, Brazil / [Soroprevalência do Mycobacterium avium subespécie paratuberculosis e vírus da leucemia bovina e fatores de risco associados em rebanhos leiteiros de Minas Gerais, Brasil]

Mycobacterium avium subesp. paratuberculosis (MAP) e o vírus da leucemia bovina (BLV) são agentes que causam grandes perdas econômicas nos rebanhos. O objetivo deste estudo foi avaliar a situação epidemiológica da paratuberculose bovina (PTB) e leucose enzoótica bovina (EBL) em rebanhos leiteiros de Lagoa Formosa, Minas Gerais, Brasil. Foram coletadas 236 amostras de sangue de vacas, as quais foram submetidas aos testes ELISA e imunodifusão em gel de ágar para detecção de anticorpos contra MAP e BLV. A soroprevalência de anticorpos contra MAP e BVL foi de 20% para os rebanhos e 6% para os animais e de 85% para os rebanhos e 50,42% para os animais, respectivamente. A presença dessas enfermidades deve servir como um alerta para os produtores e veterinários, para que concentrem maior atenção na implementação de medidas higiênico-sanitárias, incorporando elementos de vigilância com base nos riscos identificados no estudo.(AU)

Putting Crohn's on the MAP: Five Common Questions on the Contribution of Mycobacterium avium subspecies paratuberculosis to the Pathophysiology of Crohn's Disease.

For decades, Mycobacterium avium subspecies paratuberculosis (MAP) has been linked to the pathogenesis of Crohn's disease. Despite many investigations and research efforts, there remains no clear unifying explanation of its pathogenicity to humans. Proponents argue Crohn's disease shares many identical features with a granulomatous infection in ruminants termed Johne's disease and similarities with ileo-cecal tuberculosis. Both are caused by species within the Mycobacterium genus. Sceptics assert that since MAP is found in individuals diagnosed with Crohn's disease as well as in healthy population controls, any association with CD is coincidental. This view is supported by the uncertain response of patients to antimicrobial therapy. This report aims to address the controversial aspects of this proposition with information and knowledge gathered from several disciplines, including microbiology and veterinary medicine. The authors hope that this discussion will stimulate further research aimed at confirming or refuting the contribution of MAP to the pathogenesis of Crohn's disease and ultimately lead to advanced targeted clinical therapies.

Detection of latent forms of Mycobacterium avium subsp. paratuberculosis infection using host biomarker-based ELISAs greatly improves paratuberculosis diagnostic sensitivity.

Bovine paratuberculosis (PTB) is a chronic granulomatous enteritis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), responsible for important economic losses in the dairy industry. Current diagnostic methods have low sensitivities for detection of latent forms of MAP infection, defined by focal granulomatous lesions and scarce humoral response or MAP presence. In contrast, patent infections correspond to multifocal and diffuse types of enteritis where there is increased antibody production, and substantial mycobacterial load. Our previous RNA-Seq analysis allowed the selection of five candidate biomarkers overexpressed in peripheral blood of MAP infected Holstein cows with focal (ABCA13 and MMP8) and diffuse (FAM84A, SPARC and DES) lesions vs. control animals with no detectable PTB-associated lesions in intestine and regional lymph nodes. The aim of the current study was to assess the PTB diagnostic potential of commercial ELISAs designed for the specific detection of these biomarkers. The ability of these ELISAs to identify animals with latent and/or patent forms of MAP infection was investigated using serum from naturally infected cattle (n = 88) and non-infected control animals (n = 67). ROC analysis revealed that the ABCA13-based ELISA showed the highest diagnostic accuracy for the detection of infected animals with focal lesions (AUC 0.837, sensitivity 79.25% and specificity 88.06%) and with any type of histological lesion (AUC 0.793, sensitivity 69.41% and specificity 86.57%) improving on the diagnostic performance of the popular IDEXX ELISA and other conventional diagnostic methods. SPARC and MMP8 showed the highest diagnostic accuracy for the detection of animals with multifocal (AUC 0.852) and diffuse lesions (AUC 0.831), respectively. In conclusion, our results suggest that quantification of ABCA13, SPARC and MMP8 by ELISA has the potential for implementation as a diagnostic tool to reliably identify MAP infection, greatly improving early detection of MAP latent infections when antibody responses and fecal shedding are undetectable using conventional diagnostic methods.

