Systematic assessment of the reliability of quantitative PCR assays targeting IS900 for the detection of Mycobacterium avium ssp. paratuberculosis presence in animal and environmental samples.

Mycobacterium avium ssp. paratuberculosis (MAP) is the bacterium responsible for causing Johne's Disease (JD), which is endemic to dairy cattle and also incriminated in the etiology of Crohn's disease. The difficulty in diagnosing asymptomatic cows for JD makes this disease hard to control. JD is considered a priority under the One Health approach to prevent the spread of the causative agent to humans. Environmental screening is a strategic approach aimed at identifying dairy herds with animals infected with MAP. It serves as the initial step toward implementing more intensive actions to control the disease. Quantitative polymerase chain reaction (qPCR) technology is widely used for diagnosis. Given that genome sequencing is now much more accessible than ever before, it is possible to target regions of the MAP genome that allow for the greatest diagnostic sensitivity and specificity. The aim of this study was to identify among the published qPCR assays targeting IS900 the more cost-effective options to detect MAP and to validate them in the diagnostic context of JD disease. MAP IS900 is a prime target because it is a multicopy genetic element. A total of 136 publications have reported on the use of IS900 qPCR assays over the past 3 decades. Among these records, 29 used the SYBR Green chemistry and TaqMan technology was used in 107 reports. Aside from the 9 reports using commercial assays, 72 TaqMan reports cited previously published work, leaving us with 27 TaqMan qPCR designs. Upon closer examination, 5 TaqMan designs contained mismatches in primer or probe sequences. Additionally, others exhibited high similarity to environmental microorganisms or non-MAP mycobacteria. We assessed the performance of 6 IS900 qPCR designs and their sensitivity when applied to clinical or environmental samples, which varied from 4 to 56 fold overall. Additionally, we provide recommendations for testing clinical and environmental samples, as certain strategies used previously should be avoided due to poor qPCR design (e.g., the presence of mismatches) or a lack of specificity.

Improved DNA Amplification of the Hallmark IS900 Element in Mycobacterium avium subsp. paratuberculosis: a Reexamination Based on Whole-Genome Sequence Analysis.

Amplification of the IS900 multicopy element is a hallmark nucleic acid-based diagnostic test for Mycobacterium avium subsp. paratuberculosis, which causes Johne's disease in ruminants. This assay is frequently used to determine the presence of the bacterium in feces of infected cattle and sheep. Two IS900 primer sets developed in the 1990s were widely used for decades, and their use has continued in current studies. However, these primers were developed prior to the availability of complete genome sequences. Recent sequence analysis of the binding locations for one primer pair (P90/P91) identified errors and binding inefficiencies that can be easily corrected to further increase detection sensitivity. The P90 primer is missing two nucleotides that should be present near the 3' end, and it does not bind all copies of IS900 due to 5' deletions at some IS900 loci. These IS900 primer pairs, along with newly developed primers, were tested by real-time PCR on purified genomic DNA to determine which primer set performed the best and how primer design errors affect amplification efficiencies. The newly designed PCR primer set (JB5) showed increased sensitivity by two to three quantification cycles using purified genomic DNA and was similar in efficiency to 150C/921. These tests were extended using DNA from feces and tissues of infected cows, which showed similar results. Finally, a 167-bp partial duplication of IS900 was found in type I strains. Although P90 and P91 primers successfully amplify M. avium subsp. paratuberculosis DNA, their use should be discontinued in favor of more efficient primer pairs in future studies. IMPORTANCE This study is an example of how applied genomic analysis can aid diagnostic test improvements. Detection of Mycobacterium avium subsp. paratuberculosis infection of livestock prior to the appearance of clinical disease signs is very difficult but essential for identifying animals shedding the bacterium to prevent transmission of Johne's disease. Total M. avium subsp. paratuberculosis quantity in the feces as determined by real-time PCR (qPCR) using the IS900 target indicates bacterial shedding status and potential for transmission of the pathogen. However, legacy primers designed prior to the availability of complete genome sequences that are used in these tests to detect M. avium subsp. paratuberculosis were based on data from only a single copy of IS900 and not considering all copies collectively as a group. This approach resulted in primer design errors which can be easily corrected to improve test sensitivities. We tested original primers that contain these errors and their corrected versions by qPCR and showed improved sensitivity on purified genomic DNA as well as fecal and tissue samples. These findings may help detect the organism from environmental samples on farms where sensitivity is currently lacking.

