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Paratuberculosis was diagnosed in a goat herd that participated in a sanitation program against Mycobacterium avium subsp. paratuberculosis. The aim of this study was to characterise the development of gamma interferon (IFN-γ) and antibody responses as well as the occurrence of faecal shedding. Faecal culture appeared surprisingly sensitive as about 18% and 40% of the goats were positive at 9 and 15-17 months of age, respectively, and shedding was often seen prior to peripheral immune responses. Peripheral IFN-γ responses were not related to protection as clinical and high shedding goats often had high responses. An IFN-γ response usually preceded a humoral response. However, positive antibody titers could sometimes be seen simultaneously with, and even prior to, IFN-γ responses. In conclusion, faecal culture appeared as sensitive as IFN-γ testing. Furthermore, the antibody ELISA and the IFN-γ assay may perform equally well in an infected herd if surveillance is conducted annually., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The gamma interferon assay was evaluated for diagnosis of paratuberculosis in goats with special emphasis on false positive reactions. Four categories of herds were tested: (A) herds that had a history of paratuberculosis, had given positive Mycobacterium avium subsp. paratuberculosis fecal samples and were vaccinated against paratuberculosis; (B) herds that had been vaccinated but had never shown clinical signs of paratuberculosis nor given positive M. a. paratuberculosis fecal samples; and (C) non-vaccinated herds without paratuberculosis. To extend the analysis of samples from young goats free of paratuberculosis, animals less than 18 months of age from non-vaccinated herds without paratuberculosis, category D, were included. Heparinized blood was stimulated with purified protein derivate (PPD) from M. a. paratuberculosis for 24 h and plasma was assayed for the presence of gamma interferon. Results were recorded as the difference between OD values of PPD stimulated and control samples. Vaccinated animals from herds with paratuberculosis, category A, showed significant higher gamma interferon responses than animals from vaccinated herds without paratuberculosis, category B. In both these groups the responses were correlated to age with higher responses in younger animals. Some of the vaccinated animals in herds without paratuberculosis had a gamma interferon response lasting for several years, which demonstrate a long lasting interference with diagnostic testing in vaccinated goats. Only three of the 121 non-vaccinated animals free of paratuberculosis in category C had responses against PPD (corrected OD values at 0.2, 0.24 and 0.5), and none of the 255 young animals in category D had corrected OD values exceeding 0.2. This indicates that false positive reactions do not appear to the same extent in young goats as in young cattle. We conclude that the low responses of non-infected goats could make the gamma interferon assay useful in monitoring the paratuberculosis status of non-vaccinated herds. However, more information about the early gamma interferon responses of naturally infected goats and the presence of false negative samples are needed.

The main lesions of paratuberculosis in ruminants are in the small intestine. Previous studies have shown that the bacterium enters the small intestine through M cells found in the follicle-associated epithelium lining the domes of the Peyer's patches. The everted sleeve method, devised for the in vitro study of intestinal absorption, was used in this study to investigate the uptake of Mycobacterium avium subsp. paratuberculosis in goat intestine. Everted small intestinal sleeves of goat kids, prepared from areas with and without Peyer's patches, were incubated for 60 min in 3H-labeled bacterial solution. The results of this study imply that the bacteria can enter the intestinal mucosa of the jejunum, both in areas with and without Peyer's patches. These findings indicate, therefore, that M. avium subsp. paratuberculosis bacteria not only enter through M cells but also through enterocytes.

The Norwegian surveillance and control programme for paratuberculosis revealed 8 seroreactors in a single dairy cattle herd that had no clinical signs of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) infection. Paratuberculosis had been a clinical problem in goats several years previously in this herd. All 45 cattle were culled and a thorough investigation of the infection status was conducted by the use of interferon-gamma (IFN-gamma) immunoassay, measurement of antibodies, and pathological and bacteriological examination. In the IFN-gamma immunoassay, 9 animals gave positive results, and 13 were weakly positive, while 19 animals were negative. In the serological test, 10 animals showed positive reactions, and 5 were doubtful, while 30 animals gave negative reactions. There appeared to be a weak trend toward younger animals having raised IFN-gamma and older animals having raised serological tests. Histopathological lesions compatible with paratuberculosis were diagnosed in 4 animals aged between 4 and 9 years. Three of these animals had positive serological reaction and one animal gave also positive results in the IFN-gamma immunoassay. Infection was confirmed by isolation of M. a. paratuberculosis from 2 of these 4 animals. One single bacterial isolate examined by restriction fragment length polymorphism (RFLP) had the same profile, B-C1, as a strain that had been isolated from a goat at the same farm several years previously. Despite many animals being positive in one or both of the immunological tests, indicative of a heavily infected herd, none of the animals showed clinical signs and only one cow was shown to be shedding bacteria. A cross-reaction with other mycobacteria might have caused some of the immunoreactions in these animals. It is also possible that the Norwegian red cattle breed is resistant to clinical infection with M. a. paratuberculosis.

