Increased viability but decreased culturability of Mycobacterium avium subsp. paratuberculosis in macrophages from inflammatory bowel disease patients under Infliximab treatment.

Mycobacterium avium subsp. paratuberculosis (MAP) has long been implicated as a triggering agent in Crohn's disease (CD). In this study, we investigated the growth/persistence of both M. avium subsp. hominissuis (MAH) and MAP, in macrophages from healthy controls (HC), CD and ulcerative colitis patients. For viability assessment, both CFU counts and a pre16SrRNA RNA/DNA ratio assay (for MAP) were used. Phagolysosome fusion was evaluated by immunofluorescence, through analysis of LAMP-1 colocalization with MAP. IBD macrophages were more permissive to MAP survival than HC macrophages (a finding not evident with MAH), but did not support MAP active growth. The lower MAP CFU counts in macrophage cultures associated with Infliximab treatment were not due to increased killing, but possibly to elevation in the proportion of intracellular dormant non-culturable MAP forms, as MAP showed higher viability in those macrophages. Increased MAP viability was not related to lack of phagolysosome maturation. The predominant induction of MAP dormant forms by Infliximab treatment may explain the lack of MAP reactivation during anti-TNF therapy of CD but does not exclude the possibility of MAP recrudescence after termination of therapy.

In vivo and in vitro expression pattern of Toll-like receptors in Mycobacterium avium subspecies paratuberculosis infection.

Johne's disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a chronic infectious disease of ruminants. Activation of the Toll-like receptors (TLR) in response to microbial stimuli, including MAP, initiates responses in immune cells of the blood and within peripheral tissues. TLR2, 4 and 9 are believed to play a critical role in the initiation of immune responses against mycobacteria. In this study we report on the in vivo expression pattern of these receptors in sheep and cattle experimentally exposed to MAP. Experiments using the mouse macrophage cell line, RAW 264.7, and on isolated bovine monocytes were also carried out to assess the expression pattern of TLR2 and 4 in response to MAP and the non-pathogenic mycobacterial strain, M. smegmatis. Results from the in vivo study showed that there was a significant upregulation of TLR2 (P<0.05) at early time-points post-inoculation in the peripheral blood cells of sheep exposed to MAP S strain that went on to develop severe (multibacillary) disease. However, in the cattle during the initial months post-exposure to MAP C strain, TLR2 was significantly downregulated (P<0.05). TLR4 was significantly upregulated (P<0.05) at later stages (12 months post-inoculation) in MAP-exposed sheep with multibacillary disease; however significant differences in TLR4 expression were not observed in cattle. Expression of TLR9 was unchanged in MAP-exposed sheep and cattle. In vitro studies on mouse macrophages supported the findings of in vivo TLR2 gene expression increases seen in the sheep, in that the TLR2 receptor expression in response to MAP-infection was significantly increased in comparison to cells infected with a non-virulent mycobacterium, M. smegmatis. A likely role for TLR2 in the pathogenesis of Johne's disease is proposed.

Mycobacterium avium subspecies paratuberculosis: an insidious problem for the ruminant industry.

Mycobacterium avium subspecies paratuberculosis is considered as one of the most serious problems affecting the world's ruminant industry due to its significant impact on the global economy and the controversial issue that it may be pathogenic for humans. M. avium subspecies paratuberculosis is the causative agent of Johne's disease in animals and might be implicated in cases of human Crohn's disease. We provide an insight into M. avium subspecies paratuberculosis from some bacteriological, clinical, and molecular epidemiological perspectives.

Paratuberculosis in a dairy Gyr herd in the State of Paraíba, Brazil / Paratuberculose em um rebanho Gir leiteiro no Estado da Paraíba Brasil