Comparative evaluation of various in-house protocols on diagnostic performance for paratuberculosis IS900 PCR.

BACKGROUND: Paratuberculosis is a worldwide endemic infectious disease of ruminants that results in high economic losses. Public health concerns are also being raised with human Crohn's disease. Therefore, control is becoming priority for governments. Control is largely dependent on "Test and Cull" or "Test and Segregate" policy. Hence, it is critical to assure the infection before making the decision. Commercial kits are costly especially in view of resource limited areas. Present study analyzed the performance various in house DNA isolation methods and PCR master mix combinations to optimize a protocol for confirmation of paratuberculosis bacilli shedding in feces. METHODS AND RESULTS: Present study included five protocols of fecal DNA isolation (chemical, bio-chemical, physio-chemical and physical) and three reaction mixes (based on Qiagen, Genei and Thermo 2X master mixes) in nine different combinations using additives and tested their performance for IS900 PCR. Spiked fecal samples were used to select the best combination of DNA isolation method and PCR master mix (PRM). Selected combination was used to test reference (positive and negative) fecal samples and field samples. Findings revealed that combination physical method of DNA isolation and Genei based PRM (with additives; betaine DMSO and BSA) had lowest limit of detection. Sensitivity was 83% and specificity was 100% in comparison to fecal culture. High prevalence (23%) was reported for paratuberculosis on field samples. CONCLUSION: Optimized protocol has acceptable sensitivity and can easily be adopted in resource-limited laboratories. High prevalence of paratuberculosis needs immediate implementation of the control strategies.

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.

Application of Bayesian modeling for diagnostic assays of Mycobacterium avium subsp. paratuberculosis in sheep and goats flocks.

BACKGROUND: This study aimed to screen the sera of goats and sheep from flocks suspected of Mycobacterium avium subsp. paratuberculosis (MAP) infection by a newly standardized Mce-truncated ELISA (Mt-ELISA) kit for the detection of antibodies against MAP. Four diagnostic applied tests were evaluated including Indigenous plate-ELISA (IP-ELISA), Mt-ELISA, fecal Polymerase Chain Reaction (f-PCR) and fecal culture (FC). MATERIALS AND METHODS: Assuming the absence of a gold standard, latent-class models in a Bayesian framework were used to estimate the diagnostic accuracy of the four tests for MAP. RESULTS: Mt-ELISA had higher Sensitivity (Se) in sheep (posterior median: 0.68 (95% Probability Interval (PI): 0.43-0.95), while IP-ELISA recorded the highest Se in goats as 0.83 (95% PI, 0.61-0.97). The f-PCR Se estimate slightly differed between species [sheep 0.36 (0.19-0.58), goats 0.19 (0.08-0.35)], while the Se of FC was similar between species [sheep 0.29 (0.15-0.51), goats 0.27 (0.13-0.45)]. The specificity estimates for all tests were high, close to unity, and similar between species. CONCLUSION: Overall, the results showed that the Mt-ELISA method can be used for MAP detection in small ruminants' flocks.

