Paratuberculosis in red deer (Cervus elaphus): an immunohistochemical study

In the present study, we compared the utility of immunohistochemistry with serological and histological results for the characterization of Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) in tissues of affected red deer. Bacterial isolation was considered the standard reference. Samples were taken from seven clinically affected animals with typical macroscopic lesions. The enzyme linked immunosorbent assay (ELISA) and the gel diffusion tests (GD) were used for serological determinations. Samples from intestine and mesenteric lymph nodes were processed for bacterial isolation and histology. M. paratuberculosis was isolated from all the animals. Histologically, lymph nodes displayed necrosis and mineralization at the cortical and medullar areas. Ziehl-Neelsen stained bacteria were numerous inside macrophages and Langhans-type giant cells. Giant and epithelioid cells and lymphocytes were prominent at the ileal mucous membrane. The immunostaining of M. paratuberculosis was very clear inside epithelioid and giant cells. Image analysis was carried out to determine the immunostained area. There was total agreement among the methods employed. Immunohistochemistry can be very useful when the microorganism cannot be recovered from tissues or faeces.     

Molecular epidemiology of Types I/III strains of Mycobacterium avium subspecies paratuberculosis isolated from goats and cattle

Molecular characterization of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolates classifies them into three groups: cattle or Type II, sheep or Type I, and intermediate or Type III. To avoid problems associated with characterization of extremely slow growth strains, PCR-based techniques that divide the M. a. paratuberculosis strains in two main groups (cattle or Type II, and sheep or Types I/III) can be performed. The objectives of this study were to characterize the M. a. paratuberculosis isolates identified by different PCR-based tests (IS1311-PCR and restriction endonuclease analysis, PCR test based on a DNA sequence difference, and a PCR aimed at three Type I-specific loci), and to determine the clinical and epidemiological implications of Types I/III M. a. paratuberculosis strains in livestock. One hundred and fifty-eight M. a. paratuberculosis strains from domestic ruminants were analyzed. One hundred and six M. a. paratuberculosis isolates (61 from goats and 45 from cattle) were classified as Type II strains; and 52 (29 from cows, 20 from goats, and three from sheep) were included in the Types I/III. The Types I/III M. a. paratuberculosis strains were associated to Spanish native breeds. The majority of these animals had not been in direct or indirect contact with sheep flocks infected with M. a. paratuberculosis. This fact should be taken into account when implementing paratuberculosis control programs.     

Mycobacterium paratuberculosis and the bovine immune system

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease, a deadly intestinal ailment of ruminants. Johne's disease is of tremendous economic importance to the worldwide dairy industry, causing major losses due to reduced production and early culling of animals. A highly controversial but developing link between exposure to M. paratuberculosis and human Crohn's disease in some individuals has led to the suggestion that M. paratuberculosis is also a potential food safety concern. As with many other mycobacteria, M. paratuberculosis is exquisitely adapted to survival in the host, despite aggressive immune reactions to these organisms. One hallmark of mycobacteria, including M. paratuberculosis, is their propensity to infect macrophages. Inside the macrophage, M. paratuberculosis interferes with the maturation of the phagosome by an unknown mechanism, thereby evading the host's normal first line of defense against bacterial pathogens. The host immune system begins a series of attacks against M. paratuberculosis-infected macrophages, including the rapid deployment of activated gammadelta T cells, CD4+ T cells and cytolytic CD8+ T cells. These cells interact with the persistently infected macrophage and with each other through a complex network of cytokines and receptors. Despite these aggressive efforts to clear the infection, M. paratuberculosis persists and the constant struggle of the immune system leads to pronounced damage to the intestinal epithelial cells. Enhancing our ability to control this important and tenacious pathogen will require a deeper understanding of how M. paratuberculosis interferes with macrophage action, the cell types involved in the immune response, the cytokines these cells use to communicate, and the host genetic factors that control the response to infection.     