Short communication: Occurrence and differentiation of Mycobacterium avium ssp. paratuberculosis (MAP) strains from milk of cows from herd with low prevalence of MAP.

Mycobacterium avium ssp. paratuberculosis (MAP) is an important pathogen responsible for the chronic progressive granulomatous enteritis known as paratuberculosis. None of the detection methods of MAP infection based on isolation of the bacterium is 100% sensitive or specific. In this article, we describe the comparison of 2 MAP detection methods: direct isolation of genetic material and culture, in individual and pooled milk samples. The genetic types of MAP detected in the samples were also identified. The study was performed in a herd of 321 cows; apparent herd seroprevalence was 3.43%. Seven of 11 individual milk samples from seropositive cows were positive by culture (and confirmed by PCR), whereas all 11 were positive by direct PCR. Of the 62 milk pools from seronegative animals, 15 were positive by culture (and confirmed by PCR) and 13 were positive by direct PCR. Using multiplex PCR and PCR-restriction enzyme analysis (PCR-REA) methods, C (cattle) and S (sheep)-types of mycobacteria were identified. Most of the genetic material tested belonged to C-type. Detection of the MAP type occurring in an infected herd can help track the source of infection. We suggest using genetic material isolated directly from pooled milk samples for quick diagnosis, identification of MAP type, and tracking of infection, without the need to sequence the entire genome.

Assessing efficacy of N-Acetyl-l-Cysteine-Sodium Hydroxide on bacterial viability and enhanced recovery of Mycobacterium avium subsp. paratuberculosis from bovine colostrum.

The standard procedure for the improved cultural recovery of viable Mycobacterium spp. from diverse samples mainly depends on reducing the viability of background microbiota using different chemical compounds. This study was designed to i) evaluate the efficacy and comparison between N-Acetyl-l-Cysteine-Sodium hydroxide (NALC-2% NaOH) and hexadecylpyridinium chloride (0.75% HPC) treatment and exposure time on reducing the viability of undesirable microorganisms with minimal impact on colostrum consistency; and ii) assess the impact of NALC-2% NaOH on improved and enhanced recovery of Mycobacterium avium subsp. paratuberculosis (MAP) in spiked postpartum colostrum samples and consistency of colostrum. A total of 40 samples, each treated with NALC-2% NaOH for 15 min or 0.75% HPC for 5 h, were investigated for total mesophilic aerobic bacteria (MAB) and enterobacteria (EB) (CFU mL-1). The results showed that treatment of colostrum samples with NALC-2% NaOH completely eliminated EB and significantly reduced MAB (3.6 log10 CFU mL-1). Conversely, samples treated with 0.75% HPC produced a complex mixture following interaction with the colostrum protein and showed non-significant and variable results. In addition, the spiked colostrum treated with NALC-2% NaOH for 15 min revealed recovery of viable MAP cells with a minimum limit of detection of 1.36 log10 CFU 10 mL-1 where no change in the consistency of colostrum was observed. In conclusion, 15-min NALC-2% NaOH treatment of colostrum may significantly reduce the viability of undesirable microorganisms and help to enhance the efficient recovery of MAP without impacting the consistency of high quality postpartum colostrum. This rapid procedure is suitable for efficient recovery and early detection of MAP as well as preventing its transmission to neonates and young calves in MAP infected herds.

High prevalence of subclinical paratuberculosis in buffaloes (Bubalus bubalis) in Maranhão, Brazil.

Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, causing enteritis and chronic granulomatous lymphadenitis, characterized by malabsorption syndrome, its agent is the Mycobacterium avium subsp. paratuberculosis (MAP). Thus, the objective of this work was to identify and characterize MAP in buffalo herds slaughtered in Baixada Maranhense region. Samples of intestines, mesenteric lymph nodes, and ileocecal valves were collected from 115 buffaloes slaughtered at Baixada Maranhense slaughterhouses to perform the diagnosis by histopathological examination using staining with Hematoxylin and Eosin (H&E) and Ziehl-Neelsen, bacterial isolation, and real-time PCR. In the histopathology by H&E staining, there was evidence suggestive of paratuberculosis in 30% (31/115) of the buffaloes. With Ziehl-Neelsen staining, acid-fast bacilli (AFB) were visualized in 27% (26/115) of the tissue samples analyzed. MAP was isolated in 4.3% (5/115) of the fecal samples subjected to bacterial culture. The samples inoculated in HEYM with mycobactin J produced colonies identified with MAP according to their own morphological characteristics such as round, white, smooth and slightly rough, alcohol-acid staining, and slow growth with 8 weeks of incubation and mycobactin dependence. The agent confirmation was performed in five bacterial isolates (4.3%) and 15 (13%) fragments of jejunum, ileum, and mesenteric lymph node by the IS900 real-time PCR technique. The results of the present study demonstrate the subclinical occurrence of paratuberculosis in flocks of buffalo slaughtered in slaughterhouses of Baixada Maranhense.

Identification of microRNAs in bovine faeces and their potential as biomarkers of Johne's Disease.

Extracellular microRNAs (miRNAs) are detectable in the peripheral blood and have been touted as potential biomarkers for a range of maladies. The presence and biomarker potential of miRNAs in other biofluids has been less thoroughly explored, particularly in the veterinary realm. Faecal miRNAs are a case in point; while they have been identified largely in rodents and humans, they have not been reported in cattle but may have prognostic or diagnostic value for Johne's Disease (JD) in cattle, a chronic granulomatous inflammation of the ileum caused by Mycobacterium avium subspecies paratuberculosis (MAP). The aim of this study was thus to characterise the bovine faecal miRNome and to determine the utility of these transcripts as biomarkers for JD. Real-time PCR arrays consisting of 752 miRNA targets, optimised for detection of human miRNA, were used to screen RNA purified from faecal samples obtained from confirmed JD clinical cases vs. healthy controls. Two hundred and fifty-eight miRNAs were detected in bovine faeces, three of which are potentially novel orthologs of known human miRNAs. Differential abundance of three miRNA was evident in animals with clinical JD as compared to healthy controls. Our study has therefore identified a variety of miRNAs in bovine faeces and has demonstrated their utility in differentiating healthy animals from those with late-stage JD, providing potential biomarkers for MAP infection and disease progression.

Characteristics and Epidemiological Investigation of Paratuberculosis in Dairy Cattle in Tai'an, China.

Paratuberculosis, a chronic and sometimes fatal disease of ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we examined paratuberculosis cases among 2-4-year-old dairy cows at farms in Shandong Province, China. Paratuberculosis cases were diagnosed based on clinical symptoms, pathological autopsy, and histopathological inspection. Characteristics of paratuberculosis in the affected dairy cattle included poor body condition, persistent diarrhea, subcutaneous edema, granulomatous ileitis (multibacillary), mesenteric lymphadenitis, and hepatitis. Acid-fast bacilli from fecal specimens and lymphocytes were putatively identified as MAP based on Ziehl-Neelsen staining, then confirmed using polymerase chain reaction-based testing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses. Overall, only one MAP strain was isolated from a herd with symptomatic diarrhea. However, analysis of 586 serum samples from nine herds in Tai'an City revealed that 66.7% of herds and 14.2% of animals were seropositive for MAP. Our findings suggest that paratuberculosis is widely prevalent and therefore a significant threat to the dairy industry in Tai'an City, Shandong Province, China.

Synthetic cathelicidin LL-37 reduces Mycobacterium avium subsp. paratuberculosis internalization and pro-inflammatory cytokines in macrophages.

