Skin test and gamma interferon enzyme-linked immunosorbent assay results in sheep exposed to dead Mycobacterium avium subspecies paratuberculosis organisms.

Cell-mediated immunity (CMI) diagnostic tests, such as the gamma interferon enzyme-linked immunosorbent assay (IFN-gamma ELISA) and the Johnin skin test, have the potential to detect animals infected with Mycobacterium avium subspecies paratuberculosis (MAP) early in the course of the disease. While these CMI tests tend to be relatively specific in noninfected flocks, in MAP-infected flocks, these tests often identify animals that cannot be confirmed infected by any other reference test, including necropsy and culture. The aim of this study was to determine if antigen exposure by inhalation or oral ingestion of killed MAP organisms would cause a detectable CMI response in sheep. Forty-eight lambs 4 months of age were randomly divided into a control group, an orally exposed group (dosed with 1 x 10(10) autoclaved MAP organisms 3 times), and an inhalation-exposed group (dosed once with 1 x 10(5) dead organisms). Lambs were skin tested and/or bled pre-exposure and 1, 2, 3, 4, and 12 months postexposure. No significant difference was seen with either the oral- or inhalation-exposed groups of lambs versus controls with either the IFN-gamma ELISA or the skin test at any time pre- or postexposure. These results suggest that infection/invasion of MAP organisms must occur in order to have a positive skin test or IFN-gamma ELISA beyond the false-positive rate. Simple exposure is not enough to elicit a detectable CMI response.     

Differences in the immune responses in lambs and kids vaccinated against paratuberculosis, according to the age of vaccination

In order to evaluate and compare the peripheral immune responses induced by the vaccination against paratuberculosis in relation with the age of immunization, two groups of lambs and goat kids were vaccinated at 15 days and 5 months old, respectively. A heat-killed commercial vaccine was inoculated subcutaneously and humoral and cellular immune responses were measured by an ELISA and IFN-gamma assay, respectively, at 0, 30, 90, 180, 270 and 360 dpv in the lambs and 0, 30, 90 and 180 dpv in the caprine. IFN-gamma values did not show statistically significant differences between both groups, but when compared to the unvaccinated controls, this cytokine response tend to disappear earlier in animals vaccinated at 15 days old. The antibody response was always higher and more persistent in animals vaccinated at 5 months. The possibility of the incomplete degree of maturation of the immune system in 15 days old animals as the cause of the differences in the immune response to vaccination is suggested.     

Experimental infection of weaner sheep with S strain Mycobacterium avium subsp. paratuberculosis

We sought to determine whether infection of recently weaned 12-16-week-old Merino lambs with an Australian S strain M. a. paratuberculosis, at doses consistent with natural exposure, could be detected in the first few months post-inoculation. Such detection would facilitate the use of weaner sheep as sentinel animals for the presence of infectious doses of M. a. paratuberculosis on pastures. In controlled pen trials, oral doses of approximately 10(7)-10(8) viable organisms were demonstrated to be infective, whereas doses below 10(4) organisms failed to produce detectable infection. Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) was isolated from intestinal and/or lymphoid tissues collected at necropsy 7 or 14 weeks after first infection, but there were no associated gross or microscopic lesions. Skin testing with intradermal Johnin detected all three infected lambs at 13 weeks post-infection, and one of the three infected lambs at 6 weeks post-infection, with 100% specificity. Results for whole blood IFN-gamma assay showed some correlation with infection status but lacked specificity. One infected lamb gave a positive result in an ELISA for antibodies to M. a. paratuberculosis, 14 weeks post-infection and 1 week after skin testing. This was the first demonstration of experimental infection with S strain M. a. paratuberculosis in Australian Merino sheep at doses likely to be representative of natural infection. Culture from tissues in the first few months post-exposure could facilitate the use of naive weaner sheep as tracer animals to detect heavy contamination of pastures with M. a. paratuberculosis, but low-level contamination may not be detected in such a system.     