A case-control study was made of Norwegian dairy herds with high and low herd levels of antibodies against Mycobacterium avium subspecies paratuberculosis. A high proportion of the herds had a considerable number of seropositive cows, and environmental and management factors were examined for possible associations with the high serological levels of antibodies. The most important appeared to be: geographical location, red deer (Cervus elaphus) gaining access to the pastures for cattle, the observation of wild birds in the feed storage, and herds sharing common pasture with other herds of cattle. However, diagnostic tests showed that none of the animals in the case herds was infected with M a paratuberculosis.

Affinity between protein-G and immunoglobulins from red deer (Cervus elaphus), moose (Alces alces), roe deer (Capreolus capreolus), and reindeer (Rangifer tarandus tarandus) was tested in a competition binding assay. Sera from red deer, reindeer, and moose inhibited the assay less than sera from cattle (less affinity), whereas sera from roe deer showed a slightly higher affinity to protein-G than did sera from cattle. The conclusion was made that protein-G could be used instead of anti-species antibodies for these cervid species, where the aim of the screening was to look for exposure or lack of exposure to mycobacteria in the tested populations. Serologic screening of 1,373 free-ranging cervids for antibodies against Mycobacterium avium subsp. paratuberculosis was conducted. All sera were tested by a protein-G-based antigen-absorbed enzyme-linked immunosorbent assay (ELISA). Seropositive moose (10/537; 1.9%), red deer (14/371; 3.8%), roe deer (6/49; 12.2%), and semidomesticated reindeer (11/325; 3.4%) were found, whereas wild reindeer (n = 91) were seronegative. In addition, the red deer sera were tested with a commercial ELISA, by which two animals tested positive and nine were suspicious of having M. avium subsp. paratuberculosis antibodies. Tissue samples and feces from 10 moose originating from a population with a clustering of seropositive animals were investigated by histology and bacteriology with negative results. Paratuberculosis has never been diagnosed in free-ranging or farmed cervid species in Norway. Thus, further studies are indicated to prove that the present findings reflect an infection with M. avium subsp. paratuberculosis.

Milk samples from 340 individual goats in 34 dairy herds throughout Norway were examined for Mycobacterium avium subsp. paratuberculosis (M.a. paratuberculosis) by culture and immunomagnetic separation combined with PCR (IMS-PCR). The samples included three categories; (A) vaccinated dairy goats in herds with paratuberculosis; (B) vaccinated dairy goats in herds with no history of paratuberculosis; (C) unvaccinated goats in herds with no history of paratuberculosis. Viable M.a. paratuberculosis were not detected by culture in any sample, but 24 samples (7.1%) tested positive by IMS-PCR when the PCR products were visualised by dot blot hybridisation. PCR products from five milk samples originating from five different herds were sequenced; all showed 99% homology with the IS900 sequence from M.a. paratuberculosis.M.a. paratuberculosis were detected in all sampled categories. The percentage of IMS-PCR positive samples from herds where paratuberculosis had previously been reported was significantly lower than from herds where the infection had never been diagnosed (3.3 and 9.1%, respectively, P=0.048). Similar proportions of milk samples from vaccinated and non-vaccinated goats tested positive for the presence of M.a. paratuberculosis. Vaccinated goats older than 4 years tested positive more often than vaccinated animals less than 2 years old. Samples collected in May tested significantly more often positive than milk sampled during February-March (13.8 and 2.9%, respectively, P=0.001). This study showed that raw goats' milk in Norway might be contaminated with M.a. paratuberculosis.

Paratuberculosis is a chronic, granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis affecting domestic and wild ruminants. The symptoms of clinical paratuberculosis are chronic diarrhoea and progressive weight loss while subclinically infected animals mainly have decreased production. The infection is widespread throughout the world and causes substantial financial losses for the farming industry. One of the major obstacles in the control of this disease, is the difficulty of identifying subclinically infected animals. This review gives a summary of several aspects of paratuberculosis including clinical importance, pathology, immunology and properties of the infectious agent. Special emphasis will be on the available diagnostic methods, their use and limitations.