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic diarrheic intestinal infections in domestic and wild ruminants (paratuberculosis or Johne's disease) for which there is no effective treatment. Critical in the pathogenesis of MAP infection is the invasion and survival into macrophages, immune cells with ability to carry on phagocytosis of microbes. In a search for effective therapeutics, our objective was to determine whether human cathelicidin LL-37, a small peptide secreted by leuckocytes and epithelial cells, enhances the macrophage ability to clear MAP infection. In murine (J774A.1) macrophages, MAP was quickly internalized, as determined by confocal microscopy using green fluorescence protein expressing MAPs. Macrophages infected with MAP had increased transcriptional gene expression of pro-inflammatory TNF-α, IFN-γ, and IL-1ß cytokines and the leukocyte chemoattractant IL-8. Pretreatment of macrophages with synthetic LL-37 reduced MAP load and diminished the transcriptional expression of TNF-α and IFN-γ whereas increased IL-8. Synthetic LL-37 also reduced the gene expression of Toll-like receptor (TLR)-2, key for mycobacterial invasion into macrophages. We concluded that cathelicidin LL-37 enhances MAP clearance into macrophages and suppressed production of tissue-damaging inflammatory cytokines. This cathelicidin peptide could represent a foundational molecule to develop therapeutics for controlling MAP infection.

Occurrence of Mycobacterium avium subsp. paratuberculosis in coalho cheese in the State of Pernambuco, Brazil / Ocorrência de Mycobaterium avium subsp. paratuberculosis em queijo coalho do Estado de Pernambuco, Brasil

Paratuberculosis is a chronic and incurable disease that affects ruminants and other domestic animals. It is caused by Mycobacterium avium subsp. paratuberculosis (MAP) that may also be involved in some human diseases such as Crohn's disease, type 1 diabetes, sarcoidosis, multiple sclerosis, and Hashimoto's thyroiditis. The objective of this study was to investigate the occurrence of MAP DNA in samples of artisanal coalho cheese purchased in the State of Pernambuco. Forty samples of coalho cheese submitted to the Real Time Polymerase Chain Reaction (qPCR) technique were analyzed for the detection of the MAP region IS900. 11 (27.5%) were positive with a mean of 195.9 MAP colony forming unit (CFU) per gram of each sample, with a minimum of 30.3 CFU/g and a maximum of 324.2 CFU/g. Thus, this type of cheese that is one of the most consumed in this region of Brazil constitutes a source of human exposure to MAP. Further research in this area should be performed to evaluate the viability of the bacteria in this cheese type.(AU)

Paratuberculose é uma enfermidade crônica e incurável que acomete ruminantes e outras espécies de animais domésticos. É causada pelo Mycobacterium avium subsp. paratuberculosis (MAP) e ainda há a suspeita do seu envolvimento em enfermidades nos humanos como a doença de Crohn, diabetes tipo 1, sarcoidose, esclerose múltipla e tireoidite de Hashimoto. Objetivou-se com esta pesquisa investigar a ocorrência do DNA de MAP em amostras de queijo coalho artesanal adquiridas em estabelecimentos comerciais do Estado de Pernambuco. 40 amostras de queijo coalho artesanal foram submetidas a técnica de Reação em Cadeia da Polimerase em Tempo Real (qPCR) para detecção da região IS900 do MAP. 11 (27,5%) foram positivas com uma média de 195,9 unidades formadoras de colônia (UFC) de MAP por grama de queijo, com detecção mínima de 30,3UFC/g e máxima de 324,2UFC/g. Sendo assim, esse tipo de queijo que é um dos mais consumidos nesta região do Brasil constitui uma fonte de exposição humana ao MAP. Mais pesquisas nessa área devem ser realizadas para avaliar a viabilidade dessa bactéria no queijo coalho.(AU)

Characteristics of subclinical Mycobacterium avium ssp. paratuberculosis infection in a captive white-tailed deer herd.

Paratuberculosis (Johne's disease) is caused by Mycobacterium avium ssp. paratuberculosis (MAP), and affects both domestic and wild ruminants, including cattle, goats, sheep, and deer. In cattle, most infections occur during calfhood followed by a prolonged incubation period of 1-2 y or more before cows shed culturable numbers of MAP bacilli in their feces. As disease progresses, infected animals develop protein-losing enteropathy, intractable diarrhea, and weight loss. In a cohort of 32 clinically normal deer from a herd with a history of periodic clinical paratuberculosis, we found that subclinical infection was characterized by high rates of infection, common involvement of mesenteric lymph nodes, minimal lesion formation, few intralesional acid-fast bacilli, and low-level fecal shedding of MAP. The characteristics of subclinical paratuberculosis in white-tailed deer resemble those of cattle and red deer, although microscopic lesions were less common in subclinical deer than reported for subclinical cattle, and we did not see necrotizing granulomas as described in subclinical red deer and elk.