Efficacy of a killed vaccine for the control of paratuberculosis in Australian sheep flocks

A field trial was undertaken from 1999 until 2004 to determine the efficacy of a killed M. a. paratuberculosis vaccine, Gudair, for the control of ovine Johne's disease (OJD) in merino sheep run under Australian pastoral conditions. On each of three farms experiencing significant OJD losses (5-15% per annum), 200 merino lambs (age 1-4 months) were vaccinated, and 200 lambs were left unvaccinated. Animal assessments and sample collections were conducted twice yearly until 4 or 5 years of age. The impact of vaccination on mortality rate, faecal shedding of M. a. paratuberculosis (by pooled and individual faecal culture), liveweight, wool productivity, vaccine injection site lesions and cellular (BOVIGAM) and humoral (PARACHEK) immunity was examined. Vaccination stimulated cell-mediated and humoral immune responses, reduced mortalities due to OJD by 90% and delayed faecal shedding for the first year post-vaccination. Thereafter, the prevalence of shedders among vaccinates was reduced by 90%. The numbers of M. a. paratuberculosis excreted by the vaccinated groups were also reduced by at least 90% at most sampling times. However, high levels of excretion by vaccinates occurred on some occasions, and although only 7 of 600 vaccinates died from OJD, all 7 had multibacillary lesions. Thus there remains a risk that some vaccinated sheep will transfer the disease. Small reductions in liveweight were found in vaccinated lambs in the first year, but there was little effect on wool production. Vaccine injection site lesions were detected in almost 50% of sheep after 2 months, and these persisted for at least 4 years in 20-25% of vaccinates. Data from this trial enabled the registration of Gudair in Australia in 2002 and underpins the pivotal role of vaccination in the current management of OJD.     

Antigen-induced production of interferon-gamma in samples of peripheral lymph nodes from sheep experimentally inoculated with Mycobacterium avium subsp. paratuberculosis.

The production of interferon-gamma (IFN-gamma) in response to Johnin purified protein derivate was measured in samples of the prescapular lymph node (PLN) from 10 sheep, aged 2 years, and nine sheep, aged 1 year that had been inoculated orally with Mycobacterium avium subsp. paratuberculosis within their first month of life. Ten non-inoculated sheep, aged 1 year, constituted the negative control group. The results obtained in the PLN IFN-gamma assay were compared with those derived from serological tests: a complement fixation test (CFT), agar gel diffusion test (AGID) and enzyme-linked immunosorbent assay (ELISA), as well as an IFN-gamma test on samples of blood. Among the 19 inoculated sheep, 16 gave positive reactions in the PLN IFN-gamma assay on samples incubated overnight, and 18 tested positive when the assay was applied to PLN samples incubated for 48h. In comparison, three, four and seven inoculated sheep gave positive reactions in the ELISA, CFT and in the blood IFN-gamma assay on samples incubated overnight, respectively. The AGID and IFN-gamma assay on blood samples incubated for 48h detected eight inoculated animals. Twelve inoculated sheep, that tested positive in the PLN IFN-gamma assay were clinically normal, gave negative results in an IS900-based polymerase chain reaction (PCR) assay on samples of ileum and ileocaecal lymph node and had no histological evidence of paratuberculosis, but tested positive on more than two occasions in sequential serological testing before necropsy. None of the 10 non-inoculated sheep tested positive in the AGID, CFT, ELISA, blood IFN-gamma assay on samples incubated overnight and for 48h or the PLN IFN-gamma assay on samples incubated overnight, but one gave a positive result in the PLN IFN-gamma assay on samples stimulated for 48h. It is likely that the positive reactions obtained by the PLN IFN-gamma assay in the 12 inoculated sheep that tested negative in the PCR assay and histopathological examination represents immunological evidence of latent infection or previous exposure to M. paratuberculosis rather than active infection.     

