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.     

Evaluation of specificity of tuberculosis diagnostic assays in caprine flocks under different epidemiological situations

The aim of this study was to evaluate the specificity of the most widely used tuberculosis (TB) diagnostic tests, single intradermal tuberculin (SIT) and single comparative intradermal tuberculin (SCIT) tests and interferon-gamma (IFN-γ) assay in 937 animals from eight TB-free caprine flocks under different epidemiological situations. Maximum specificity was found using SCIT test (99.4-100% depending on the interpretation criteria) while SIT test and IFN-γ assay showed a slightly lower overall specificity (97.6-99.2% and 96.4-98.4% respectively). Specificity of the SIT test in a Corynebacterium pseudotuberculosis infected flock was significantly (P<0.05) lower if a severe interpretation criterion was applied. Similarly, specificity values of SIT test and particularly IFN-γ assay in a paratuberculosis (PTB)-vaccinated flock were lower than those observed in non-vaccinated flocks. Higher proportion of false positive reactors to TB tests (SIT and IFN-γ assay) were observed among animals positive in the PTB-ELISA in PTB vaccinated flock. These results demonstrate that TB diagnostic tests show an adequate specificity when performed in goats from TB-free flocks in most situations. However, certain factors such as C. pseudotuberculosis infection and paratuberculosis vaccination can have a negative impact in the most sensitive tests.     

Development of an improved ESAT-6 and CFP-10 peptide-based cytokine flow cytometric assay for bovine tuberculosis

Control of bovine tuberculosis (bTB) continues to be a problem world-wide because of difficulties in identifying infected animals at all stages of infection. The use of the IFN-γ release assays (IGRA) as an ancillary test with the tuberculin skin tests has improved the ability to identify infected animals. However, infected animals may still be missed. The objective of the present study was to evaluate a rapid flow-cytometric assay based on intracellular cytokine staining as an alternative to the in vitro IFN-γ release assay (IGRA). Antigen-specific cells producing IFN-γ were identified after a 6 h stimulation with PPD-B, PPD-A and ESAT-6/CFP-10. Defined groups of animals naturally infected with Mycobacterium bovis (Mbv), animals infected with non-tuberculous mycobacteria (NTM), and uninfected control animals were analysed to evaluate the sensitivity and specificity of the optimized assay. Both antemortem and postmortem diagnostic tests were carried out to verify the status of infection. We show that IFN-γ is induced in T cells from whole blood samples from cattle infected with Mbv 6 h post stimulation with PPD-B, PPD-A and ESAT-6/CFP-10, whereas non-infected animals did not respond. Four colour flow cytometric analysis demonstrated responding cells were CD45R0⁺CD69⁺CD4⁺ memory T cells. Also, the response to stimulation with ESAT-6/CFP-10 can be used to distinguish between cattle infected with Mbv and cattle exposed to NTM. Although further studies are needed, the results indicate that detection of intracellular IFN-γ may represent an important alternative approach for improved method of detection of cattle secreting IFN-γ below levels of detection in culture medium.     

Faecal shedding detected earlier than immune responses in goats naturally infected with Mycobacterium avium subsp. paratuberculosis

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.     

Use of the johnin PPD interferon-gamma assay in control of bovine paratuberculosis

Although the interferon-gamma (IFN-γ) assay for measurements of cell-mediated immune (CMI) responses to paratuberculosis PPD (johnin) has been available for close to 20 years, the assay has not yet emerged as the long desired test to identify infected animals at an early time point. Among other issues, this relates to problematic interpretation of the test results and maybe an over-expectation of what can be deducted from this kind of test given the chronic nature and slow development of infection of paratuberculosis. Over a number of years a modified IFN-γ assay with addition of recombinant bovine IL-12 to the PPDj stimulation of blood samples from the heifer group in more than 20 Danish dairy herds which also perform surveillance of MAP antibodies in milk have been performed. The results indicate that IFN-γ assay results are specific for paratuberculosis, but the IFN-γ assay result of an individual animal cannot establish whether the animal is infected or predict the future progression of disease in this animal. The IFN-γ assay should thus be used on a group of animals to test the level of exposure to paratuberculosis bacteria the animals have experienced, and thereby assist in maintaining rational in-herd management procedures and in the establishment of paratuberculosis status of a given herd. Indeed, for any diagnostic test applied in paratuberculosis, both the diagnostic target condition and the purpose of the diagnostic testing must be considered before any meaningful estimates of sensitivity or specificity can be given.     