Reactions to Gudair® vaccination identified in sheep used for biomedical research.

CASE REPORT: We report Gudair® vaccination (against ovine Johne's disease, Mycobacterium avium subsp. paratuberculosis) site reactions in sheep used as a surgical model in biomedical research and discuss the actual and potential impact these lesions may have on surgical procedures, particularly in otology. Nine female Merino-cross sheep (Ovis aries) were enrolled in a project designed to investigate the healing capabilities of the malleus bone in the middle ear. The sheep were 12-18 months of age. Eight sheep had lesions near the base of the right ear that were discovered when surgery was performed. The size of the lesions varied and all lesions had a thick capsule containing various amount of caseous material. Two lesions had a draining tract where purulent material was apparent at the lowest point. The prescapular lymph nodes were not palpable in any of the sheep. Aerobic growth of various organisms was reported from four sheep lesions when the purulent material was transferred to a broth media. Histopathological examination revealed intralesional Mycobacteria and focal caseous necrosis or granulomatous dermatitis and cellulitis in seven of the eight lesions. Mild necrotising to granulomatous dermatitis and cellulitis was described in the lesion where organisms were not found. CONCLUSIONS: The lesions were confirmed at the end of the study to be associated with the vaccination and did not cause any known adverse effects on the research. However, it is important to acknowledge the risk of contamination these lesions could have on a sterile surgical site.

Deciphering the virulence of Mycobacterium avium subsp. paratuberculosis isolates in animal macrophages using mathematical models.

Understanding why pathogenic Mycobacterium avium subsp. paratuberculosis (Map) isolates cause disparate disease outcomes with differing magnitudes of severity is important in designing and implementing new control strategies. We applied a suite of mathematical models: i) general linear, ii) and neurofuzzy logic, to explain how the host of origin of several Map isolates, Map genotype, host, macrophage-based in vitro model and time post-infection contributed to the infection. A logistic growth ordinary differential equation (ODE) model was applied to estimate within macrophage growth rates for the different Map isolates. The models revealed different susceptibilities of bovine and ovine macrophages to Map infection and confirmed distinct virulence profiles for the isolates, judged by their ability to grow within macrophages. Ovine macrophages were able to internalize Map isolates more efficiently than bovine macrophages. While bovine macrophages were able to internalize Map isolates from cattle with more efficiency, ovine macrophages were more efficient in internalizing ovine isolates. Overall, Map isolates from goat and sheep grew minimally within macrophages or did not grow but were able to persist by maintaining its initial population. In contrast, the ability of the bovine isolates and the non-domesticated animal isolates to grow to higher CFU numbers within macrophages suggests that these isolates are more virulent than the sheep and goat isolates, or that these isolates are better adapted to infect domestic ruminants. Overall, our study confirms the different virulence levels for the Map isolates and susceptibility profiles of host macrophages, which is crucial in increasing our understanding of Map infection.

Prevalence of Mycobacterium avium subspecies paratuberculosis IS 900 DNA in biopsy tissues from patients with Crohn's disease: histopathological and molecular comparison with Johne's disease in Fars province of Iran.

