Factors affecting the performance of P22 ELISA for the diagnosis of caprine tuberculosis in milk samples.

Caprine tuberculosis (TB) is a zoonosis caused by members of the Mycobacterium tuberculosis complex (MTBC). Caprine TB eradication programmes are based mainly on intradermal tuberculin tests and slaughterhouse surveillance. However, the use of serological test has been extended as a potential diagnostic tool in goats through the use of serum, plasma, or even milk samples. Milk production and the antibodies (Ab) present in milk can vary depending on several circumstances. In the present study, different factors that may affect the performance of humoral TB diagnosis were analysed using goat milk samples: 1) lactation stage, 2) a recent previous skin test (booster effect) and 3) the effect of freeze-thaw cycles on milk samples preserved with azidiol. TB-infected animals (n = 44) were selected to evaluate the evolution of the Ab levels during the 6-month lactation period, along with its potential effect on the P22 ELISA results. In general, no significant changes (p = 0.079) were observed throughout the study as regards Ab levels in milk samples between consecutive analysis although the reactivity to P22 ELISA decreased when samplings were performed at the last two months of the lactation. Regarding the booster effect, the quantitative results showed a significant variation (p < 0.001) for both milk and serum samples when serological tests were carried out 15 days after the skin test. Finally, there were no significant differences (p = 0.99) in the P22 ELISA results when using milk samples preserved with azidiol that had undergone freeze-thaw cycles.

Effect of the topical administration of corticosteroids and tuberculin pre-sensitisation on the diagnosis of tuberculosis in goats.

BACKGROUND: Caprine tuberculosis (TB) is a zoonosis caused by members of the Mycobacterium tuberculosis complex (MTBC). Caprine TB control and eradication programmes have traditionally been based on intradermal tuberculin tests and slaughterhouse surveillance. However, this strategy has limitations in terms of sensitivity and specificity. Different factors may affect the performance of the TB diagnostic tests used in goats and, subsequently, the detection of TB-infected animals. In the present study, the effect of two of the factors that may affect the performance of the techniques used to diagnose TB in goats, the topical administration of corticosteroids and a recent pre-sensitisation with tuberculin, was analysed. METHODS: The animals (n = 151) were distributed into three groups: (1) a group topically treated with corticosteroids 48 h after intradermal tuberculin tests (n = 53); (2) a group pre-sensitised with bovine and avian purified protein derivatives (PPDs) 3 days before the intradermal tuberculin test used for TB diagnosis (n = 48); and (3) a control group (n = 50). All the animals were tested using single and comparative intradermal tuberculin (SIT and CIT, respectively) tests, an interferon-gamma release assay (IGRA) and a P22 ELISA. RESULTS: The number of SIT test reactors was significantly lower in the group treated with corticosteroids when compared to the pre-sensitised (p < 0.001) and control (p = 0.036) groups. In contrast, pre-sensitisation with bovine and avian PPDs did not cause a significant reduction in the number of SIT and CIT test reactors compared with the control group. In fact, a higher number of reactors was observed after the prior tuberculin injection in the pre-sensitised group (p > 0.05). No significant effect was observed on IGRA and P22 ELISA due to corticosteroids administration. Nevertheless, a previous PPD injection affected the IGRA performance in some groups. CONCLUSIONS: The application of topical corticosteroid 24 h before reading the SIT and CIT tests can reduce the increase in skin fold thickness and subsequently significantly decrease the number of positive reactors. Corticosteroids used can be detected in hair samples. A previous pre-sensitisation with bovine and avian PPDs does not lead to a significant reduction in the number of intradermal tests reactors. These results are valuable in order to improve diagnosis of caprine TB and detect fraudulent activities in the context of eradication programs.

Tuberculosis caused by Mycobacterium caprae in a camel (Camelus dromedarius).

BACKGROUND: Animal tuberculosis (TB) is distributed worldwide and has a wide range of wild and domestic reservoirs. Few studies concerning TB in camelids have been published in the last decade, particularly as regards Old World Camelids (OWC), but the increase in reports of TB outbreaks in these species in recent years suggests a high susceptibility to the infection. CASE PRESENTATION: We studied a dromedary camel (Camelus dromedarius) herd (n = 24) in which a Mycobacterium caprae infection was detected. The TB infection was confirmed in one animal at necropsy through the detection of TB lesions, mainly in the abdominal organs, and the subsequent isolation of M. caprae (SB0157 spoligotype). The whole herd was additionally tested using cellular and humoral based diagnostic techniques. The intradermal tuberculin test results were compared with those obtained using P22 ELISA for the detection of specific antibodies against the M. tuberculosis complex. The TB infected animal was a positive reactor to both the intradermal tuberculin tests and P22 ELISA, while the others were negative to all the diagnostic tests. CONCLUSION: The present study found M. caprae infection in OWC. This is the first report of M. caprae infection in an OWC not living in a zoo. Since the animal was born in the herd and fed with goat's milk, this practice was suspected to be the potential source of TB infection, which was not confirmed in the other animals present in the herd. Moreover, our results highlight that the intradermal tuberculin test and the P22 ELISA could be valuable tools for the diagnosis of TB in OWC.