Molecular characterisation of Mycobacterium avium subsp. paratuberculosis in Australia.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (Map) causes Johne's disease (JD), a chronic enteritis widespread in ruminants, resulting in substantial economic losses, especially to the dairy industry. Understanding the genetic diversity of Map in Australia will assist epidemiological studies for tracking disease transmission and identify subtype characteristics for use in development of improved diagnostic typing methods. Here we investigated the phylogenetic relationships of 351 Map isolates and compared different subtyping methods to assess their suitability for use in diagnostics and accuracy. RESULTS: SNP-based phylogenetic analysis of 228 Australian isolates and 123 publicly available international isolates grouped Type S and Type C strains into two distinct lineages. Type C strains were highly monomorphic with only 20 SNP differences separating them. Type S strains, when aligned separately to the Telford strain, fell into two distinct clades: The first clade contained seven international isolates while the second clade contained one international isolate from Scotland and all 59 Australian isolates. The Australian Type B strain clustered with US bison strains. IS1311 PCR and Restriction Enzyme Analysis (REA) intermittently generated incorrect results when compared to Long Sequence Polymorphism (LSP) analysis, whole genome SNP-based phylogenetic analysis, IS1311 sequence alignment and average nucleotide identity (ANI). These alternative methods generated consistent Map typing results. A published SNP based assay for genotyping Map was found to be unsuitable for differentiating between Australian and international strain types of Map. CONCLUSION: This is the first phylogenetic analysis of Australian Map isolates. The Type C lineage was highly monomorphic, and the Type S lineage clustered all Australian isolates into one clade with a single Scottish sheep strain. The Australian isolate classified as Type B by IS1311 PCR and REA is likely to be descended from bison and most closely related to US bison strains. Limitations of the current typing methods were identified in this study.

Therapeutic management of Mycobacterium avium subspecies paratuberculosis infection with complete resolution of symptoms and disease in a patient with advanced inflammatory bowel syndrome.

BACKGROUND: A 26-year-old male had a history of frequent bowel movements, mushy stool with mucus and loss of 25 kg body weight in 6 months was diagnosed as a case of inflammatory bowel disease (IBD). The patient did not respond to routine and standard treatment for IBD. His condition was steadily deteriorating, and he was in a very precarious state when he reported to us. METHODS: Upon laboratory investigation by using IS900 specific PCR [which is specific for Mycobacterium avium subspecies paratuberculosis (MAP)], the blood and stool samples were found negative. However, the presence of low titer MAP-antibodies by indigenous ELISA were found followed by detection of the typical acid-fast MAP bacilli (with 3 + or 4 + grade) microscopically. The MAP stool culture was positive after 6 months incubation. The biotyping by IS1311 specific polymerase chain reaction restriction enzyme (PCR-RE) confirmed infection with 'Indian Bison Type Genotype', a dominant biotype infecting the domestic livestock population of India. Standard anti-MAP therapy was initiated under supervision of the treating physician. The drug of choice in prescribed treatment regimen included Isoniazid (5 mg/kg), Rifampicin (10 mg/kg), Ethambutol (15-25 mg/kg) once a day for 24 weeks and Clarithromycin (250 mg)/Levofloxacin (250 mg) twice a day for 6 weeks. RESULTS: Following treatment, the patient started improving progressively with reduction in bowel movement frequency and gained body weight with an enhanced appetite propensity. Upon follow-up of the patient after 1 year of treatment, stool-microscopy and stool-culture were found negative for MAP. Till the recent past, the patient was further monitored for disease relapse, if any. CONCLUSIONS: This patient has experienced a complete resolution of IBD using a combination of anti-MAP antibiotics. The initial detection of heavy shedding of acid-fast MAP bacilli and typical colony morphology with its characterization obtained from culturing of stool sample indicated the infection of MAP. Interestingly, the present case is one more example of the linkage of demonstrable MAP infection treated with anti-MAP therapy in the presence and then absence of disease in the human host.

Bio-typing of Mycobacterium avium subspecies paratuberculosis isolates recovered from the Himalayan sheep and goats.

Information on bio-type profile of Mycobacterium avium subspecies paratubeculosis (MAP) in sheep flocks and goat herds of Himalayan region is not reported earlier. The aim of our study was to determine the bio-type of MAP infecting livestock of this region. A total of 71 faecal samples (sheep-57, goats-14) were screened by Ziehl-Neelsen (ZN) staining and IS900 PCR, and then processed for culture on Herrold's egg yolk medium (HEYM) having mycobactin J (MJ). Out of 71 faecal samples, MAP colonies were seen only in four samples (sheep-3 and goat-1). Isolates were confirmed as MAP on the basis of slow growth, acid fastness, MJ dependency, IS900 and IS1311 PCR. All the IS900 and IS1311 PCR positive samples were bio-typed by IS1311 PCR-REA (restriction endonuclease analysis), which confirmed all four isolates as 'bison type.' In IS1311 based phylogeny of MAP isolates by ClustalW method of the MegAlign program of DNASTAR Lasergene software, the four sequences of MAP isolates (NCBI sequence nos. MH988763, MH988765, MH988766 and MH988764) did not show any distinct clustering/grouping pattern. However, these four isolates showed a bit of closeness to the MAP sequences (KC990353.1 and KC990352.1) of 'bison type' isolated from wood bison in Canada. In conclusion, this is the first report on isolation and bio-type profile of MAP infecting sheep and goats of Himalayan region. Study will help in devising prevention and control strategies against spread of MAP infection in livestock population of Himalayan region.