BACKGROUND: Crohn's disease is a chronic enteritis of humans that affects the gastrointestinal tract, especially the terminal ileum, cecum and colon. The etiology of this disease is still unknown but seems to be multifactorial. There are reports about the potential link between Crohn's disease in humans and the causative agent of Johne's disease in ruminants. Because of the prevalence of Johne's disease in the Fars Province of Iran, the aim of this study was to investigate the prevalence of MAP in the biopsy tissues of patients affected by Crohn's disease in this area. METHODS: The study was performed from April 2015 to June 2017 at Namazi Hospital, Shiraz University of Medical Sciences, and School of Veterinary Medicine, Shiraz University, Shiraz, Iran. Intestinal biopsies of 30 patients (12 male and 18 female; mean age, 34 years; range 4-77 years) with the confirmed diagnosis of Crohn's disease and 30 patients diagnosed as non-inflammatory bowel disease (19 male and 11 female; mean age, 38 years; range 13-68 years) were studied by molecular, histopathological and histochemical methods. Also, similar numbers of adult goats affected by Johne's disease were studied, comparatively. DNA extractions of tissue specimens were subjected to PCR to amplify a 413-bp sequence of the IS900 gene. RESULTS: Using IS900-PCR, the overall prevalence of MAP in patients affected by Crohn's disease and non-inflammatory bowel disease were 47 and 13%, respectively. In addition, the prevalence of MAP in goats affected by Johne's disease was 70%. Using acid-fast histochemical staining, only 7% of Crohn's disease patients were weakly positive as paucibacillary and 43% of Johne's disease cases were moderate to strongly positive as multibacillary. Histopathologically, granulomatous enteritis (83 and 90%), lymphoplasmacytic enteritis (17 and 14%), edema and lymphangiectasia (67 and 96%), and vasculitis (20 and 73%) were common findings in Crohn's and Johne's diseases, respectively. CONCLUSION: Our findings demonstrate a remarkable association between MAP and CD in this population, and support an etiologic relationship between MAP infection in humans and the development of CD. MAP infection in human tissue may display species-specific pathologic findings, as occurs with other zoonotic pathogens.

Detection of microRNA in cattle serum and their potential use to diagnose severity of Johne's disease.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, which is characterized by chronic progressive granulomatous enteritis. The infection leads to wasting and weight loss in the animals and eventually death, causing considerable production losses to the agricultural industry worldwide. Currently available ELISA- and PCR-based diagnostic tests have limited sensitivity and specificity during early MAP infection in cattle, suggesting that there is an urgent demand for alternative diagnostic tests. Circulating microRNA (miRNA) have recently gained attention as potential biomarkers for several diseases in humans. However, knowledge and use of miRNA as biomarkers in diseases of ruminants, including Johne's disease, are very limited. Here we used NanoString nCounter technology (NanoString, Seattle, WA), a digital platform for amplification-free and hybridization-based quantitative measurement of miRNA in the sera of noninfected and naturally MAP-infected cattle with different severity of infection. Using probes developed against human miRNA, 26 miRNA were detected in cattle serum; 13 of these miRNA were previously uncharacterized for cattle. Canonical discrimination analysis using 20 miRNA grouped animals into 4 distinct clusters based on their disease status, suggesting that the levels of these miRNA can reflect disease severity. A model was developed using a combination of 4 miRNA (miR-1976, miR-873-3p, miR-520f-3p, and miR-126-3p), which distinguished moderate and severely infected animals from noninfected animals. Our study demonstrated the ability of the NanoString nCounter technology to detect differential expression of circulating miRNA in cattle and contributes to widely growing evidence that miRNA can be used as biomarkers in infectious diseases in cattle.

Relationship between the pathology of bovine intestinal tissue and current diagnostic tests for Johne's disease.

Johne's disease is an enteric disease caused by the intracellular pathogen Mycobacterium avium subsp. paratuberculosis (MAP). Upon translocation from the lumen of the small intestine, mycobacteria have the ability to thwart innate defense mechanisms and persist within the macrophage in the lamina propria. In an effort to understand how the pathology of disease is reflected in current diagnostic tests, immunofluorescent (IFA) labeling was performed to quantitate macrophage and MAP numbers in the ileum of infected cattle and correlate results with common methods for diagnosis of MAP infection; including ELISA, IFN-γ assay, RT-PCR, culture of MAP, and histological classification of tissue sections. Predictive models for clinical and subclinical disease states, histopathology acid-fast (AF), MAP location, granulomatous inflammation and type classifications, as well as macrophage, MAP and macrophages with intracellular MAP IFA labeling were successfully developed. The combination of macrophage number and ELISA were the best predictors of clinical disease state, while macrophage number was the best and only significant predictor of subclinical disease state. Fecal culture and number of MAP were the best predictors of granulomatous inflammation, and of combined AF, MAP location and granuloma type, respectively. Additionally, fecal culture and tissue culture were the best predictors of numbers of macrophages and MAP, respectively, while both ELISA and tissue culture were the best predictors of number of macrophages with intracellular MAP.