This paper describes the clinical, pathological, and microbiologic aspects of paratuberculosis (Johne's disease) in a dairy Gyr herd in the State of Para�ba, northeastern Brazil. An eight years old cow with chronic unresponsive diarrhea was clinically examined and euthanized for pathological evaluation. Fecal samples from all 160 animals over 12 months of age from the herd were collected for isolation of Mycobacterium avium subsp. paratuberculosis. Clinically, the index case cow was severely dehydrated, cachectic, with profuse mucous diarrhea. The main post-mortem findings were emaciation and thickened intestinal wall. Microscopically, the intestinal lamina propria and submucosa were infiltrated by macrophages, epithelioid cells, and Langhans giant cells with numerous alcohol-acid resistant bacilli in the cytoplasm. Two fecal samples displayed growth in slants of Herrold's egg-yolk agar supplemented with mycobactin J, 150 days after incubation. No growth was noticed in slants without mycobactin J. Microscopic examination of the isolated microorganisms stained by Ziehl-Neelsen revealed considerable amounts of alcohol-acid resistant bacilli, morphologically compatible with Mycobacterium spp. Based on the clinical signs, gross and histological lesions, growth time, bacterial morphology in Ziehl-Neelsen staining, and dependence of mycobactin J, the first diagnosis of paratuberculosis in Zebu cattle was made.

Objetivou-se descrever os aspectos clínicos, anátomo-histopatológicos e microbiológicos da paratuberculose em um rebanho Gir leiteiro no Estado da Paraíba. Uma vaca de oito anos que apresentava diarréia persistente, refratária a tratamento foi necropsiada para estudo anátamo-histopatológico. Também foram coletadas amostras de fezes de todos os 160 animais do plantel, com idade superior a 12 meses, para tentativa de isolamento de Mycobacterium avium subsp. paratuberculosis. Ao exame clínico, o animal caso índice apresentou caquexia, diarréia profusa e desidratação grave. À necropsia, o animal apresentou-se emaciado e, ao exame detalhado do trato digestivo, foi observado espessamento da parede e superfície mucosa do íleo e intestino grosso. À microscopia, verificou-se intensa infiltração de macrófagos espumosos associado a raras células epiteliódes e gigantes do tipo Langerhans na lâmina própria e submucosa. À coloração de Ziehl-Neelsen foram observadas miríade de bacilos álcool-ácido resistentes no citoplasma destas células. Houve crescimento de colônias bacterianas em duas das 160 amostras de fezes após 150 dias de incubação em tubos com meio Herrold's egg-yolk suplementados com micobactina J e ausência de crescimento nos tubos com mesmo meio, mas sem suplementação. Os microrganismos isolados foram corados pelo Ziehl-Neelsen observando-se presença de grande quantidade de bacilos álcool-ácido resistente, com morfologia compatível ao gênero Mycobacterium. Baseado na história clínica, achados anátomo-histopatológicos e histoquímicos (Ziehl-Neelsen), e microbiológicos, firmou-se o primeiro diagnóstico de paratuberculose em Zebu na Paraíba.

Is alpha-dystroglycan the missing link in the mechanism of enterocyte uptake and translocation of Mycobacterium avium paratuberculosis?

Mycobacterium avium paratuberculosis (MAP) is an important animal pathogen with a potential role in human disease. MAP is recognized to cause severe diarrheas and wasting syndrome in patients infected by the human immuno-deficiency virus. Recently, there is also growing evidence that MAP is somehow involved into the patho-mechanisms of Crohn's disease. However, the mechanism how MAP binds to the intestinal mucosa and consecutively invades and translocates the intestinal epithelial cells is still unknown. Here it is suggested, that MAP enters the intestinal cells via the dystrophin-glycoprotein-complex (DGC) in a similar manner as known from Mycobacterium tuberculosis in peripheral Schwann cell invasion. Recent approaches to identify the mechanism of intestinal MAP uptake revealed several molecules which are therefore thought to be involved in MAP cell invasion. Nevertheless, there is no comprehensive connection so far to link all identified mechanisms together. Since the DGC has a direct association to all identified molecules and mechanisms and therefore seems to be the missing link, it is hypothesized now, that MAP binds to alpha-dystroglycan and exploits an endogenous recycling mechanism to control the dystroglycan expression levels to enter and translocate the intestinal cells. Since there are options to modify dystroglycan this might be a potential new target to prevent or even treat intestinal MAP infections.

Paratuberculosis and Type I diabetes: is this the trigger?

Type 1 diabetes mellitus (T1DM) is an autoimmune disease. The etiology of T1DM is incompletely understood but environmental agent(s) are thought to trigger T1DM in the genetically at risk. Exposure to cow's milk early in life is a recognized risk factor in the development of T1DM. Mycobacterium avium ss. paratuberculosis (MAP) is the cause of bovine Johne's disease and also is thought to act as an immune antigen in Crohn's disease and other granulomatous diseases. MAP is shed in cow's milk and has been shown to survive pasteurization. Genetic susceptibilities, epitope homologies and epidemiologic studies are presented that support MAP as a causative agent of T1DM in the genetically at risk.