Differences in the peripheral immune response between lambs and adult ewes experimentally infected with Mycobacterium avium subspecies paratuberculosis

The peripheral immune response, and its relationship with the outcome of the infection according to the age of the animal, has been investigated in young lambs and adult ewes experimentally infected with two different doses of Mycobacterium avium subspecies paratuberculosis (Map). Sixteen 1.5-month-old lambs out of 24 and 23 adult ewes out of 30 were orally challenged with an ovine Map field isolate. Animals were divided into two groups: HD, infected with a higher dose of Map and LD, with a lower dose. The remaining animals were used as uninfected control groups. Animals were euthanized at 110-120 and 210-220 days post-infection (dpi). Along the experiment, the humoral response and the specific and non-specific IFN-γ production were assessed. An intradermal skin test (IDT), using avian PPD, was also performed at 90 and 195 dpi. Samples of intestine and related lymphoid tissue were taken for histological, bacteriological and PCR studies. The Ab and IFN-γ production as well as the IDT response appeared earlier and with more intensity in the adult ewes compared to the lambs. The basal non-specific IFN-γ levels increased only in the adult ewes from the HD group. Animals from the LD and HD groups were positive to PCR; however, lesions consistent with paratuberculosis were exclusively observed in the HD group, both in lambs and in adult sheep, but they only progressed to more advanced stages in the former. These results suggest that the peripheral immune response induced by Map infection in the adult ewes is more efficient to control the progression of the infection than in lambs. This could likely be due to the existence of previous contacts with Map or other mycobacteria in the adult sheep compared to the young lambs.     

Immunoperoxidase studies of cell mediated immune effector cell populations in early Mycobacterium avium subsp. paratuberculosis infection in sheep

Immunoperoxidase (IPX) labelling for CD4, CD8, TCR-gammadelta, WC1, CD1b, IFN-gamma, CD45R, CD56 and lysozyme was used to investigate changes in cell mediated immune effector cell populations in the intestinal Peyer's patches (PP) and mesenteric lymph nodes of lambs, 2 and 4 months after experimental infection with low doses of sheep strain Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis). The organism was cultured from the tissues of each infected lamb, but histological lesions were not present. This infection model was considered to be more representative of natural M. a. paratuberculosis infection than previous studies. Infected sheep had significantly more CD4+ cells in the mucosa, domes and interfollicular areas of the terminal ileum, and in the interfollicular areas of the jejunal Peyer's patch. Infected sheep also had significantly increased numbers of TCR-gammadelta+ cells in the mucosa and interfollicular areas of the jejunal Peyer's patch, and increased numbers of WC1+ cells in the ileal Peyer's patch. These findings are consistent with previous findings in sheep given higher doses of cattle strain M. a. paratuberculosis. Significantly fewer CD1b+ cells were present in the paracortical areas of the mesenteric lymph nodes of infected sheep, and the reduction was greater in sheep infected for 4 months compared to sheep infected for only 2 months. Down-regulation of CD1b expression may be important for the continued survival and multiplication of M. a. paratuberculosis as specific adaptive immunity develops. Across all sheep, jejunal Peyer's patches had higher numbers of CD4+, CD8+, TCR-gammadelta+, WC1+ and CD45R+ cells, and lower numbers of CD56+ fibres compared to ileal Peyer's patches. These findings confirm and extend the peculiarities of the terminal ileal Peyer's patch in the young ruminant, with possible implications for the early establishment of M. a. paratuberculosis infection.     

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.     

Comparison of three serological tests and an interferon-gamma assay for the diagnosis of paratuberculosis in experimentally infected sheep

OBJECTIVE: To compare the diagnostic performance of a complement fixation test, an agar gel immunodiffusion test, an enzyme-linked immunosorbent assay, and a whole-blood interferon-gamma assay for paratuberculosis in 14 sheep experimentally infected with Mycobacterium avium subsp paratuberculosis. EXPERIMENTAL DESIGN: Longitudinal study. RESULTS: The IFN-gamma assay detected more experimentally infected sheep, and earlier, than any of the serological tests. None of the antibody assays was able to detect all sheep with histologically confirmed paratuberculosis. CONCLUSIONS: The superior performance of the IFN-gamma assay in detecting infected sheep in this small experimental population warrants its further evaluation in a larger population of sheep naturally exposed to M a paratuberculosis.     