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.

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.

First molecular epidemiological study of Mycobacterium avium subsp. paratuberculosis in cattle and buffalo from different regions of Brazil.

Paratuberculosis is an incurable disease in ruminants with great worldwide economic impact, caused by Mycobacterium avium subsp. paratuberculosis (MAP). The objective of this study was to carry out a study of the molecular epidemiology of the MAP using the restriction enzyme analysis (REA) technique of IS1311 MAP region in biological samples of feces, intestinal tissue, and mesenteric lymph nodes of cattle and buffaloes from six Brazilian states. In total, 109 samples of feces and tissues of cattle and buffaloes were collected from animal paratuberculosis suspected. Twenty-five samples were positive in the detection of the DNA of the IS900 region of MAP and it was possible to type 18 strains in the analysis of the region IS1311, being 100% of them identified as belonging to subtype Bison MAP strain. This is the first epidemiological molecular study of MAP in Brazil. The results indicate that paratuberculosis is widespread in cattle and in buffaloes in several regions of Brazil, and the subtype Bison MAP strain was the only one identified in the samples analyzed in this study, demonstrating the similarity between the strains from different states tested. These results provide the necessary support for the implementation of paratuberculosis control strategies in cattle and buffaloes in Brazil.

Prevalence of Mycobacterium avium subsp. paratuberculosis and Escherichia coli in blood samples from patients with inflammatory bowel disease.

Mycobacterium avium subsp. paratuberculosis (MAP) and adherent-invasive Escherichia coli (AIEC) have been implicated as primary triggers in Crohn's disease (CD). In this study, we evaluated the prevalence of MAP and E. coli (EC) DNA in peripheral blood from 202 inflammatory bowel disease (IBD) patients at various disease periods and compared against 24 cirrhotic patients with ascites (CIR) (non-IBD controls) and 29 healthy controls (HC). MAP DNA was detected by IS900-specific nested PCR, EC DNA by malB-specific nested PCR and AIEC identity, in selected samples, by sequencing of fimH gene. CD patients with active disease showed the highest MAP DNA prevalence among IBD patients (68 %). Infliximab treatment resulted in decreased MAP detection. CIR patients had high individual and coinfection rates (75 % MAP, 88 % EC and 67 % MAP and EC), whilst HC controls had lower MAP prevalence (38 %) and EC was undetectable in this control group. EC DNA prevalence in IBD patients was highly associated with CD, and 80 % of EC from the selected samples of CD patients analyzed carried the fimH30 allele, with a mutation strongly associated with AIEC. Our results show that coinfection with MAP and AIEC is common and persistent in CD, although the high MAP and EC detection in CIR patients suggested that colonization is, at least, partially dependent on increased gut permeability. Nevertheless, facilitative mechanisms between a susceptible host and these two potential human pathogens may allow their implication in CD pathogenesis.

Application of multiple laboratory tests for Mycobacterium avium ssp. paratuberculosis detection in Crohn's disease patient specimens.

The difficulties involved in detecting and enumerating Mycobacterium avium subsp. paratuberculosis (MAP) as a pathogen potentially involved in Crohn's disease (CD) are well known. This study aimed to improve this situation through the application of multiple laboratory diagnostic tests to detect and isolate this bacterium from different specimens collected from CD-patients and non-CD subjects as controls. A total of 120 samples (terminal ileum and colon biopsies, blood and stool) were obtained from 19 CD-patients and from 11 individuals who did not have a clinicopathological diagnosis of CD (non-CD controls) attending for ileocolonoscopy. All samples were processed by staining techniques, culture on both solid and liquid media, and Insertion Sequence 900/F57 real-time PCR. The MAP frequency in CD-patients was found in a significantly greater proportion than in non-CD subjects; the most positive samples were biopsies from CD-patients tested by real-time PCR. MAP detection in biopsies, and in the other samples, by applying multiple and validated laboratory diagnostic tests, could be a marker of active infection, supporting MAP involvement in CD.