Characterization of Mycobacterium paratuberculosis antigenic proteins

The characterization of a purified antigen from Mycobacterium paratuberculosis, recently made commercially available for use in serodiagnosis by enzyme-linked immunosorbent assay (ELISA), of paratuberculosis in cattle was described. This assay had 89% specificity and 83% sensitivity for M paratuberculosis infection. The protein/polypeptide composition of the purified antigen was compared with that of a crude protoplasmic extract of strain 18 M paratuberculosis used in the agar-gel immunodiffusion test and ELISA and with that of sonicated strain 19698 M paratuberculosis organisms grown on Dorset-Henley synthetic liquid medium. The sonicated M paratuberculosis contained 27 major proteins/polypeptides; the crude protoplasmic extract, 18; and the purified antigen contained 14 proteins/polypeptides, using sodium dodecyl-sulfate polyacrylamide-gel electrophoresis analysis. The serologic reactivity of these proteins/polypeptides were defined, using the enzyme-linked immuno-electrotransfer blot technique. The sonicated M paratuberculosis contained 20 serologically reactive proteins/polypeptides (34,000 to 84,000 daltons); the crude protoplasmic extract contained 3 (37,000 to 45,000 daltons); and the purified extract contained a diffuse polypeptide band (34,000 to 38,000 daltons). Identification by enzyme-linked immuno-electrotransfer blot technique of M paratuberculosis antigens reactive in the ELISA will allow us to further study these antigens in the ELISA to improve sensitivity and specificity of the diagnostic test.     

Binding of bovine growth hormone to. Mycobacterium avium ss paratuberculosis

Mycobacterium avium ss paratuberculosis causes a chronic progressive enteritis in cattle and other ruminants referred to as Johne's disease. It also has been suggested by some as possibly being associated with Crohn's disease in humans. In a previous study we observed that incubation of bovine monocytes with recombinant bovine growth hormone (bGH) altered the ingestion and intracellular growth of M. avium ss paratuberculosis in vitro. This led us to investigate whether bGH also has a direct effect on M. avium ss paratuberculosis. We observed that addition of bGH (5 microg/ml) had a direct inhibitory effect on the growth of M. avium ss paratuberculosis in Middlebrook 7H9 broth. In contrast, the growth of Mycobacterium smegmatis was unaffected, even at a bGH concentration of 50 microg/ml. Using 125I-bGH we observed high affinity binding (Kd = 1.32 nM) of bGH to M. avium ss paratuberculosis, with an estimated 204 binding sites per bacillus. To the best of our knowledge, this is the first report of a mammalian hormone binding to this important enteric pathogen.     

Temporal patterns and quantification of excretion of Mycobacterium avium subsp paratuberculosis in sheep with Johne's disease

OBJECTIVES: To determine the frequency of excretion of Mycobacterium avium subsp paratuberculosis in Merino sheep with Johne's disease and to quantify excretion in a group of Merino sheep. DESIGN: A pen and laboratory experiment. PROCEDURE: Seven sheep selected from an affected flock on the basis of acid-fast bacilli in the sheep's faeces were housed and total daily faecal output was collected, weighed and subjected to culture for M avium subsp paratuberculosis. An end-point titration method was used to enumerate viable M avium subsp paratuberculosis in a 15 day pooled sample from five sheep that had acid-fast bacilli in their faeces while housed. RESULTS: Four sheep with subclinical multibacillary Johne's disease excreted M avium subsp paratuberculosis each day for 11 days of cultural observation. A further three sheep were intermittent excreters but lacked other evidence of infection with M avium subsp paratuberculosis. The average number of viable bacteria excreted was 1.09 x 10(8) per gram of faeces while total daily excretion was 8.36 x 10(10) viable M avium subsp paratuberculosis per sheep. Examination of faecal smears stained with Ziehl Neelsen was an unreliable means of assessing daily excretion in individual animals except in those with severe lesions. CONCLUSION: Excretion of M avium subsp paratuberculosis in Merino sheep with multibacillary Johne's disease occurred daily, proving that environmental contamination can be continuous on farms with endemic ovine Johne's disease. Faecal culture is a useful method for detecting infection as it does not appear to be affected by the timing of collection of a sample from sheep with multibacillary disease however, to maximise the sensitivity of disease surveillance using faecal culture, sampling rates should be adjusted to take account of the proportions of multibacillary and paucibacillary cases.     

Development of ELISA to detect antibodies specific to Mycobacterium avium subsp. paratuberculosis with truncated 34 kDa proteins

To develop ELISA to detect antibodies specific to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the carboxyl termini of the 34 kDa proteins of M. paratuberculosis and Mycobacterium avium subsp. avium (M. avium) were expressed in Escherichia coli expression system. Antibodies specific to M. paratuberculosis were detected with the truncated 34 kDa protein of M. paratuberculosis in ELISA after pre-absorption of serum samples with the truncated 34 kDa protein of M. avium. All the serum samples from cattle confirmed to be infected with M. paratuberculosis were positive and those from healthy cattle were negative in the present ELISA system. These results indicate that the established ELISA detects antibodies specific to M. paratuberculosis with high specificity and sensitivity and is an useful tool for the screening of Johne's disease.     

