Presence Of Mycobacterium Avium Research Articles

Detection of Mycobacterium avium subsp. paratuberculosis in liver of slaughtered cattle, sheep and goats by PCR and culture

The presence of Mycobacterium avium subsp. paratuberculosis (MAP) in tissues other than intestines and associated lymph nodes is a potential public health concern. Therefore, the presence of MAP in the liver of slaughtered cattle, sheep and goats with unknown Johne’s disease status was evaluated. Liver samples were collected randomly from 470 mature animals including 200 cattle, 135 sheep and 135 goats. The existence of MAP DNA from obtained liver samples was determined using PCR, thereafter PCR-confirmed samples were cultured. Based on liver PCR results, IS900 genes were detected in 11/200 (5.5%), 3/135 (2.2%), 2/135 (1.48%) liver samples of cattle, sheep and goats, respectively. In cattle, sheep and goats, 6 (54.5%), 1 (33.3%) and 1 (50%) PCR-positive samples showed positive results in culture, respectively. Our results revealed that MAP can be detected and cultured from the liver of slaughtered cattle, sheep and goats with unknown Johne’s disease status. Hence, given concerns about a potential zoonotic role for MAP, there is a crucial need to detect animals with MAP disseminated infection in the liver before slaughter.

Mycobacterium avium subsp. paratuberculosis down-regulates mRNA expression of iron-induced macrophage Ferroportin 1

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease. The mechanisms by which MAP is able to adapt to the innate host response are still unclear. We examined Ferroportin 1 (FPN1) mRNA expression levels via real-time PCR of the mouse macrophage cell line J774 that was incubated in the presence of Mycobacterium avium subsp. paratuberculosis (MAP) or MAP crude protein extract. Infection with live MAP decreased FPN1 mRNA levels in a multiplicity of infection (MOI)-dependent fashion. Macrophages infected with MOIs of 20:1 and 15:1 did not show any change in FPN1 gene expression, whereas MOIs of 10:1 and 5:1 induced a decrease of 50 and 80%, respectively. Macrophages treated with 50, 100, 150 and 200 µg/mL of MAP crude extract (ATCC19698) decreased FPN1 mRNA expression by 25%. Additionally, up-regulation of FPN1 mRNA by an iron overload treatment of 400 µM of ferric nitrilotriacetate (FeNTA) was abrogated by live MAP (MOI 20:1) by approximately 70%. Our data revealed an inhibitory effect of MAP on FPN1 mRNA and suggested a bacterial mechanism that may play a role in host iron regulation.

Mycobacterium avium subsp. paratuberculosis down-regulates mRNA expression of iron-induced macrophage Ferroportin 1

Veterinaria México OA ISSN: 2448-6760Cite this as:Landeros Sánchez B, Gutierrez Pabello JA, Medina Basulto GE, Renteria Evangelista TB, Díaz Aparicio E, Oshima S. Mycobacterium avium subsp. paratuberculosis down-regulates mRNA expression of iron-induced macrophage Ferroportin 1. Veterinaria México OA. 2016;3(1). doi: 10.21753/vmoa.3.1.361Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease. The mechanisms by which MAP is able to adapt to the innate host response are still unclear. We examined Ferroportin 1 (FPN1) mRNA expression levels via real-time PCR of the mouse macrophage cell line J774 that was incubated in the presence of Mycobacterium avium subsp. paratuberculosis (MAP) or MAP crude protein extract. Infection with live MAP decreased FPN1 mRNA levels in a multiplicity of infection (MOI)-dependent fashion. Macrophages infected with MOIs of 20:1 and 15:1 did not show any change in FPN1 gene expression, whereas MOIs of 10:1 and 5:1 induced a decrease of 50 and 80%, respectively. Macrophages treated with 50, 100, 150 and 200 µg/mL of MAP crude extract (ATCC19698) decreased FPN1 mRNA expression by 25%. Additionally, up-regulation of FPN1 mRNA by an iron overload treatment of 400 µM of ferric nitrilotriacetate (FeNTA) was abrogated by live MAP (MOI 20:1) by approximately 70%. Our data revealed an inhibitory effect of MAP on FPN1 mRNA and suggested a bacterial mechanism that may play a role in host iron regulation.Figure 1. Infection by Mycobacterium avium subsp. paratuberculosis (MAP) down-regulates macrophage fpn1 gene expression. Murine macrophages, J774, were infected with MAP at different multiplicities of infection (MOIs) for 6 hours. Total RNA was extracted and gene expression was measured by real-time PCR. Results were normalized to GAPDH and expressed as units of relative expression (URE). Results are the mean ± standard deviation of 3 independent experiments with 3 replicates each. Statistical significance, P = 0.05.