Mycobacterium avium subsp. paratuberculosis is not discerned in diabetes mellitus patients in Hyderabad, India.

Mycobacterium avium ssp. paratuberculosis (MAP) is an obligate intracellular pathogen. It causes chronic intestinal inflammation in ruminants known as Johne's disease and is associated with human Crohn's disease. Furthermore, association of MAP with other autoimmune diseases, such as type-1 diabetes, has been established in patients from Sardinia (Italy) which is a MAP endemic and genetically isolated region. Due to largest livestock population and consequently high MAP prevalence amidst a very high diabetes incidence in India, we sought to test this association on a limited number of patient samples from Hyderabad. Our results of ELISA with MAP lysate and MAP-specific protein MAP3738c as well as PCR/real-time PCR of MAP-specific sequences IS900 and/or f57 indicated that, in contrast to Sardinian diabetic patients, MAP infection in blood is not discerned in diabetic patients in Hyderabad. The association of a mycobacterial trigger with diabetes therefore could well be a population-specific phenomenon, highly dependent on genetic repertoire and the environment of susceptible populations. However, a larger study is needed in order to confirm this.

Comparison of 2 PCR assays on environmental samples cultured for Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis, a chronic, contagious, and incurable enteric disease of ruminants. An in-house IS900 PCR assay validated for MAP detection in sheep has been shown to have a higher sensitivity than a commercial PCR and fecal culture. We have now compared the performance of this in-house IS900 PCR assay with a commercial ISMap02 PCR assay for the detection of MAP DNA in bovine dairy farm environmental samples. We purposefully selected 30 culture-positive, 62 culture-negative, and 62 non-interpretable environmental samples. We applied the IS900 PCR assay directly to the frozen inoculum of these samples. Inocula were incubated in an automated system, and growth was confirmed by an acid-fast bacilli stain and the IS900 PCR assay. Among culture-positive samples before incubation, the IS900 PCR assay yielded significantly more positive results than the ISMap02 PCR assay; however, among culture-negative samples, the IS900 PCR assay yielded positive results both before and after incubation. The ISMap02 PCR assay did not flag positively among the culture-negative samples either before or after incubation. The IS900 PCR assay is a sensitive method that can be used to detect MAP DNA in environmental samples before incubation. The ISMap02 PCR assay is a specific method used to detect MAP DNA in environmental samples both before and after incubation.

Diagnostic performance of faecal and tissue multiplex qPCR IS900/F57 for the detection of Mycobacterium avium subspecies paratuberculosis in cattle.

Mycobacterium avium subspecies paratuberculosis (MAP) is responsible for bovine-paratuberculosis (bPTB), which causes high production losses in cattle. A cross-sectional study was conducted in 228 cattle to evaluate the validity and diagnostic utility of a multiplex real-time PCR (qPCR) on faecal and intestinal samples [ileocaecal valve (ICV) and ileocaecal lymph nodes (ICLN)], using intestinal tissue culture as a reference test. Based on the sensitivity, specificity, and likelihood ratios (LR) obtained, the diagnostic value of faecal qPCR for confirming MAP infection was moderate (sensitivity 50.3%, specificity 93.5%, positive LR 7.8), and low to rule it out (negative LR 0.5). In areas with a prevalence of >23% the credibility of positive results was higher than 70%. In the case of negative results, their credibility was higher than 90% in herds with an infection rate below 19%, so faecal qPCR would be very useful in these areas to certify the absence of infection. For post-mortem diagnosis, qPCR on ICV samples showed good diagnostic accuracy to confirm the disease (sensitivity 71.7%, specificity 93.3%, positive LR 10.8), with a credibility higher than 70% in animals from areas or herds with a prevalence of infection greater than or equal to 18%. The best strategy to rule out the disease was the parallel combination of both tissues (ICV + ICLN) (sensitivity 81.3%, specificity 89.5%, negative LR 0.2) with a credibility of over 95% in animals from areas with an infection prevalence of 0-20%. Faecal and tissues qPCR techniques can be used to monitor bPTB, the interpretation of results, according to epidemiological situation of the herd or area, are shown.