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.     

Temporal Mycobacterium paratuberculosis infection in T-cell receptor (TCR)-alpha and TCR-delta-deficient mice

Mycobacterium paratuberculosis is the causative agent of paratuberculosis (Johne's disease), a chronic inflammation of the terminal portion of the ileum in ruminants. The predominance of cell-mediated immunity in early stages of the disease suggests that T lymphocytes are essential to protect the host from infection with M. paratuberculosis. In this study, we investigated the role of alphabeta and gammadelta T cells in resistance to M. paratuberculosis infection using a T-cell receptor (TCR) knockout mouse model. Weanling TCR-alpha-deficient, TCR-delta-deficient, and C57BL/6 control mice (5-6 weeks of age) were acclimated for 2 weeks and then inoculated intraperitoneally with 10(8)CFU/ml of M. paratuberculosis (either strain 19698 or strain Ben). Groups of mice within each treatment group were euthanized at 1, 3 and 6 months post-inoculation. Sections of spleen, liver, ileum and mesenteric lymph node were prepared for bacterial culture and histologic examination. At all time points of infection and regardless of bacterial strain, TCR-alpha-deficient mice had higher levels of M. paratuberculosis colonization in their tissues compared to TCR-delta-deficient mice or C57BL/6 control mice. Lesions were located predominately in the liver and the ileum, depending upon period of infection, and lesion scores were higher for TCR-alpha-deficient mice compared to the other treatment groups. These results suggest that alphabeta T cells play a major role in resistance to infection with M. paratuberculosis and that gammadelta T cells may play a lesser role and potentially confound protective immune responses.     

Detection methods of Mycobacterium paratuberculosis

Mycobacterium paratuberculosis is receiving increasingly wider interest of scientific groups worldwide. This slow-growing mycobacterium does not only evoke paratuberculosis--an infectious cattle disease that brings huge economic losses--but it is also regarded as a potential cause of human Crohn;s disease. It is very difficult to diagnose precisely this kind of animal infection in its very early stages, as well as to detect occurrence of M. paratuberculosis cells in the environment, including food of animal origin. This paper reviews currently known and employed diagnostic techniques for M. paratuberculosis cell detection and identification.     

Heat-shock protein-specific T-cell responses in various stages of bovine paratuberculosis.

Bovine paratuberculosis is characterized by a chronic inflammation of the small intestine, caused by infection with Mycobacterium avium ssp. paratuberculosis. Research regarding diagnostic as well as immunopathogenic aspects of paratuberculosis are hampered by the lack of specific antigens. The aim of the present study was to evaluate the potential of mycobacterial heat-shock proteins, as specific antigens, to measure cell-mediated immune responses during various stages of the disease. In a cross-sectional study, peripheral blood mononuclear cells of 179 cows in different stages of M. avium ssp. paratuberculosis infection, vaccinated against paratuberculosis or noninfected, were used to evaluate lymphoproliferative responses to mycobacterial heat-shock protein of 70 kD (HSP70) and 65 kD (HSP65). In addition, lymphoproliferative responses were measured using purified protein derivate (PPD) preparations from M. avium ssp. paratuberculosis, M. avium and M. bovis as antigens. Responses to HSP70 were higher in the vaccinated animals and in asymptomatic animals that shed the organism in their faeces. Compared with these animals, responses were lower in cows with clinical signs of paratuberculosis. Mycobacterial HSP65 induced less prominent responses compared with HSP70, but showed a similar pattern with regard to the stages of disease. Vaccinated and shedding animals also showed the highest responses to PPD derived from M. avium ssp. paratuberculosis (PPD-P). Observations with short-term cell lines raised to PPD-P and to HSP70 indicated that the similarity between those two antigens was not due to the presence of HSP70 in PPD-P. In conclusion, our study indicated that, as for PPD antigens the mycobacterial heat-shock protein-specific cell-mediated immune responses decrease when comparing the asymptomatic stage to the clinical stage in bovine paratuberculosis. Furthermore, this study shows that HSP70, being a well-defined antigen in comparison with PPD antigens, can be used to monitor cell-mediated immune responses in studies regarding the immunopathogenesis of bovine paratuberculosis.     

