Serological, bacteriological and molecularbiological survey of paratuberculosis (Johne's disease) in Austrian cattle

Paratuberculosis (Johne's disease) is a chronic infectious disease of ruminants, caused by Mycobacterium avium subspecies paratuberculosis (MAP). Because of its long incubation period, high economic losses, difficulties in diagnosis and possible links to Morbus Crohn in humans, paratuberculosis is one of the most important diseases of ruminants today. An abattoir-based nationwide survey on the occurrence of MAP in the Austrian cattle population was performed using serology (SVANOVIR-ELISA) as well as culture, ZN-stain and IS900-PCR on faeces and lymph node samples. A total of 756 Austrian slaughter cattle were serologically, bacteriologically and molecularbiologically tested for the occurrence of MAP and specific antibodies respectively. Samples were collected following a statistical plan to obtain balanced specimens from the whole country. Nineteen per cent of the animals tested were serological positive, 10.1% gave an inconclusive result and 70.9% showed no specific antibodies against MAP. Only in four individuals MAP could be detected by stain, bacteriology or Polymerase Chain Reaction. The calculated prevalence of 19.0% positive cattle, each representing one farm, showing specific antibodies against MAP is rather high in terms of animal-level but low in herd level prevalence compared with other countries. When this study is compared with a similar study performed in Austria 1999, a significant increase of positive cattle and farms could be seen in Austria.     

Genotypes of Mycobacterium avium subsp. paratuberculosis from South American countries determined by two methods based on genomic repetitive sequences

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of Johne's disease or paratuberculosis of ruminants and has been associated with Crohn's disease in humans. In this study, the genotypes of MAP obtained so far in South American countries using a combination of the subtyping methods Mycobacterial Interspersed Repeats Units-Variable Number of Tandem Repeats (MIRU-VNTR) and Multilocus Short Sequence Repeats (MLSSR) were analyzed. Through this analysis, seven different MIRU-VNTR genotypes and seven MLSSR genotypes have been detected. If both methods were combined, nine different genotypes were found. Results revealed the predominance of MIRU-VNTR genotype 1 (INMV 1) and MLSSR genotype A (7 g-10 g-4ggt) among MAP isolates from different host species in South America. These predominant MAP genotypes are also commonly detected in Europe and the United States. This predominance could be the result of higher animal infection ability or better culturability on solid media used for isolation. Further studies on molecular epidemiology of MAP must be carried out in South America to increase our knowledge of the global distribution of MAP.     

The histopathologic diagnosis of subclinical Johne's disease in North American bison (Bison bison)

The morphologic changes of subclinical Johne's disease in North American Bison (Bison bison) are characterized by microgranulomas composed of epithelioid macrophages and individual multinucleate giant cells of Langhans'-type occasionally containing individual cytoplasmic acid-fast bacilli compatible with Mycobacterium avium paratuberculosis. The microgranulomas are best visualized in the mesenteric lymph nodes of infected subclinical animals. Macrophages that can be confused with infection-associated epithelioid macrophages in the mesenteric lymph nodes are pigment-carrying cells from the intestinal tract. Mesenteric lymph node biopsy may be a useful diagnostic tool for detection of mild subclinical infection in individual ruminants from herds of unknown infection status. The biopsy may also be useful for Johne's disease surveillance during test-and-cull programs.     

Mycobacterium paratuberculosis monoassociated nude mice as a paratuberculosis model

In this study, a paratuberculosis (Johne's disease) model was developed by intragastrically dosing gnotobiotic athymic nude mice with Mycobacterium paratuberculosis. The mice infrequently shed bacilli from their intestinal tracts during the first 4 months after inoculation. Following this time, increasing numbers of M. paratuberculosis (greater than 4.0 log10 bacilli per fecal pellet by 40 weeks) were recovered from the feces of the 12 mice that remained in the isolator. A similar pattern of recovery of M. paratuberculosis was obtained from the ileum, cecum, colon, and liver. Histopathologic lesions and acid-fast bacilli were rare during the first 4 months of infection and then, with time, increased in prevalence and severity. Mice maintained for 7 months or longer exhibited severe granulomatous inflammation and large numbers of acid-fast bacilli in the gastrointestinal tract and liver (up to 10(8) log10 colony forming units per gram wet weight). Five mice maintained for 7 months or more developed clinical signs consistent with those seen in paratuberculosis (weight loss, chronic diarrhea); three of these mice eventually died or became moribund and were euthanatized. M. paratuberculosis monoassociated mice released increased levels of tumor necrosis factor activity into their sera, as compared to uninfected control mice, when they were injected with bacterial lipopolysaccharide. The clinical signs, fecal shedding of M. paratuberculosis, granuloma formation, and progressive bacillary multiplication observed with these mice are consistent with naturally occurring M. paratuberculosis infection of ruminants (Johne's disease). This model will be useful for future studies of immunoregulation and antimicrobial therapy of paratuberculosis.     