Presence of Mycobacterium avium subsp. paratuberculosis (MAP) in Brazilian patients with inflammatory bowel diseases and in controls.

Mycobacterium avium subsp. paratuberculosis (MAP) has attracted the interest of researchers because of similarities between paratuberculosis and Crohn's disease (CD). The aim of this study was to evaluate the frequency of MAP through cultures, histology and polymerase chain reaction (PCR) on intestinal biopsies from Brazilian CD patients. Quantitative real time PCR (qRT-PCR) was performed on positive samples. Analytical cross-sectional study with control group at two federal universities. Fresh samples were collected from 25 patients; five with CD, eight with ulcerative colitis (UC) and 12 controls with non-inflammatory bowel disease (nIBD). Formalin-fixed paraffin-embedded (FFPE) samples from 143 patients were also collected: 44 CD, 49 UC and 56 nIBD. None of the fresh samples was positive for MAP. Five FFPE samples (one CD, two UC and two nIBD) and three fresh samples (one in each group) were positive through IS900-PCR. qRT-PCR was performed on these eight samples. Among the FFPE samples, there were 192.12 copies/μl in the CD group, 72.28 copies/μl in UC and 81.43 copies/μl in nIBD. Among the fresh samples, there were 432.99 copies/μl, 167.92 copies/μl and 249.73 copies/μl in the CD, UC and nIBD groups, respectively. The highest bacterial load was in the CD group. This study does not provide evidence for a role of MAP in the etiology of CD, although MAP DNA was detected in all three patient groups. This is the first report of MAP presence in human intestinal biopsies in Brazil.

Survey on the presence of Mycobacterium avium subsp. paratuberculosis in ground beef from an industrial meat plant

Paratuberculosis of ruminants is characterised by chronic enteritis but, at advanced stages of the disease, a systemic dissemination of Mycobacterium avium subsp. paratuberculosis (MAP) in tissues and organs can occur. MAP has been recovered from lymph nodes and muscles of clinical and sub-clinical cows. In most countries, dairy and beef cattle infected with paratuberculosis are routinely sent to slaughter and the consumption of their meat could be a possible route of human exposure to MAP. However, few studies on MAP in ground beef are currently available. During the period November 2013–March 2014 we carried out a survey on the ground beef produced in an industrial meat processing plant. One-hundred and forty samples of ground meat were analysed by IS900-qPCR and culture (VersaTrek System®). The limit of detection (LOD) of qPCR was 630 MAP cells/g (107 CFU/g) while the LOD for culture was 170–230 MAP cells/g (62–115 CFU/g). No samples were positive by direct IS900 qPCR, while two samples were positive by liquid culture. Our data suggest that the presence of live MAP in raw minced meat is possible. In order to avoid exposure for humans through the consumption of contaminated meat, proper cooking of meat is recommended.

Mycobacterial pseudotumor of the skin

Inflammatory pseudotumors have a diverse etiology, mycobacterial pseudotumor (MP) being one of them. MP is a rare entity; it has been reported infrequently in various organs and is extremely rare in the skin. We report a cutaneous MP in an immunosuppressed liver transplant recipient. The lesion consisted mostly of spindle cells, with small numbers of lymphocytes. Conventional acid-fast bacilli (AFB) stain revealed a large number of acid-fast bacilli within spindled histiocytes and the presence of Mycobacterium avium was determined by polymerase chain reaction. Given that the patient had a prior history of cutaneous squamous cell carcinoma resected and reconstructed in the same area, establishing the diagnosis was challenging. Immunohistochemical staining for lysosome-associated membrane protein was strongly positive, suggesting the presence of numerous mature lysosomes within infected spindle cells. Mycobacterial spindle cell pseudotumors can mimic malignant or benign neoplasms and should be considered in differential diagnosis of spindle cell lesions, especially in immunocompromised patients. Further studies are needed to determine mechanisms that permit the survival of mycobacteria within the lesions and that cause this unusual manifestation of infection.