Detection by immunomagnetic PCR of Mycobacterium avium subsp. paratuberculosis in milk from dairy goats in Norway

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.     

Evaluation of diagnostic tests for Johne's disease in young cattle

OBJECTIVE: To investigate the development of immune responses in calves experimentally and naturally infected with Mycobacterium paratuberculosis and to evaluate the potential for diagnostic tests to detect infected calves. DESIGN: Sequential testing of four treatment groups of calves over a 2 year period. PROCEDURE: Twenty-nine calves were allocated to four groups. Group D calves were orally dosed with M paratuberculosis, group N calves naturally exposed to M paratuberculosis, group V calves vaccinated for M paratuberculosis, and group C were control calves (not infected or vaccinated). Blood and faecal specimens were collected from each calf at monthly intervals to 18 months of age and then every 2 months until they were slaughtered between the ages of 21 and 29 months. Specimens were tested using absorbed EIA, IFN-gamma EIA and faecal culture. The infection status of the calves was confirmed by extensive histopathological examination and tissue culture. RESULTS: M paratuberculosis infection was confirmed in 10 calves, comprising six of eight orally dosed calves, three of five naturally exposed calves and one of nine vaccinated calves. The six artificially infected calves and one naturally infected calf were detected shedding M paratuberculosis in their faeces. Results with positive absorbed EIA were obtained from one artificially infected calf, one naturally infected calf and three vaccinated calves. All calves including controls had positive results on at least one occasion using the IFN-gamma EIA. In addition, seven calves had positive bovine tuberculosis results using the IFN-gamma EIA, even though bovine tuberculosis has been eradicated from Australia. CONCLUSION: Detection of M paratuberculosis infection in young cattle continues to be difficult using current tests.     

Control of paratuberculosis (Johne's disease) in goats by vaccination

After several years of unsuccessful efforts to eradicate paratuberculosis in goats in Norway by conventional methods such as general hygienic precautions and the isolation and slaughtering of clinically affected and serologically positive animals, a vaccination programme was initiated in 1967. The vaccine used consists of two live attenuated strains of Mycobacterium paratuberculosis suspended in a mixture of liquid paraffin, olive oil and pumice powder. The vaccine may be stored at 4 degrees C for two weeks, the dose is 1 ml and the goat kids are vaccinated at the age of two to four weeks. The efficacy of the vaccine has been judged mainly by post mortem examination of vaccinated and unvaccinated goats in the period 1967-82. During this period about 131,000 goats were vaccinated and, based on the post mortem examination of 15,219 goats, the infection rate was reduced from 53 to 1 per cent. Moreover, infection occurred almost exclusively in goats which for some reason or other had not been vaccinated or which had been too old when vaccinated. The results of these examinations showed that the adjuvanted vaccine with live M paratuberculosis bacteria offers a high degree of protection against paratuberculosis in goats.     

Nested PCR on blood and milk for the detection of Mycobacterium avium subsp paratuberculosis DNA in clinical and subclinical bovine paratuberculosis

OBJECTIVE: To determine the potential of PCR on blood and milk to detect cattle infected with Mycobacterium avium subsp paratuberculosis. PROCEDURE: A nested PCR method probing for IS900 was developed and compared to ELISA serology in 11 clinically infected and 46 subclinically infected, lactating Holstein cows from a herd with confirmed paratuberculosis (Johne's disease). RESULTS: When compared to serum ELISA the nested blood- and milk PCRs were equal in identifying DNA from clinically infected animals. The PCR procedures also gave positive DNA results with some subclinically infected animals when these only gave suspicious or negative results in the ELISA test. Most clinically and subclinically infected animals were detected with milk PCR. CONCLUSION: Since there may well be a haematological phase in paratuberculosis, nested PCR testing of blood and milk samples shows potential to detect animals subclinically infected with M a paratuberculosis. More subclinically infected animals need to be tested and confirmed infected before estimates of sensitivity and specificity can be made.     