Mycobacterium avium Subspecies paratuberculosis in Asymptomatic Zoo Herbivores in Poland.

Mycobacterial infections are significant issues in zoo animals, influencing animal welfare, conservation efforts, and the zoonotic potential of pathogens. Although tuberculosis is recognised to be highly dangerous, paratuberculosis can also lead to animal losses and is potentially dangerous for humans. The aim of the current study was to confirm whether Mycobacterium avium spp. paratuberculosis (MAP) infections are currently present in zoos in Poland. Faeces samples (n = 131) were collected from different animal species from eight zoos in Poland. The faeces were decontaminated and inoculated into Herrold's Egg Yolk Media. The species was determined using commercial DNA testing. The IS900 was checked using RT-PCR. The culture was positive in seven samples: five with M. avium, one with Mycobacterium fortiatum, and one without any identified Mycobacterium species. RT-PCR confirmed MAP genetic material in nine animals. Our findings represent the first confirmation of MAP in bongo (Tragelaphus eurycerus), indicating that it is present in Polish zoological gardens. Fortunately, the disease can be monitored more easily due to recent legislation (the Animal Health Law).

Diagnosis of paratuberculosis in cattle: microbiological culture, serology and PCR.

The aim of this study was to confirm clinical diagnosis of paratuberculosis in two cows showing suggestive clinical signs of the disease. Based on clinical signs, in culture and in IS900 PCR results from the individual milk samples it was possible to diagnose paratuberculosis in the cows studied.

First isolation of Mycobacterium avium subsp Paratuberculosis from commercial pasteurized milk in Argentina.

Mycobacterium avium subsp paratuberculosis was isolated from two out of seventy samples (2.86 %) of pasteurized and ultra-pasteurized milk. The isolates were positives to IS900 PCR and showed a C17 RFLP pattern, the most prevalent in Argentina. The present study is the first report of Mycobacterium avium subsp paratuberculosis culture from pasteurized milk in Argentina.

First identification of Mycobacterium avium subsp. paratuberculosis in wild ruminants in a zoo in Mexico.

Background and Aim: Paratuberculosis (PTB) is an infectious disease that induces chronic enteritis in ruminants. It is caused by Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we evaluated the presence of MAP using bacteriological, molecular, and anatomopathological studies, based on the clinical suspicion of PTB in a zoo, in an area housing 10 scimitar-horned oryx (Oryx dammah), five giraffes (Giraffa camelopardalis), and three blue wildebeests (Connochaetes taurinus). Materials and Methods: From November 2016 to June 2017, fecal samples were collected from individuals of the three species on four occasions, resulting in a total of 56 fecal samples. In addition, five small intestine samples were collected from the necropsies of three adult scimitar-horned oryx females and two oryx calves. MAP identification was performed through isolation in Herrold's medium with egg yolk, mycobactin, and sodium pyruvate, Ziehl-Neelsen staining, IS900 polymerase chain reaction (IS900 PCR), and anatomopathological examination of intestine samples. Results: Diffuse granulomatous enteritis with abundant acid-fast bacilli was found in two out of five intestine samples from adult scimitar-horned oryx females. MAP was isolated in 7/56 (12.5%) of the fecal samples from four scimitar-horned oryx, one giraffe, and two wildebeest samples. Two out of 5 (40%) samples obtained from scimitar-horned oryx tested positive. IS900 PCR yielded five positive samples (two fecal samples and three small intestine samples). MAP isolates were classified as Type C (Cattle) using type-specific PCR. Conclusion: These results demonstrated the presence of MAP in the area evaluated and indicated the importance of both sampling live animals and conducting postmortem examinations. The use of bacteriological and histopathological diagnostic techniques demonstrated in this study will provide insight into the health status and prevalence of paratuberculosis in wild ruminants under human care.