Evaluation of two cocktails containing ESAT-6, CFP-10 and Rv-3615c in the intradermal test and the interferon-γ assay for diagnosis of bovine tuberculosis

The intradermal tuberculin tests and the interferon-gamma (IFN-γ) assay are the principal tests used worldwide for the ante-mortem diagnosis of bovine tuberculosis. The conventional reagent currently in use in these tests is purified protein derivative (PPD) tuberculin obtained from Mycobacterium bovis culture. The components of PPD are poorly characterized and difficult to standardize. To overcome this issue, antigens specific to the Mycobacterium tuberculosis complex are being studied. Here we have assessed the biological potency of ESAT-6, CFP-10 and Rv-3615c presented as peptide or recombinant protein cocktails in comparison with the standard bovine PPD used routinely in Spanish eradication campaigns. The study was performed in cattle (n=23) from a herd with natural M. bovis infection. Animals were simultaneously injected with PPD and the peptide and protein cocktails. The percentages of cattle reacting positively to single intradermal test were 60.9% (bovine PPD), 47.8% (peptide cocktail) and 60.9% (protein cocktail), with no significant difference between the actual skin fold thickness increases (p>0.05). The IFN-γ assay detected 60.9% of animals when stimulation was performed with bovine PPD, but decreased to 52.2% when stimulation was performed with the peptide cocktail and to 47.8% when stimulation was performed with the protein cocktail. However, no significant differences were found between IFN-γ responder frequencies (p>0.05). These results show a potential use of these defined reagents for in vivo tuberculosis diagnosis.     

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.     

Immunohistochemical expression of interferon-γ in different types of granulomatous lesions associated with bovine paratuberculosis

Animals infected with Mycobacterium avium subsp. paratuberculosis (Map) show a variety of lesions, from focal forms, seen in subclinical stages to diffuse lesions in clinical cases. The purpose of this study was to evaluate the local expression of IFN-γ by immunohistochemistry in relation with the type of lesion in naturally Map-infected cows. The number of immunolabelled cells, −the majority morphologically consistent with lymphocytes-, was higher in focal and diffuse paucibacillary forms than in diffuse multibacillary lesions, where they appeared closely related to epithelioid cells. Diffuse multibacillary lesions had the lowest numbers, but higher than controls, and positive cells were intermingled among the macrophages. The peripheral IFN-γ production was higher in all Map infected cows and a positive correlation was found with the number of immunolabelled cells in the intestine. The findings of this study show that IFN-γ would play a role in the development of the different types of lesions in paratuberculosis, and also points out the importance of adequate sampling of lymphoid tissue containing samples when studying the local immune response in which IFN-γ expression may be involved, especially in cases where focal lesions are present.     

Assessment of in vivo and in vitro tuberculosis diagnostic tests in Mycobacterium caprae naturally infected caprine flocks

Caprine tuberculosis in Spain is mainly caused by Mycobacterium caprae although the progression of the disease and lesion severity is similar to that caused by Mycobacterium bovis. In this study, the sensitivity of the gamma-interferon (IFN-γ) assay using an antigen cocktail containing early secretory antigenic target-6kDa (ESAT-6) and culture filtrate protein 10 (CFP-10) peptides for stimulation was determined and compared with those obtained in single intradermal tuberculin (SIT) and single intradermal cervical comparative tuberculin (SICCT) tests and IFN-γ assay using purified protein derivative (PPD) in three different flocks infected with M. caprae under different epidemiological conditions. Correlation between specific IFN-γ production and severity of lesions was also evaluated. Sensitivities of the diagnostic tests varied greatly in the three flocks studied, with higher values in those where higher lesion scores were observed. The results show that IFN-γ assay applied in goats using PPD or the ESAT-6/CFP-10 peptides cocktail for stimulation yielded similar sensitivity values. A significant yet weak positive correlation between specific IFN-γ production and lesion scores was detected after the stimulation with PPDs (p=0.004) whereas when the blood samples were stimulated with ESAT-6/CFP-10 peptides, the correlation was not significant (p>0.05). Therefore, specific-IFN-γ production after the stimulation with PPDs or ESAT-6/CFP-10 was not an accurate indicator of lesion severity in naturally tuberculosis infected goats with M. caprae.     