Cytokine gene expression in ileal tissues of cattle infected with Mycobacterium paratuberculosis

Cytokine gene expression in ileal tissues of cattle infected with Mycobacterium paratuberculosis was evaluated. The effects of infection with M. paratuberculosis on cytokine production may influence immune regulation at the site of colonization, resulting in the chronic inflammatory state associated with the latter stages of this disease. Ileal samples were obtained at necropsy from noninfected control cows (n=8) and clinically infected cows (n=7) and processed for immunohistochemistry and in situ hybridization. Cows infected with M. paratuberculosis were in the latter stages of disease with clinical signs such as weight loss, watery diarrhea, and inappetence. Among cytokines we studied, interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, and interferon-gamma (IFN-gamma) were expressed significantly more in infected animals than in noninfected control animals. The expression of tumor necrosis factor-alpha (TNF-alpha), however, was not different between the two groups of cattle. In addition, immunohistochemical staining demonstrated that the number of resident macrophages in the ileum of infected animals was three times greater than that of noninfected cows. In contrast to this, ileal tissues from noninfected control animals contained 1.5 times more neutrophils than the ileal tissues from cows infected with M. paratuberculosis. These data demonstrate that localized ileal cytokine production is different between cows chronically infected with M. paratuberculosis and noninfected control cows.     

Use of the polymerase chain reaction assay for the detection of Mycobacterium avium subspecies paratuberculosis in blood and liver biopsies from experimentally infected sheep

OBJECTIVE: To evaluate a polymerase chain reaction-based assay for the detection of Mycobacterium avium subsp paratuberculosis in blood and liver biopsies from subclinically infected sheep. STUDY DESIGN: A direct PCR assay for the detection of M a paratuberculosis was applied to liver biopsy specimens and to samples of blood that were sequentially collected over 53 weeks from 14 sheep infected experimentally with the organism. RESULTS: Of 117 blood samples from the 14 experimentally infected sheep, two tested positive in the PCR assay. Both positive results were obtained in two subclinically infected sheep that had paratuberculosis later confirmed by histological examination at necropsy. However, the assay failed to detect the target DNA in samples of blood from five other sheep with histologically confirmed paratuberculosis. Similarly, the PCR assay on liver biopsy specimens collected 32 weeks after administration of M a paratuberculosis gave only two positive results, both of which were obtained in sheep with histological evidence of paratuberculosis. CONCLUSIONS: The PCR assay on blood and liver biopsies does not provide a useful method for the diagnosis of M a paratuberculosis infection in subclinically infected sheep.     

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.     

Protection from parainfluenza-3 virus and persistence of infectious bovine rhinotracheitis virus in sheep vaccinated with a modified live IBR-PI-3 vaccine.

Ewes (N = 7) and their lambs (N = 12) were vaccinated with a commercial modified live infectious bovine rhinotracheitis-parainfluenza type 3 virus vaccine. Both the vaccinated ewes and lambs and a group of unvaccinated ewes (N = 8) and their lambs (N = 13) were subsequently challenged with virulent parainfluenza type 3 virus. Although absolute immunity to infection and clinical response was not conferred, the clinical response was less severe in vaccinated lambs. Vaccinated animals also shed parainfluenza type 3 virus in nasal secretions for a shorter time than nonvaccinated animals. Some vaccinated lambs developed a persistent infectious bovine rhinotracheitis virus infection that was recrudesced by treatment with dexamethasone. It was concluded that vaccination was of benefit in reducing the severity of infection with parainfluenza type 3 virus. However, the inclusion of infectious bovine rhinotracheitis virus in a vaccine for sheep respiratory tract disease is highly questionable as it might increase the risk factor associated with vaccination. The consequences of the persistence of infectious bovine rhinotracheitis virus are now known.     