DETECTION OFMYCOBACTERIUM AVIUMSUBSPECIESPARATUBERCULOSISBY IS900-BASED PCR ASSAYS FROM AN ALPACA (VICUGNA PACOS) KEPT IN A GERMAN ZOOLOGICAL GARDEN

An alpaca (Vicugna pacos) bred and kept in a German zoological garden exhibited clinical signs consistent with paratuberculosis. The presence of Mycobacterium avium ssp. paratuberculosis (MAP) was confirmed in feces and in the ileocecal lymph node (ILN) by IS900-based polymerase chain reaction (PCR) assays and culture. A bacterial burden of 7.6 x 10(6) MAP/g in feces and 4.4 x 10(7) MAP/g in lymph node tissue was determined by real-time PCR. For further characterization, a conventional PCR was developed. After sequencing of the 864-bp PCR amplicon covering nucleotide positions 13 to 876 within the IS900, the alpaca isolate shared 100% nucleotide homology with the bovine MAP-K10 IS900 reference sequence (GenBank: AE16958), indicating a cattle strain. This report supports the present occurrence of MAP in German camelid populations and highlights the need to expand routine MAP surveillance to South American camelids held in European zoos.

Sale of Raw Milk in Northern Italy: Food Safety Implications and Comparison of Different Analytical Methodologies for Detection of Foodborne Pathogens

The safety of raw milk sold in Northern Italy was investigated in relation to hygiene quality parameters and presence of Salmonella spp., Listeria monocytogenes, thermotolerant Campylobacter, and Verocytotoxin producing Escherichia coli O157:H7. The performance of different analytical methods used-official culture method (ISO), modified Bacteriological Analytical Manual cultural method (mBAM), and polymerase chain reaction (PCR)-was evaluated. The presence of Mycobacterium avium subsp. paratuberculosis (Map) was investigated only by PCR. All samples met regulations for alkaline phosphatase and inhibitory substance, while 18% and 44.8% of samples collected from vending machines had, respectively, somatic cell count (SCC) >300,000/mL and total bacterial count (TBC) >50,000 CFU/mL. The correlation between hygienic quality parameters in samples collected from bulk tank and vending machines showed a significant increase of TBC in vending machines meaning that raw milk was mishandled during distribution and sale. All pathogens investigated were detected in raw milk sold at vending machines; a total of five samples (5%) had at least one pathogen, of which two were detected by PCR and three by mBAM. None of the samples was positive by cultural ISO methods. Even if the comparison of analytical methods showed that none performs significantly better than the others, testing a higher volume of milk (25 versus 210 mL) affects significantly the detection rate of pathogens. Three samples (3%) were positive for Map, suggesting that raw milk is a significant source of Map exposure for consumers. The observed TBC increase and the detection of several pathogenic bacteria pose questions on the safety of raw milk; the use of ISO seems inefficient in detecting a low contamination level of pathogens in milk and consequently not appropriate as official method for testing. In order to ensure consumer's safety, a new approach for the raw milk chain is required.

The presence of Mycobacterium avium subsp. avium in common pheasants (Phasianus colchicus) living in captivity and in other birds, vertebrates, non-vertebrates and the environment

Although avian mycobacteriosis is not prevalent among domestic fowl used for intensive husbandry, it has been described in both free living birds and birds in captivity, e.g., zoological gardens and small fowl flocks. In this study, we examined 305 pheasants from six flocks as well as 70 other birds belonging to 14 species and 97 other vertebrates caught in a closed area. We also investigated the prevalence of mycobacteria in non-vertebrates (earthworms) and soil in two pheasant flocks. Mycobacterium avium subsp. avium (M. a. avium) was isolated in four flocks from 17 (5.6%) pheasants. In one M. a. avium-infected pheasant co-infection with M. a. hominissuis was diagnosed. Granulomatous inflammatory lesions were observed in liver and spleen in only four M. a. avium-infected pheasants originating from two flocks. From the other 38 pheasants other mycobacterial species were isolated, such as M. fortuitum, M. terrae, M. triviale, M. chelonae, M. scrofulaceum, M. smegmatis, M. flavescens, M. diernhoferi and non-identifiable mycobacterial species. In the group of 70 birds of other species, we identified M. a. avium in two (2.9%) goshawks (Accipiter gentilis). We did not isolate M. a. avium from any of the other 97 vertebrates, the 391 environment samples or 97 earthworms.