两种奶牛结核病检测方法的比较

本研究中采用常规皮内变态反应(TST)对广西南宁、柳州共计2 818头黑白花奶牛进行牛结核病检测,采用抗原特异性IFN-γ试验方法对PPD检测结果为阳性、可疑的牛以及部分阴性牛进行复检。将抗原特异性IFN-γ试验与TST检测结果相比,其敏感性为53.33%,特异性为70.49%,符合率为67.11%。本研究证实单独使用PPD皮内变态反应方法在牛结核病的诊断方面存在非特异性强等缺点,易造成假阳性牛的误杀;用抗原特异性IFN-γ试验对TST试验阳性和可疑牛进行复检可以提高特异性,该方法在临床上有重要应用价值。     

Evaluation of the gamma-interferon assay for eradication of tuberculosis in a goat herd

Objective To evaluate the usefulness of the gamma-interferon assay in the diagnosis of caprine tuberculosis in comparison with a single intradermal tuberculin test, and to obtain a group of animals free from this infection in a herd with a high prevalence.
Design An immunological study involving four serial comparative gamma-interferon and single intradermal tuberculin tests.
Animals A herd of 87 goats of Guadarrama breed.
Procedure Serial testing and segregation of animals.
Results We found that the number of infections detected by the gamma-interferon test was considerably greater than the number detected by the single intradermal tuberculin test. A group of 10 animals was negative to both tests in two consecutive rounds and three kids were negative in the last round of testing.
Conclusions Gamma-interferon assay is appropriate for diagnosis and eradication of tuberculosis in goats. This test is able to detect early Mycobacterium bovis infection. Avian reactors with simultaneous increased reaction to bovine PPD in the gamma-interferon assay (designated as avian^B reactors) should be considered test positive for M bovis . By serial testing with the gamma-interferon and the single intradermal tuberculin tests, and a policy of segregation of kids at birth, it is possible to achieve a group of animals test negative for tuberculosis from a herd of goats with high immunoreactivity to this infection.     

Enhancement of the interferon gamma assay to detect paratuberculosis using interleukin-7 and interleukin-12 potentiation

A limitation to the widespread application of interferon gamma (IFN-γ) response assays has been logistical difficulties, as they must be performed within hours of blood collection. Detection of specific IFN-γ responses to Mycobacterium avium subspecies paratuberculosis (MAP) as part of the cell-mediated immune response of ruminants with Johne's disease could aid in diagnosis and control programs. In this study, a modified protocol was developed in which cultures were supplemented with interleukin (IL)-7, a survival factor required to maintain resting T cells, alone or in combination with IL-12 to potentiate IFN-γ responses and extend blood storage time. The combination of IL-7 and IL-12 was synergistic, giving IFN-γ responses greater than with IL-12 alone, for sheep blood stored up to 2 days. In a cohort of naturally infected sheep, the same number of animals was identified as test positive using the modified assay (performed after 2 days with IL-7/IL-12 supplementation) as the standard IFN-γ assay performed on the day of blood collection. The modified assay offered greatest advantage in the detection of early stages of paratuberculosis infection, for sheep with low grade and paucibacillary lesions, and at early time points post-infection. The potentiation protocol allowed for practical shipment of blood samples from farm to laboratory, extending permissible transit times and applicability of IFN-γ testing to detect Johne's disease.     