[Pathogenesis and immune reactions of paratuberculosis]. / Pathomechanismen und Immunreaktionen bei der Paratuberkulose.

Mycobacterium avium subspecies paratuberculosis (M. ptb) is known as the cause of paratuberculosis for over a century but the knowledge on biology of the organism and pathogenesis of the disease is still limited. There are several reasons for the present lack of progress, these are (i) the extremely slow growth of the bacterium, a feature which has also protected the organism against researchers, (ii) confusion over its taxonomy and identification, (iii) limited possibilities for the application of molecular biology techniques, and (iv) the extremely long incubation period in natural infection for which no suitable laboratory model exists. Despite these discouraging facts, recent research efforts have led to important findings, which have shown that a better understanding of the disease may contribute to the improvement of control strategies. This presentation focuses mainly on the unique nature of M. ptb within the mycobacteria and the central role of the macrophage in pathogenesis and immune response. More details can be found in a number of excellent recent reviews (see list of references).

Construction and screening of Mycobacterium paratuberculosis insertional mutant libraries.

Mycobacterium paratuberculosis causes Johne's disease, a common wasting disease in ruminants. As a first step in studying virulence mechanisms, libraries of random mutants were produced in two M. paratuberculosis strains by using the conditionally replicating shuttle phasmid phAE94 which contains the transposon Tn5367. Two thousand mutants were screened for auxotrophy, carbon source preference, and altered cell wall. Genes interrupted by insertion were identified for seven mutants isolated from the screening process. Two mutants had insertions in putative genes involved in synthesis of the cell wall.

Lack of support for a common etiology in Johne's disease of animals and Crohn's disease in humans.

The superficial similarity of Johne's disease to Crohn's disease led to the hypothesis that, like the former. Crohn's disease was caused by Mycobacterium paratuberculosis. Detailed pathologic comparisons, however, reveal little similarity between these two entities, including the lack of important extraintestinal manifestations. Attempts to recover M. paratuberculosis by culture have only rarely succeeded and the significance of spheroplasts that appear more frequently on culture is seriously in question. Five immunocytochemistry studies have failed to find mycobacterial antigens in diseased tissues and the five most recent polymerase chain reaction (PCR) attempts to find genomic evidence of M. paratuberculosis were uniformly negative. Numerous serologic studies failed to demonstrate antibody to M. paratuberculosis and attempts to show cell-mediated immunity were also unrewarding. Inoculation of numerous experimental animals with Crohn's disease tissue has failed to induce Johne's disease, and inoculation of various animal species with M. paratuberculosis has equally failed to result in Crohn's disease. Controlled studies of the treatment of Crohn's disease with antimycobacterial agents have generally resulted in no improvement, and most studies that have shown a positive response are either uncontrolled or include broad-spectrum antibiotics that may be acting on pathogens other than mycobacteria. Finally, although Johne's disease is common in farm animals, and infected animals shed M. paratuberculosis in large numbers, no record of zoonotic transmission has been recorded.

Paratuberculosis in saiga antelope (Saiga tatarica) and experimental transmission to domestic sheep.

Mycobacterium paratuberculosis was isolated in low numbers from the small intestine and associated mesenteric lymph nodes of a saiga antelope (Saiga tatarica) using routine culture techniques in spite of histologic evidence of high numbers of acid-fast bacteria in these tissues. Two newborn domestic sheep were fed the ground intestinal tissue containing acid-fast bacteria and the progression of the experimental disease was followed by fecal culture, immunodiffusion (AGID) and lymphocyte stimulation (LST) tests. One experimentally infected sheep developed progressive clinical illness 1 yr postinoculation. Few M. paratuberculosis were isolated from feces or tissues although an extensive granulomatous mycobacterial enteritis, lymphadenitis and lymphangitis were observed containing large numbers of typical acid-fast organisms. No clinical illness was observed in the second inoculated sheep after 18 mo of observation, although infection was demonstrated at necropsy. Both sheep developed AGID and LST reactions indicative of paratuberculosis. This study demonstrated that a difficult to culture isolate of M. paratuberculosis was responsible for paratuberculosis in captive wild ruminants and was transmissible to domestic sheep. Diagnosis of paratuberculosis in four of eight of the imported saiga antelope and in eleven of their 18 offspring indicates the importance of this disease in management of captive wild ruminants and the ease with which this organism can be transmitted.