Association of single nucleotide polymorphisms in the ANKRA2 and CD180 genes with bovine respiratory disease and presence of Mycobacterium avium subsp. paratuberculosis1

The objective was to determine whether single nucleotide polymorphisms (SNPs) in the ANKRA2 and CD180 genes are associated with incidence of bovine respiratory disease (BRD) and presence of Mycobacterium avium subsp. paratuberculosis (MAP) in cattle. Two independent populations were used. The first population (BRD-affected; N = 90) was composed of 31 half-sib progeny, from a Brahman × Angus sire, that were treated for BRD. Untreated offspring from the sire were selected to serve as controls. The second population (MAP-infected) of 330 animals of unknown parentage was evaluated for the presence of MAP in ileocecal lymph node and classified as positive or negative. Markers in both genes were assessed for association in these two populations. In the BRD-affected population, five SNPs in the ANKRA2 gene were significantly associated (P < 0.05), and two SNPs were highly associated (P < 0.01) with incidence of BRD. In addition, two SNPs in the CD180 gene were found to be associated with this trait. In the MAP-infected population, one SNP in the ANKRA2 gene was significantly associated (P < 0.05) with the presence or absence of MAP, and a SNP in the CD180 gene was highly associated (P < 0.01) with the trait. Haplotypes, using significant markers, showed a positive association with both incidence of BRD (P = 0.0001) and with the presence of MAP (P = 0.0032). Markers in the ANKRA2 and CD180 genes are associated with the ability of the animal to cope with pathogens.

Have They Proven that Mycobacteria Are Present “in the Mist?”

In the December 2009 issue of Microbe (page 545), Marcia Stone discusses the recent paper by Feazel et al. (“Opportunistic pathogens enriched in showerhead biofilms,” Proc. Natl. Acad. Sci. USA 106:16393–16399, 2009) concerning the suggested presence of Mycobacterium avium in waters derived from showerheads, in an article entitled “Mycobacteria in the mist.” It is noted in this article that these organisms are “strongly enriched” in waters from the showerhead environment, that “microbial communities” were being studied, and that that the paper authors “relied on metagenomics, using amplified ribosomal RNA (rRNA) genes, to identify organisms.” A review of the original PNAS paper suggests that none of these conclusions can be supported. Bulk extraction approaches were used to recover nucleic acids from the natural microbial assemblages used in these analyses. No evidence has been provided that would relate the recovered sequences to microbes, more specifically to in situ active microbes, the “microbial community,” or to make it possible to “identify organisms.”

Detection of Mycobacterium avium in pet birds

The present study is a report on the presence of Mycobacterium avium in four birds of the psittaciform order kept as pets. Anatomopathological diagnosis showed lesions suggestive of the agent and presence of alcohol-acid resistant bacilli (AARB) shown by the Ziehl-Neelsen staining. The identification of Mycobacterium avium was performed by means of PRA (PCR Restriction Analysis). DNA was directly extracted from tissue of the lesions and blocked in paraffin. The role of this agent in pet bird infection is discussed, as well as its zoonotic potential.

Dynamics of endemic infectious diseases of animal and human importance on three dairy herds in the northeastern United States

Endemic infectious diseases in dairy cattle are of significant concern to the industry as well as for public health because of their potential impact on animal and human health, milk and meat production, food safety, and economics. We sought to provide insight into the dynamics of important endemic infectious diseases in 3 northeastern US dairy herds. Fecal samples from individual cows and various environmental samples from these farms were tested for the presence of major zoonotic pathogens (i.e., Salmonella, Campylobacter, and Listeria) as well as commensal bacteria Escherichia coli and enterococci. Additionally, the presence of Mycobacterium avium ssp. paratuberculosis was tested in fecal and serum samples from individual cows. Test results and health and reproductive records were maintained in a database, and fecal, plasma, DNA, and tissue samples were kept in a biobank. All bacteria of interest were detected on these farms and their presence was variable both within and between farms. The prevalence of Listeria spp. and L. monocytogenes in individual fecal samples within farm A ranged from 0 to 68.2% and 0 to 25.5%, respectively, over a period of 3 yr. Within farm B, continuous fecal shedding of Salmonella spp. was observed with a prevalence ranging from 8 to 88%; Salmonella Cerro was the predominant serotype. Farm C appeared less contaminated with Salmonella and Listeria, although in the summer of 2005, 50 and 19.2% of fecal samples were positive for Listeria and L. monocytogenes, respectively. The high prevalence of E. coli (89 to 100%), Enterococcus (75 to 100%), and Campylobacter (0 to 81%) in feces suggested they were ubiquitous throughout the farm environment. Fecal culture and ELISA results indicated a low prevalence of Mycobacterium avium ssp. paratuberculosis infection in these farms (0 to 13.6% and 0 to 4.9% for culture-positive and ELISA-positive, respectively), although the occasional presence of high shedders was observed. Results have major implications for food safety and epidemiology by providing a better understanding of infectious disease dynamics on dairy farms. Comprehensive understanding of these infections may lead to better farm management practices and pathogen reduction programs to control and reduce the on-farm contamination of these pathogens and to prevent their further entry into the food-chain.