An experimental oral infection of goats with a caprine isolate of Mycobacterium a. subsp. paratuberculosis was used to investigate immunological and bacteriological events during the subclinical phase of infection. Seven goats at 5-8 weeks of age were given a bacterial suspension in milk-replacement three times weekly for 9 weeks. Six animals were kept as controls. Cellular recall responses against M. a. paratuberculosis were analysed by means of a lymphocyte proliferation test, an IFN-gamma assay and an IL-2 receptor assay. All inoculated animals had detectable CMI responses from 9 weeks post-inoculation and through the 2 years of study, although the responses were highest during the first year. Antibodies against M. a. paratuberculosis could be detected from weeks 15-20 in four of the seven animals, and one additional animal became antibody positive at week 35, while two inoculated animals did not produce significant antibody titres during the experiment. At about 1-year post-inoculation, two animals became faecal shedders, while two others started to excrete bacteria into faeces about 2 years post-inoculation. The appearance of M. a. paratuberculosis in faeces was not associated with a decline in cellular responses as far as could be assessed using the current methods for measuring CMI. Pathological lesions due to M. a. paratuberculosis infection and presence of bacteria were recorded in the intestine and/or mesenteric lymph nodes of five animals while lymph node changes suggestive of paratuberculosis were observed in one animal. Only the two animals with no signs of an active infection at necropsy showed a considerable decline in the cellular parameters during the last year of the study, particularly in the IFN-gamma assay. The two animals with the highest levels of M. a. paratuberculosis responsive CD8+ lymphocytes in the circulation about 1-year post-inoculation had no detectable lesions in the distal ileum and colon at necropsy, while high numbers of gammadelta T-cells responsive to M. a. paratuberculosis in the circulation were associated with disseminated lesions in the distal ileum and colon.

The Norwegian surveillance and control programme for paratuberculosis revealed 8 seroreactors in a single dairy cattle herd that had no clinical signs of Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) infection. Paratuberculosis had been a clinical problem in goats several years previously in this herd. All 45 cattle were culled and a thorough investigation of the infection status was conducted by the use of interferon-γ (IFN-γ) immunoassay, measurement of antibodies, and pathological and bacteriological examination. In the IFN-γ immunoassay, 9 animals gave positive results, and 13 were weakly positive, while 19 animals were negative. In the serological test,10 animals showed positive reactions, and 5 were doubtful, while 30 animals gave negative reactions. There appeared to be a weak trend toward younger animals having raised IFN-γ and older animals having raised serological tests. Histopathological lesions compatible with paratuberculosis were diagnosed in 4 animals aged between 4 and 9 years. Three of these animals had positive serological reaction and one animal gave also positive results in the IFN-γ immunoassay. Infection was confirmed by isolation of M. a. paratuberculosis from 2 of these 4 animals. One single bacterial isolate examined by restriction fragment length polymorphism (RFLP) had the same profile, B-C1, as a strain that had been isolated from a goat at the same farm several years previously. Despite many animals being positive in one or both of the immunological tests, indicative of a heavily infected herd, none of the animals showed clinical signs and only one cow was shown to be shedding bacteria. A cross-reaction with other mycobacteria might have caused some of the immunoreactions in these animals. It is also possible that the Norwegian red cattle breed is resistant to clinical infection with M. a. paratuberculosis.

In Norway, paratuberculosis has been frequently diagnosed in goats, while cattle have been almost free of the infection. This difference in prevalence between goats and cattle has led to speculations about the existence of a Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolate that is non-pathogenic for cattle. There is little information available on genotypic variation of M. a. paratuberculosis isolated from animals in Norway. In the present study, genotypic information on 51 isolates from goats and four isolates from cattle in Norway was obtained by use of IS900 restriction fragment length polymorphism (RFLP) analysis. All isolates from cattle and 84% of the isolates from goats had the same RFLP pattern (B-C1). Five RFLP patterns not previously detected were found. No genotypic variation that could explain a difference in host origin was found between the isolates from cattle and the majority of the Norwegian goat isolates. This lack of difference indicates that the most common M. a. paratuberculosis isolates in Norway may infect both cattle and goats. [ABSTRACT FROM AUTHOR]