Accidental self-inoculation with Mycobacterium paratuberculosis bacterin (Johne's bacterin) by veterinarians in Wisconsin.

We surveyed Wisconsin veterinarians to assess the frequency and severity of accidental self- and other human exposure to Mycobacterium paratuberculosis bacterin (Johne's bacterin). Of 199 veterinarians administering the bacterin to cattle, 22 reported one or more exposures, including 19 needle-stick exposures, 8 skin surface exposures, and 2 oral mucosa exposures. The mean incidence of needle sticks was 5.5/100 veterinarians/year of bacterin use or 1/1,000 doses administered. The mean total doses given in the needle-stick exposure group was 276 +/- 318 vs 80 +/- 268 in the group without needle-stick exposure, and the mean number of months administering the bacterin was 21.7 and 16.1, respectively; 63% of needle-stick exposures took place during the injection process. Five adverse reactions were reported, and each resulted from needle-stick exposure. The only systemic reaction followed an exposure to the original bacterin formulation of sonically ruptured M paratuberculosis in Freund incomplete adjuvant. The remaining reactions were to the current formulation of whole killed M paratuberculosis in mineral oil and ranged from a small nodule persisting for 4 to 6 months to painful inflammation of a finger persisting for 24 months. We anticipate an increase in incidence of these minimally debilitating injuries as the use and distribution of the bacterin expands. For hand wounds, we recommend conservative management. Surgical intervention should be considered if a granuloma persists and causes the patient functional difficulty.

Intracellular iron storage and the pathogenesis of paratuberculosis. Comparative studies with other mycobacterial, parasitic or infectious conditions of veterinary importance.

The distribution of iron and mycobacteria was examined in the intestinal tract of ruminants with naturally-occurring M. paratuberculosis infection and compared with mycobacterial infections in several species. This distribution was compared with that of iron in chronic lesions caused by other microbial or parasitic agents. In the clinical form of paratuberculosis in cattle, sheep and goats there was marked lymphangiectasis and a high proportion of the granulomatous lesions contained siderotic macrophages with a high mycobacterial content. In cattle with preclinical lesions of granulomatous enteropathy, the greatest number of acid-fast organisms was present in siderotic, non-differentiated, ileo-caecal macrophages; concurrent mast cell-associated allergic enteropathy was also apparent in the duodenum, proximal and mid-ileum of most animals. In paratuberculosis-affected herds, a high proportion of non-productive cows were without classical granulomatous change but had cultural or immunological evidence of M. paratuberculosis infection and similar allergic catarrhal enteropathy of the upper intestinal tract. Interstitial haemorrhage of the ileocaecal valve, with the accumulation of haemosiderin and ferritin in undifferentiated macrophages was observed in some of these cattle and also in others with experimentally-induced copper deficiency and acute ostertagiasis. Colonisation of the ileo-caecal or caecal glandular crypts by large, apparently saprophytic acid-fast organisms indicated regional tolerance to such organisms in all cattle. In other mycobacterioses such as bovine or avian tuberculosis, undifferentiated, siderotic macrophages containing mycobacteria were also seen in early granulomas, but epithelioid and giant cell differentiation invariably led to the disappearance of intracellular iron and a reduction in mycobacterial numbers. In possums in which epithelioid and giant cells did not occur in response to M. bovis infection, siderosis persisted in many macrophages and overwhelming mycobacterial multiplication occurred. These studies indicate that, in most infections with mycobacteria, differentiation of macrophages radically reverses their iron acquisitive properties, creating an intracellular environment unsuitable for mycobacterial multiplication. It seems likely that allergically mediated microvascular haemorrhage, local tolerance of commensal mycobacteria and attenuation of the macrophage siderosis reversal mechanism provide unique conditions for early, uninhibited, intracellular multiplication of M. paratuberculosis in the ileo-caecal valve of certain mature ruminants.