Mycobacterium paratuberculosis and milk

The possibility that milk from cattle with Johne's disease could be a potential vehicle of transmission of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) to humans has been the focus of a UK government-funded research programme at Queen's University, Belfast since 1993. The main findings of this research programme are reported and practical advice about the most appropriate methods for the isolation/detection of this organism in milk is given. The findings of several milk surveys during which optimised sensitive detection methods were employed (decontamination with 0.75% cetyl pyridinium chloride for 5 h prior to culture and a novel immunomagnetic PCR technique) have revealed that detectable levels of M. paratuberculosis are present in bulked raw cows' milk in the UK at both the farm level and at dairy processing plants prior to pasteurisation. Furthermore, results of three different experimental approaches to assess the effect of pasteurisation time/temperature conditions on the viability of M. paratuberculosis (laboratory pasteurisation studies, a national survey of commercially pasteurised milk, and processing of naturally infected milk through commercial-scale pasteurising plant) provide firm evidence that this organism is capable of surviving commercial milk pasteurisation on occasion. Hence, both raw and pasteurised cows' milk are potential vehicles of transmission of M. paratuberculosis to humans.     

Use of a polymerase chain reaction to subtype Mycobacterium avium subspecies paratuberculosis, an increasingly important pathogen from farmed deer in New Zealand

AIMS: To review the number of microbiologically-confirmed cases of Johne's disease in farmed deer since 2000, and determine the prevalence of the bovine and ovine subtypes of Mycobacterium avium subsp paratuberculosis (M. paratuberculosis), using a highly specific polymerase chain reaction (PCR) test on samples from infected herds. METHODS: The number of cases of M. paratuberculosis in farmed deer identified by culture or IS900 PCR was documented. A highly specific PCR test was applied to subtype M. paratuberculosis from BACTEC 12B cultures selected on the basis of one culture per deer herd, to give a wide coverage of herds in New Zealand. RESULTS: From January 2001 to October 2005, M. paratuberculosis was isolated from 1,141 farmed deer, and has now been identified by microbiological testing in over 600 deer herds in New Zealand. The bovine subtype of M. paratuberculosis was shown by a highly specific PCR test to be present in 91/95 herds examined; the ovine subtype was found in the remaining four herds. CONCLUSIONS: Since 2000, there has been a substantial increase in both the number of microbiologically-confirmed cases of Johne's disease in farmed deer and the number of infected herds. Johne's disease is now widespread and common in deer herds throughout New Zealand. Whilst the bovine subtype of M. paratuberculosis predominates in deer herds in New Zealand in which Johne's disease has been confirmed, the occasional finding of the ovine subtype highlights the need to consider both sheep and cattle as potential sources of infection for farmed deer.     

Effect of egg yolk on the detection of Mycobacterium avium subsp. paratuberculosis using the ESP II liquid culture system.

Rapid diagnosis of paratuberculosis in infected cattle is important for the successful control of Johne disease within herds. Thus, improving culture methods for Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) will aid in the identification of asymptomatic animals. Egg yolk is a component of the media used for growing M. paratuberculosis, but its requirement as a supplement has not been reported. Using the ESP II liquid culture system, 2 different sources and 5 concentrations (3.3%, 1.6%, 0.8%, 0.4%, and 0%) of egg yolk were analyzed. Egg yolk source did not affect either recovery rate or time to detection, but both parameters were significantly improved when the 3.3% egg yolk concentrations (final volume) were used over media containing no egg yolk. This study also assessed the recovery of M. paratuberculosis from fecal samples that were cultured multiple times using Herrold egg yolk agar (HEY). Specimens containing greater than 70 cfu/g feces could routinely be identified as positive for M. paratuberculosis after only 1 culture attempt, whereas specimens with fewer bacteria were only intermittently positive, even after 5 replicate cultures. Therefore, this study indicates that the sensitivity of the Trek Diagnostic ESP II liquid culture system for M. paratuberculosis is affected by egg yolk concentration and that single culture attempts using HEY solid media may not identify specimens containing low numbers of bacteria.     

In situ hybridization method for studies of cell wall deficient M. paratuberculosis in tissue samples

Cell wall deficient forms of mycobacteria may be important in the pathogenesis of Crohn's disease and sarcoidosis. However, no method has been available to localize this type of organisms in tissue sections. We developed an in situ hybridization method for the demonstration of Mycobacterium paratuberculosis spheroplasts (cell wall deficient forms) in paraffin embedded tissue sections.M. paratuberculosis spheroplasts were prepared by treatment with glycine and lysozyme. Pieces of beef were injected with the prepared spheroplasts. The samples were fixed in buffered formalin and paraffin embedded. A M. paratuberculosis-specific probe derived from the IS900 gene was used. Specificity was controlled by using an irrelevant probe and by hybridizing sections with spheroplasts from other bacteria.Beef samples injected with M. paratuberculosis spheroplasts were the only samples that hybridized with the probe. Beef samples containing acid-fast or spheroplast forms of M. smegmatis and M. tuberculosis as well as the acid-fast forms of M. paratuberculosis did not hybridize with the probe. Unrelated bacterial controls, i.e. Helicobacter pylori and Escherichia coli were also negative in the assay. In situ hybridization with the IS900 probe provides a specific way to localize M. paratuberculosis spheroplasts in tissue sections and may be useful for studies of the connection between M. paratuberculosis and Crohn's disease and sarcoidosis. The assay may also be valuable for studies on Johne's diseased animals.     