From farm to fork--Mycobacterium avium ssp. paratuberculosis (MAP) as zoonotic agent?

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of the paratuberculosis (Para Tb) in ruminants. In addition, this pathogen has been suspected to be implicated in the pathogenesis of Morbus Crohn disease (MC), causing chronic inflammatory intestine changes of humans. The participation of MAP in this illness is discussed intensively and has very contradictory opinions. On the one hand several times succeeded in proving MAP DNA in changed human tissues as well as, in recent time, the bacteria has been isolated from patient's blood. On the other hand there are many publications which support the opposite opinion. In critical evaluation of already available data, therefore the hypothesis can be formulated that MAP could possibly take part in the MC of humans. The reliable verification of this hypothesis will only be possible, if the diagnostic procedures can be refined upon the substantial deficit concerning the sensitivity and/or specificity of the diagnostic methods. In addition, till now there is lack of optimized statistically case control studies. The conceivable transmission of the bacteria to humans by the direct animal contact has been considered as possible vector, furthermore, MAP has been detected in pasteurised milk and other food of animal origin. The prevalence data, usually estimated by ELISA for milk cattle stock show over 80% prevalence in many counties of the Federal Republic of Germany with an individual case prevalence ranging between 1% and 17% in different stocks. Comparable data are present also from other countries as well as for small ruminants. MAP has been concerned as a global problem, moreover the high spreading rate of MAP in wild animal populations as well as the considerable ability of the bacteria to survive in different stages of the infectious- and contamination-cycle, which might hardly be broken through. Thus it requires intensive research efforts for the development of the methodical diagnostic process as basis for valid epidemiological investigations of animals, humans and food.     

Intracellular invasion and persistence: survival strategies of Streptococcus suis and Mycobacterium avium ssp. paratuberculosis

Streptococcus (S.) suis and Mycobacterium avium ssp. paratuberculosis (MAP) differ substantially in their host specificity and tissue tropism. S. suis is a facultative pathogen in swine, which mainly colonises the upper respiratory tract and can cause meningitis, septicemia, arthritis and pneumonia. In contrast, MAP is an obligatory pathogen causing paratuberculosis in ruminants, and shows high tropism for the intestinal tract. Both pathogens are able to invade and persist in host cells. In S. suis, the significance of invasion for pathogenesis is a matter of controversial discussions. In vitro it has been shown that S. suis is internalized by epithelial cells and survives intracellularly for at least 24 h. However, at present there is no evidence that S. suis invades epithelial cells also in vivo. In MAP, on the other hand, persistence in macrophages is generally considered a crucial step in pathogenesis, but it remains to be elucidated, how it contributes to pathophysiology of the disease. The two pathogens exemplify how intracellular invasion and persistence might play different roles in pathogenesis. In S. suis, intracellular life may represent only a transient retreat phase, whereas in MAP it is the predominant in vivo niche of the pathogen.     

Comparative evaluation of positive tests to Mycobacterium avium subsp. paratuberculosis in clinically healthy sheep and goats in south-west Greece using molecular techniques, serology, and culture

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of paratuberculosis, which affects mainly ruminants although there is a growing concern about its possible implication in Crohn's disease in humans especially in connection with environmental spread and risks to the food chain. Retail cheese may represent a significant source of human exposure to MAP and the aim of this study was to assess MAP status in clinically healthy sheep and goats in Greece, comparing techniques routinely used in the positive diagnosis of the disease. From a total of 30 flocks, 632 sheep and goats had faecal, serum, and whole-blood samples examined by culture, complement fixation test (CFT), and polymerase chain reaction (PCR) targeted at IS900, IS1245, and IS6110. PCR produced positive results in 21% of the animals tested, with 5.6%, 3.9%, and 11.5% being identified as MAP, Mycobacterium avium subsp. avium, and Mycobacterium tuberculosis complex, respectively. CFT produced positive and suspicious results in 4.4% and 14.4% of the cases. Faecal cultures were negative in all but a single case that was identified as restriction fragment length polymorphism (RFLP)-type BC1. Agreement between results obtained by PCR and CFT was poor with isolated cases although an assessment of the MAP positive tests produced similar results for both methods. The findings indicate the need for additional measures of control, although the costs may be substantial if public health protection justifies elimination of MAP from livestock.     