IS900 Restriction Fragment Length Polymorphism (RFLP) Analysis of Mycobacterium avium subsp. paratuberculosis Isolates from Goats and Cattle in Norway

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.     

Expansion of intracellular IFN-γ positive lymphocytes during Mycoplasma agalactiae infection in sheep

A method to assess the expansion of antigen-specific intracellular IFN-γ positive T cell subsets during the infection will be helpful for a better understanding of mycoplasmal infections physiopathology in the sheep. We analysed the percentage of antigen-specific lymphocytes positive for intracellular IFN-γ during the infection of sheep with Mycoplasma agalactiae by culturing peripheral blood mononuclear cells of infected or uninfected animals with irradiated M. agalactiae. The expansion of antigen-specific IFN-γ positive lymphocytes in infected sheep was initially sustained by CD4⁺ T cells at day 15 after infection, when antigen specific IgG start to be detectable, followed by CD8/IFN-γ double positive cells. γδ T-cells were not expanded at any time point analysed. IFNγ⁺ T cells disappear 60 days after infection, suggesting that antigen specific IFNγ⁺ T cells, mainly detected in the early phase of the disease, could be useful to understand the role of cell-mediated immunity during M. agalactiae infection.     

Evaluation of the gamma interferon test for diagnosis of paratuberculosis in goats

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.     

Description of the Infection Status in a Norwegian Cattle Herd Naturally Infected by Mycobacterium avium subsp. paratuberculosis

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.     

基于结核菌素与CFP10／ESAT6的牛IFN-γ检测法在牛结核病诊断中的比较研究

本研究通过比较三种刺激物（牛结核菌素、禽结核菌素和牛型结核菌特异性抗原CFP10／ESAT6对结核菌素皮内变态反应阳性牛的IFN-γ刺激反应,探讨IFN-γ检测法在我国牛结核病诊断中的应用前景。无菌采集22头结核菌素皮内变态反应阳性牛的血液．肝素抗凝。每1mL全血与1mL RPMI1640完全培养基混合均匀,并加入0.1mL（20μg）PHA（阳性对照孔）、0．1mL（20μg PHA）CFP10／ESAT6融合蛋白、0．1mL（2000U）＆结核菌素（PPD／B）、0．1mL（2500u）禽结核菌素（PPD／A）或等量PBS（无刺激阴性对照）,37℃培养过夜。次日用夹心ELISA法检测各刺激组0．1mL培养上清的IFN-γ,以OD630表示IFN-γ浓度。结果,特异性抗原CFP10／ESAT6刺激组与牛结核菌素（PPD／B）刺激组的IFN-γ反应具有良好的相关性．相关系数为0．84。但CFP10／ESAT6刺激组IFN-γ浓度与牛和禽PPD的比较反应（以两刺激组IFN-γ浓度差值表示）间无相关性,相关系数为-0．11。分析禽PPD组的IFN-γ反应,发现实验牛中有少数牛对禽PPD有反应。以OD630=0．17为阳性反应切割值,牛PPD检出阳性牛21头,CFP10／ESAT6检出20头,牛和禽PPD比较反应（以OD值差值表示）检出14头,禽PPD阳性反应3头。扣除禽PPD阳性反应牛后,牛和禽PPD比较反应与牛PPD刺激组的相关系数增至0．54。结果表明,牛PPD的IFN-γ释放反应检测灵敏度最高。当出现牛型结核菌与环境分枝杆菌混合感染时．应用牛和禽PPD比较反应检测牛结核的准确度低,混合感染牛被误判为结核阴性牛。而基于CFP10／ESAT6的IFN-γ释放反应不受环境分枝杆菌的影响,检测具有良好的特异性与敏感性。     

Characterization of the long-term immune response to vaccination against Mycobacterium avium subsp. paratuberculosis in Danish dairy cows