Paratuberculosis recognized as a problem at last: a review

This article attempts to review briefly current opinions on Johne's disease, or paratuberculosis, in ruminants caused by Mycobacterium avium subsp. paratuberculosis. Paratuberculosis has been known to be prevalent in domestic livestock, such as cattle, goats, and sheep, for more than a century. Despite this knowledge only minor efforts have been made to control the disease and, with the attention being focussed on the eradication of other diseases, the problem of paratuberculosis has been neglected in most countries in the past decades. However, recent epidemiological surveys performed in Europe showed a high prevalence of paratuberculosis in cattle and sheep, indicating that the situation has become quite alarming. In addition, the possible role of M. avium subsp. paratuberculosis in the aetiology of Crohn's disease in humans is still debated, as discussed in this article. Therefore, there is suddenly a renewed interest in paratuberculosis, and the disease is recognized as a significant problem. As a consequence, there is a need for reliable diagnostic tools for large-scale use to allow the introduction of programmes to control and eventually eradicate the disease. The current status and the possibilities for such programmes are discussed.     

Incidence of Mycobacterium paratuberculosis in raw sheep and goats' milk in England, Wales and Northern Ireland

A blind survey of 104 raw sheep and goats' milk samples (90 goat, 14 sheeps) from bulk tanks on farms throughout England, Wales and Northern Ireland was carried out over a 5-month period (January-May 1998) in order to determine the incidence of Mycobacterium paratuberculosis. Each milk sample (100 ml) was divided into two 50ml portions. One portion was decontaminated with 0.75% hexadecylpyridinium chloride for 5h before culture on slopes of Herrold's egg yolk medium and in BACTEC radiometric medium. The second portion was subjected to immunomagnetic separation followed by IS900 PCR (IMS-PCR). The IMS-PCR assay was employed in order to provide a more rapid indication of the presence of M. paratuberculosis in each milk sample than is possible by culture. Information on the Johne's disease status of the sheep and goat herds that took part in the survey was not sought at the time of milk sampling. However, it subsequently emerged that at least some of the herds whose bulk milk was tested during this study were previously or currently infected with Johne's disease. Overall, during this survey one raw goats' milk sample tested positive for the presence of M. paratuberculosis by IMS-PCR (<1% of milk samples tested) but no viable M. paratuberculosis were isolated by culture. The results of this study suggest that bulk raw sheep and goats' milk from these regions of the UK may not represent significant vehicles of transmission of M. paratuberculosis to humans.     

A technique for the purification of Mycobacterium paratuberculosis from the ileal mucosa of infected cattle

Mycobacterium paratuberculosis, the causative agent of paratuberculosis, produces considerable economic loss in the cattle industry in many countries. The slow growth of M. paratuberculosis has hindered investigations of the antigenic composition of the organism and the development of species-specific antigen for serological detection of this disease. This paper describes a simple method for the isolation of large quantities of viable M. paratuberculosis from the intestinal mucosa of infected cattle by a combination of trypsin digestion, deoxyribonuclease/lysozyme treatment and differential centrifugation. Purity was about 99% and yield between 10(5)-10(9) bacteria/g tissue.     

Disseminated Mycobacterium paratuberculosis infection in a cow

A cow with chronic diarrhea and weight loss caused by localization of Mycobacterium paratuberculosis in the intestinal tract (Johne's disease) had gross and microscopic changes indicative of a disseminated infection. A direct association between the remote lesions and the intestinal infection was shown by isolation of M paratuberculosis from renal tissue, detection of intracellular M paratuberculosis antigen(s), using an indirect immunoperoxidase method, and by the characteristic granulomatous nature of the lesions. This case illustrates the potential for extra-intestinal lesions in M paratuberculosis infection of cattle and should cause veterinarians to consider mycobacterial disease when confronted with multinodular lesions of the bovine kidney. The immunoperoxidase method was useful in determining the cause of the inflammatory lesion in which intact organisms were not evident.