Mitochondrial DNA variation and genetic relationships in Spanish donkey breeds (Equus asinus)

In this study, the mitochondrial DNA diversity of six Spanish donkey breeds and two African donkey populations (one from Morocco and the other from Zimbabwe) was analysed. A total of 79 animals were sequenced for 313 bp of the cytochrome b gene, and 91 individuals for 383 bp of the D‐loop region or control‐region. Sequence comparisons and phylogenetic analyses of both Spanish and African populations revealed low diversity. Only six and seven haplotypes respectively were found in cytochrome b and the D‐loop region. Relatively low nucleotide diversity (π) values were detected in these populations. The π values, from the D‐loop region, ranged from 0.0006 to 0.0169 for the Catalana and Andaluza breeds, respectively. The obtained results seem to confirm the existence of two divergent maternal lineages of African origin (Equus asinus africanus and E. a. somaliensis). In this paper the genetic relationships between these breeds are analysed and compared with those obtained in other European populations. Also, the data on the genetic relationships between the populations, obtained from nuclear DNA (microsatellites) and mitochondrial DNA (cytochrome b and D‐loop region) is argued and interpreted.     

Ultrastructure of Sarcocystis spp. from donkeys (Equus asinus) in Egypt

The fine structure of Sarcocystis spp. from donkeys (Equus asinus) in Egypt is described. Sarcocysts were found in the oesophagus, diaphragm and heart of 18 of 20 donkeys. Only one type of mature muscle cyst was found. Sarcocysts were 120-410.6 X 48.4-50.2 microns. The primary cyst wall had numerous 3.3-3.7 microns villi. Each villus contained 20-60 fibrillar elements which extended from the ends and sides of the villi throughout the ground substance, where they became tightly packed. The bundles of fibrillar elements formed junctions with the pellicles of the metrocytes. Ultrastructurally, Sarcocystis spp. of the donkey was similar to sarcocysts previously described from the horse.     

Detection of Mycobacterium avium subsp paratuberculosis in formalin-fixed paraffin-embedded intestinal tissue by IS900 polymerase chain reaction

OBJECTIVES: To evaluate and compare methods for DNA extraction from formalin-fixed, paraffin-embedded tissues and methods for detection of Mycobacterium avium subsp paratuberculosis by IS900 PCR for confirmation of Johne's disease in ruminants. DESIGN: A laboratory study. PROCEDURE: Three methods of DNA extraction of differing complexity and two PCR protocols using different pairs of IS900 primers were compared. Sensitivity and specificity were assessed using samples from ruminants with and without histological evidence of Johne's disease. RESULTS: The simplest method of DNA extraction, which involved two cycles of boiling and freezing followed by centrifugation, gave more consistent results than two methods that required solvent extraction of paraffin, proteinase digestion and DNA purification. The sensitivity of detection of M avium subsp paratuberculosis in paraffin blocks stored for 1 to 6 years from 34 cases of Johne's disease in sheep, cattle and goats was 88% for a 229 bp IS900 PCR assay and 71% for a 413 bp assay, using the detection of acid-fast bacilli by Ziehl Neelsen staining of histological sections from the same blocks as the gold standard test. PCR results correlated with the abundance of acid-fast organisms in the tissues. No false positive reactions were detected. CONCLUSION: PCR for identification of M avium subsp paratuberculosis in formalin-fixed, paraffin-embedded intestinal tissues from ruminants is a rapid and useful method. A simple method of sample preparation is effective. Amplification of short fragments of IS900 is more effective than amplification of longer fragments.     

The detection of Mycobacterium paratuberculosis in bovine faeces by isolation and the comparison of isolation with the examination of stained smears by light microscopy

The detection of Mycobacterium paratuberculosis organisms in bovine faeces by isolation was compared with that by the microscopical examination of Ziehl-Neelsen stained faecal smears for the presence of clumps of acid-fast M. paratuberculosis organisms. Faeces were obtained from cattle naturally or experimentally infected with M. paratuberculosis as well as from uninfected cattle. Microscopical examination was an unreliable method for the detection of M. paratuberculosis organisms, since the organisms were only detected in 99 (=55.9%) of 177 culturally positive faecal samples. 1111 addition, clumps of acid-fast organisms indistinguishable from M. paratuberculosis were also observed iin three of 18 samples from cattle free from Johne's disease and in 18 of 37 culturally negative samples from paratuberculous cattle. When M. paratuberculosis organisms were added to faeces from an uninfected cow, results showed that isolation attempts should be positive when 15 or more M. paratuberculosis organisms per gram of faeces are present.     