Vaccination of cattle against Mycobacterium avium subsp. paratuberculosis (MAP) provides partial protection by delayed shedding of MAP and reduced numbers of clinically affected animals. The duration of vaccine induced immune response is not known. The primary objective of this study was therefore to characterize the long-term effect of whole-cell based vaccination against MAP on the immune response. A secondary objective was to evaluate whether immunodiagnosis of MAP and Mycobacterium bovis infections is affected by MAP vaccination. Two studies were performed: (1) A retrospective longitudinal study including 895 vaccinated and 2526 non-vaccinated dairy cows in 9 Danish dairy herds aiming at characterizing the long-term antibody-response to vaccination; and (2) a cross-sectional study of responses in the IFN-γ assay carried out in 140 vaccinated animals in two herds to evaluate the effect of vaccination on the cell-mediated immune response and to evaluate a possible interference with the diagnosis of M. bovis infections. The results showed that 37% of samples from vaccinated animals and 5% of samples from non-vaccinated animals, respectively, were test positive in the milk antibody ELISA. The prevalence of antibody responses of the vaccinated animals was relatively constant from 2 to 6 years of age, but decreased in older animals. Among the 140 vaccinated animals 88% tested positive with the IFN-γ test to johnin PPD and 50% responded to PPDb with IFN-γ production above a similar cut-off. Although Denmark is free of M. bovis, two of the vaccinated animals responded with higher IFN-γ levels when cultured with PPDb compared to PPDa. In conclusion, immunization with whole-cell MAP vaccines elicits both humoral and cell-mediated immune reactions, which may interfere with surveillance and diagnosis of both MAP and M. bovis infections using currently available tests.     

Assessment of diagnostic tools for eradication of bovine tuberculosis in cattle co-infected with Mycobacterium bovis and M. avium subsp. paratuberculosis

The intradermal tuberculin (IDTB) test and the interferon-gamma (IFN-gamma) assay are used worldwide for detection of bovine tuberculosis in cattle, but little is known about the effect of co-infecting agents on the performance of these diagnostic tests. This report describes a field trial conducted in a cattle herd with dual infection (bovine tuberculosis and paratuberculosis) during 3.5 years. It has been based on a strategic approach encompassing serial parallel testing (comparative IDTB test, the IFN-gamma assay and serology of paratuberculosis) that was repeated 8 times over the period, and segregation of animals into two herds. The IDTB test detected 65.2% and the IFN-gamma test detected 69.6% of the Mycobacterium bovis culture-positive cattle. However, the IDTB test performed better during the first part of the trial, while the IFN-gamma test was the only method that detected infected animals during the following three samplings. The number of false positive reactors with the IDTB and/or the IFN-gamma tests was remarkably high compared to other reports, and could be caused by cross-reactivity with M. avium subsp. paratuberculosis. Also, the M. bovis isolates from cattle and wildlife from the same property were characterised using molecular techniques to disclose an epidemiological link. The IDTB test may not be appropriate to eradicate bovine tuberculosis in herds with dual mycobacterial infections. This report highlights the need to use several diagnostic techniques for the accurate detection of M. bovis infected animals in these herds.     

Abnormalities in the axial skeleton of a Risso's dolphin,Grampus griseus

As the first step in the development of a cervine IFN-γ assay for the diagnosis of tuberculosis in deer, cervine IFN-γ cDNA was amplified by polymerase chain reaction using primers based on the bovine IFN-γ sequence. A high level of amino acid homology was found between the cervine and the ovine and bovine sequences (94% and 91% respectively). There was less identity with the porcine, human, mouse and rat sequences (78%, 62%, 37% and 39%, respectively). The amino terminus of the mature IFN-γ protein, which is critical for interaction with its receptor and for triggering biological activity, is highly conserved between the cervine, bovine and ovine proteins. A monoclonal antibody-based sandwich enzyme immunoassay (EIA) specific for bovine IFN-γ also detects ovine but not cervine IFN-γ. This suggests that the antibodies recognise epitopes common to the bovine and ovine protein but not cervine IFN-γ. Seven amino acid residues that were common to the bovine and ovine sequence differed in the cervine sequence, suggesting that the specificity of the monoclonal antibodies may be dependent on one or more of these residues. The possibility of the development of an EIA for cervine IFN-γ as a commercial in vitro diagnostic assay for tuberculosis in deer is discussed.