Mycobacterium avium subsp. paratuberculosis in Scavenging Mammals in Wisconsin

The presence of Mycobacterium avium subsp. paratuberculosis (MAP) in non-ruminant wildlife has raised questions regarding the role of these species in Johne's disease transmission. In this study we tested 472 tissues from 212 animals of six different species of scavenging mammals. All animals were taken from within a 210-square-mile area in Dane and Iowa counties of south central Wisconsin from September to May in 2003-04 and tested for the presence of MAP. We detected MAP-specific DNA in 81 of 212 (38%) scavenging mammals, in 98 of the 472 (21%) tissues; viable MAP was cultured from one coyote's ileum and lymph node tissue. Despite the low numbers of viable MAP isolated in this study, our data adds to the increasing evidence demonstrating the potential for transmission and infection of MAP in nonruminant species and provides possible evidence of interspecies transmission. The apparently high exposure of nonruminant wildlife provides potential evidence of a spill-over of MAP to wildlife species and raises the question of spillback to domestic and wild ruminants. These results demonstrate the importance of understanding the role of wildlife species in developing management strategies for Johne's disease in domestic livestock.

Outbreak of avian tuberculosis in 48-week-old commercial layer hen flock.

This report describes an outbreak of tuberculosis in a flock of 38,500 48-wk-old layer hens. Clinical characteristics of the process included a gradual drop in egg production and feed intake, as well as an increased mortality rate. Two well-defined clinical features were observed. On the one hand, a number of birds displayed good body condition and continued to lay but presented granulomatous nodular lesions, particularly in the infraorbital sinus, liver, and intestine. In contrast, other hens were emaciated and presented granulomatous lesions of various sizes throughout the internal organs. The lesions observed in the various organs of all the affected birds consisted of granulomas containing acid/alcohol-fast bacilli. The presence of Mycobacterium avium was confirmed through polymerase chain reaction techniques. This report describes the epidemiologic and histopathologic characteristics of the outbreak.

Primera descripción concomitante de tuberculosis y paratuberculosis en ovinos lecheros en Argentina

The authors present the results of a comparative intradermal tuberculin test performed on dairy sheep to determine rates of reaction to bovine and avian purified protein derivative (PPD). Test reactions were interpreted by palpation and measurement with a calliper. Results were analysed by plotting the values obtained. Agar gel immunodiffusion tests, growth in culture and histopathological examinations were also performed to allow differential diagnosis between tuberculosis and paratuberculosis. In the first tuberculin test, performed on 288 sheep, 74 animals showed a positive reaction to avian PPD (25.69%), two animals showed a positive reaction to bovine PPD (0.69%), and one animal was positive with respect to both tuberculins (0.34%). In the second test, involving 247 sheep, 55 demonstrated a positive reaction to avian PPD (22.26%), whilst none reacted to bovine PPD. The presence of Mycobacterium avium subsp. paratuberculosis was confirmed by culture and serological testing. In addition, lesions compatible with those caused by M. bovis were found by histopathological examination. These findings highlight the importance of the accurate diagnosis of tuberculosis and paratuberculosis for the effective control of both diseases.

Determination of the presence of Mycobacterium avium on Guam as a precursor to reintroduction of indigenous bird species

Eight of 11 native forest bird species on Guam were extirpated by the introduction of the Brown Tree Snake Boiga irregularis. Emergency measures necessary to rescue the Guam subspecies of Micronesian Kingfisher Halcyon cinnamomina cinnamomina from extinction involved translocation and captive breeding in American mainland zoos. Soon after the establishment of a captive breeding population, the kingfisher demonstrated a high degree of susceptibility to avian tuberculosis (ATB), a disease that proved to be a major threat to the preservation of the species. The cause of ATB is Mycobacterium avium which produces a prolonged course of infection in kingfishers and other birds. Kingfishers infected with M. avium are difficult to detect until late in the course of the disease, thereby potentially posing a risk of transmitting ATB to the Guam captive population of Guam Rails Gallirallus owstoni, if kingfishers are repatriated. M. avium is considered to be ubiquitous in nature. However, there are no reported mortalities due to ATB in any bird species on Guam. In this study, six of twenty-one cultures yielded Mycobacterium spp., two of which were further identified as M. avium. Since this study demonstrates that M. avium already exists on Guam, repatriating kingfishers to Guam poses no threat of introducing a new pathological agent to the island's ecosystem. Strict quarantine procedures along with rigorous animal husbandry protocols should minimize risks of repatriating infected kingfishers to Guam, and prevent transmission of ATB to the captive population of Guam Rails and other bird populations on Guam.