Divergent cytokine responses of macrophages to Mycobacterium avium subsp. paratuberculosis strains of Types II and III in a standardized in vitro model

Based on epidemiological and clinical observations, different strains of Mycobacterium avium subsp. paratuberculosis (MAP) are suspected to significantly differ in their virulence for ruminants. In the pathogenesis of paratuberculosis, macrophages represent the principal target cell for MAP. In order to judge the ability of different MAP-genotypes to modulate macrophage responses, the cytokine responses of the monocyte cell line THP-1 were studied after challenge with three different MAP strains under standardized conditions. The bovine field isolate J1961 (major Type II) and the ovine field isolate JIII-86 (Type III) were compared with the laboratory adapted reference strain ATCC 19698 (Type II). Strains were shown by three different typing methods (IS900-RFLP-, MIRU-VNTR-, and SSR-analysis) to substantially differ in several genotypic features. Macrophage function was assessed by quantifying mRNA of the cytokines TNF-α, IL-1?, and IL-10 by quantitative RT-PCR. Secreted TNF-α protein was measured by a cytotoxicity test, IL-1? and IL-10 using ELISA tests. The three MAP strains of various genotypes differ in their effect on human macrophages depending on challenge dose and infection time. These differences concerned both the mRNA level and secreted protein amounts of proinflammatory cytokines TNF-α, IL-1β and anti-inflammatory cytokine IL-10. Type III strain produced less IL-10 and IL-1β mRNA and protein but more TNF-α protein at 2h than the Type II strains. In summary, our results support the hypothesis that strain characteristics might have relevance for the host response towards MAP and, consequently, for the pathogenesis of paratuberculosis.     

Antigen-specific regulatory T cells in bovine paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP) is an intracellular pathogen that survives in the host's intestinal macrophages and causes chronic enteritis in ruminants. The subclinical stage of MAP infection is accompanied by loss of pro-inflammatory T(H)1 response, and a predominant, but ineffective, antibody-mediated T(H)2 response. How MAP interacts with the bovine immune system and suppresses T(H)1 responses is unclear. Studies carried out in our lab and others indicate that when peripheral blood mononuclear cells (PBMCs) from subclinical MAP-infected cattle are stimulated with MAP-antigen, IL-10 is up-regulated and leads to suppression of IFN-gamma expression in MAP-antigen-reactive effector T cells. IL-10 up-regulation and reduction in IFN-gamma would favor MAP survival and proliferation in macrophages. Depletion studies in PBMCs from MAP-infected cattle also revealed that the MAP responsive T-cell population that produces IL-10 is CD4(+) and CD25(+). Therefore, we hypothesize that cattle infected with MAP develop regulatory T (Treg) cells capable of producing IL-10 that in turn limits peripheral and tissue-specific T(H)1 immune responses. The aim of this review is to summarize current thinking regarding Treg cells and provide preliminary evidence that infection of cattle with MAP may lead to development of Treg cells.     

Regulatory T cells in cattle and their potential role in bovine paratuberculosis

The intracellular bacterium Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in wild and domestic ruminants. Johne's disease presents as a chronic enteritis with severe inflammation of intestinal tissues, characterized by widespread infiltration of macrophages, the target cell of MAP. Clinical signs of Johne's disease are typically accompanied by a loss of peripheral CD4+ T cell responses to MAP antigens and an increase in anti-MAP serum IgG levels. Recently, it was proposed that regulatory T cells might develop over the lengthy course of subclinical MAP infection. In the past five years, significant progress in defining bovine regulatory T cells has been made. These studies grew out of observations that IL-10 is produced by PBMCs in response to MAP antigen stimulation and that neutralization of this IL-10 could enhance IFN-γ production from MAP-antigen reactive effector T cells. Depletion studies revealed that MAP responsive cell populations producing IL-10 were largely CD4+ and CD25+, although monocytes have also been shown to produce IL-10 in response to MAP. In addition, evidence for a regulatory population of γδ T cells has also begun to accumulate. We summarize current thinking regarding regulatory T cells in MAP infection and provide data suggesting a potential link between regulatory T cells, bovine leukemia virus, and MAP.     