Effect of an inactivated paratuberculosis vaccine on the intradermal testing of goats for tuberculosis

The effect of an inactivated paratuberculosis vaccine on the diagnosis of tuberculosis (TB) in goats was investigated in a herd with a history of clinical paratuberculosis but which was free of TB. Cohorts of animals in 2006, 2008 and 2009, were vaccinated once at 1 month of age, and 50% of the 2006 cohort served as unvaccinated controls. The goats were aged 8 months, 20 months and 3.5 years old at the time of the survey. All animals were assessed using a single intradermal injection of bovine tuberculin purified protein derivative (PPD) (SID test), or using both bovine and avian PPD (CID test). An interferon (IFN)-γ assay using both bovine and avian PPD was carried out on the 2006 cohort and was interpreted according to three different 'cut-off' points. No unvaccinated (control) animals tested positive to any of the assays, confirming that the herd was TB-free. The SID test had a low specificity in vaccinated animals at 8 and 20 months of age, whereas the CID test demonstrated 100% specificity in animals ≥20 months-old. The specificity of IFN-γ assay was less than maximal for vaccinated animals 3.5 years old as small numbers of false positives were detected, although this depended on the chosen cut-off point. The study findings demonstrate that the use of an inactivated paratuberculosis vaccine in goats <1 month-old in a TB-free herd does not result in false positives to a CID test for TB when performed in animals ≥20 months-old.     

Serologic enzyme-linked immunosorbent assay responses of calves vaccinated with a killed Mycobacterium paratuberculosis vaccine

The purpose of this study was to document the effect of calfhood vaccination for Mycobacterium paratuberculosis on a serologic ELISA. Fifteen calves vaccinated with a killed paratuberculosis vaccine and 5 unvaccinated control calves were tested from the first through the fifteenth month of life. Age of vaccination ranged from 5 to 40 days. Blood samples were collected prior to vaccination and periodically thereafter. Serum antibody was analyzed by use of the ELISA. All calves were ELISA-negative prior to vaccination. Thirteen of 15 vaccinated calves became ELISA-positive between 2 and 6 months after vaccination. The unvaccinated cohort remained ELISA-negative. Wide-spread use of vaccine may interfere with diagnosis of paratuberculosis and with control programs that are based on serologic tests that measure humoral antibody.     

Lymphocyte blastogenesis, complement fixation, and fecal culture as diagnostic tests for paratuberculosis in North American wild ruminants and domestic sheep

The efficacy of the lymphocyte blastogenesis and complement-fixation tests and fecal culture for detection of Mycobacterium paratuberculosis infection was assessed in bighorn sheep (Ovis canadensis), elk (Cervus elaphus nelsoni), mule deer (Odocoileus hemionus), white-tailed deer (O virginianus), bighorn X mouflon (O musimon) hybrid sheep, and domestic sheep. Spontaneously infected bighorns were tested at the time of capture; experimentally infected animals were tested monthly for 12 months or periodically for 36 months. Lymphocyte blastogenesis tests were conducted with peripheral blood mononuclear cells and protein antigens of M avium, M bovis, and M paratuberculosis. Best diagnostic results were obtained when M avium purified-protein derivative was used as antigen and 20% bovine fetal serum was incorporated in the culture medium; a positive test was defined as a stimulation index greater than or equal to 3.5. Test sensitivity and specificity, respectively, were 82% and 94% in hybrid sheep and were 72% and 100% in domestic sheep. Sensitivity and specificity, respectively, were 39% and 94% in elk and 53% and 92% in deer. When infection was determined in spontaneously infected bighorns by culture of M paratuberculosis and/or the presence of acid-fast bacilli in characteristic microscopic lesions, sensitivity was 75% and specificity was 87%. Fecal cultures and the complement-fixation tests seldom correctly identified infected animals.