驴Zfx基因CDS序列克隆及序列分析

本研究旨在克隆驴Zfx基因CDS序列,并对其进行生物信息学分析。根据GenBank中公布的牛Zfx基因mRNA序列（登录号：D84097.1）设计特异性引物,利用RT-PCR技术获取驴Zfx基因CDS序列,并对驴Zfx基因CDS区核苷酸序列和蛋白质结构进行生物信息学分析。结果表明,驴Zfx基因CDS区序列长度为2 403 bp,编码800个氨基酸;驴与马、牛、家犬、白犀牛、马鹿、猫、人、绵羊、黑猩猩的Zfx基因CDS区核苷酸序列同源性分别为99.6%、95.0%、95.3%、97.9%、93.5%、94.9%、95.0%、95.7%和94.9%。对驴及其他9种哺乳动物Zfx基因CDS区的核苷酸序列构建系统进化树,结果显示,驴与马亲缘关系很近,与白犀牛的亲缘关系次之;同时又与聚在一类的家犬、猫亲缘关系较近,而与绵羊、牛的亲缘关系稍远。Zfx蛋白理论等电点（pI）为5.19,不稳定系数为42.94,消光系数为35 810,属于不稳定的亲水性蛋白,含有60个磷酸化位点,无信号肽及跨膜结构,属于非分泌蛋白。Zfx蛋白α-螺旋、延伸链和无规则卷曲分别为23.12%、20.25%和56.63%,三级结构主要为α-螺旋和无规则卷曲。本试验成功克隆获得驴Zfx基因CDS序列,为后期深入研究该基因及其编码蛋白的结构功能提供依据。     

A survey for Mycobacterium avium subspecies paratuberculosis in the Royal Zoological Society of Antwerp

Paratuberculosis is a chronic intestinal disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Map). Very little is known about the status of paratuberculosis in European zoos. In this study, the presence of Map in the animal collection of the Royal Zoological Society of Antwerp (RZSA) was investigated. Faecal and post mortem samples from 48 ruminants were used to set up cultures. DNA from faeces, tissue and positive cultures were tested by IS900 polymerase chain reaction (PCR). Additionally, 448 serum samples were tested with an ELISA kit. All culture samples were negative whereas PCR gave three positives on biopsy samples and one positive on faecal samples. With the ELISA, 21 sera could be classified as positive. There is evidence that Map is present in the RZSA but no high level faecal shedders could be detected. Further investigations are required in other European Zoos in order to complete the picture of Map infections.     

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

Tropical Animal Health and Production - Paratuberculosis is an incurable disease in ruminants with great worldwide economic impact, caused by Mycobacterium avium subsp. paratuberculosis (MAP). The...     

Evaluation of clinical pathology parameters in fecal PCR-positive or PCR-negative goats for Johne’s disease

Tropical Animal Health and Production - Johne’s disease (JD) is an economically important infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). This...     

A testing scheme for the detection of Mycobacterium avium subsp. paratuberculosis in bovine feces utilizing the ESP para-JEM liquid culture system.

A testing scheme for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in broth cultures of bovine fecal samples carried out in ESP para-JEM System was evaluated. The scheme included acid-fast staining (on signal-positive and signal-negative samples), and confirmation by PCR for 2 MAP-specific targets and subculture of all acid-fast positive PCR-negative samples. Two hundred and fifty bovine fecal samples were evaluated for the presence of MAP using this scheme. Thirty-seven (15%) of 250 fecal samples had a positive culture result when the proposed testing scheme was used, compared to 14 (6%) positive results when using the standard ESP para-JEM protocol (requiring samples to have a positive signal from the system, a positive acid-fast stain, and a positive IS900 PCR result), and 20 (8%) positives when conventional culture was performed on Herrold egg yolk (HEY) media. A preliminary comparison of real-time and conventional PCR on DNA extracted from 15 MAP-positive broth cultures by 3 different protocols suggested that conventional PCR may be a better choice for the confirmation of the presence of MAP in the liquid cultures than real-time PCR.     

Recent research on bovine paratuberculosis in South Korea

Bovine paratuberculosis (Johne's disease), a chronic and debilitating disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a major cause of chronic ruminant enteritis. A national eradication program has been in place in South Korea since the first report of the disease in 1967; however, only limited data on bovine paratuberculosis in South Korea are available. Some research, such as investigations of the reactivity of animal sera against MAP antigens, has been done in localized areas and in limited animal species. Compared with the worldwide situation, the development of diagnostic methods in South Korea has shown similar results even though some data were obtained from international collaborative studies. MAP is considered by some to be zoonotic, noting an association with Crohn's disease, although this issue is still controversial; however, research into this association is limited. Decisions based on disease priorities have hampered active progress in research on the disease. In this paper, we reviewed the available results generated from South Korea compared with global research. Finally, we propose a theme for future research.