Accuracy of tuberculosis diagnostic tests in small ruminants: A systematic review and meta-analysis.

Tuberculosis (TB) in small ruminants is a neglected disease despite its major impact on goat and sheep production and the global public health. The awareness of the role of small ruminants in the epidemiology of animal TB has increased in the last two decades, however, there is a lack of standardization of procedures and robust quantitative estimates on the accuracy of diagnostic TB tests in the scientific literature. To address this knowledge gap, all the available information regarding the use of ante-mortem diagnostic techniques in small ruminants was collected and summarized through a systematic review process. Furthermore, a random-effects meta-analysis was conducted to separately estimate the sensitivity (Se) and specificity (Sp) of cell-based tests among the retrieved studies in goats. Studies included in the meta-analysis were also evaluated using the Quality Assessment of Diagnostic Accuracy Studies included in systematic reviews adapted for animal diagnostic tests (VETQUADAS). Median pooled Se estimates of the single intradermal tuberculin (SIT) test (ranged from 0.51 to 0.59), the comparative intradermal tuberculin (CIT) test (ranged from 0.30 to 0.50) and the interferon-gamma (IFN-γ) release assay (IGRA) (ranged from 0.66 to 0.72) were lower than that reported previously in cattle, regardless the interpretation criteria and the reporting of MAP infection or vaccination. However, the specificity was adequate for all the tests (ranged from 0.95 to 0.99), except for the SIT test in MAP vaccinated herds (ranged from 0.78 to 0.90). This study provides an overview of the accuracy of diagnostic tests for TB in goats, however, the considerable between-study heterogeneity found hampered the conclusive interpretation of the pooled Se and Sp estimates. Therefore, further studies in small ruminants are necessary to optimize the diagnostic Se, which could help to design effective control strategies, accelerate the eradication of TB in these species and harmonize test procedures.

Evaluation of a new enzyme-linked immunosorbent assay for the diagnosis of tuberculosis in goat milk.

Caprine tuberculosis (TB) is a zoonosis with sanitary and economic repercussions. Caprine TB control programs are based on a test and cull strategy using the intradermal tuberculin tests and slaughterhouse surveillance. However, this approach is not always feasible and may have a limited sensitivity under specific circumstances. In this study, performance of a new experimental test based on the P22 protein complex (P22 ELISA) was evaluated in two TB-infected herds using milk and serum samples and compared with cell-based diagnostic tests. Samples from a low (n = 62, herd 1) and a high (n = 52, herd 2) TB prevalence herd were selected. Moreover, bulk tank milk samples from both herds were analysed using the P22 ELISA. At the end of the study, a group of animals (n = 21) was euthanized and subjected to post-mortem analysis and bacteriological culture. Significant differences (p < .001) on the qualitative and quantitative (ODs) results were observed between herds using both serum and milk samples in the P22 ELISA. The correlation observed in the quantitative results obtained in serum and milk samples was very strong in animals from flock 2 (rs = 0.91) and moderate in animals from flock 1 (rs = 0.46). Among the slaughtered animals, the P22 ELISA detected a higher proportion of lesion-culture positive animals than cell-based diagnostic tests (61.9 and 66.7% using milk and serum samples, respectively). The P22 ELISA using milk samples demonstrated a similar sensitivity compared with serum samples, suggesting it might be a valuable test for TB control in dairy goats.

Specificity of serological test for detection of tuberculosis in cattle, goats, sheep and pigs under different epidemiological situations.

BACKGROUND: Serum antibody detection has potential as a complementary diagnostic tool in animal tuberculosis (TB) control, particularly in multi-host systems. The objective of the present study was to assess the specificity (Sp) of an enzyme-linked immunosorbent assay (ELISA) based on the new multiprotein complex P22 for the detection of specific antibodies against the Mycobacterium tuberculosis complex (MTC) in the four most relevant domestic animals acting as MTC hosts: cattle, goat, sheep and pig. We used sera from an officially TB-free (OTF) country, Norway, and from a non-OTF one, Spain. The samples included sera from goats that had been vaccinated against M. avium subsp. paratuberculosis (MAP) and sheep from a herd in which Corynebacterium pseudotuberculosis had been isolated. RESULTS: In cattle, the Sp ranged from 92.5 (IC95% 90.7-94) to 99.4% (IC95% 98.3-99.8) depending on the cut-off used and the origin of the samples (Spain or Norway). Sp in cattle (cut-off point 100) was significantly higher (P < 0.05) for Norwegian samples. By contrast, Sp in goats was consistently low at the 100 cut-off [30.9 (CI95%23.4-39.5)-78% (CI95% 68.9-85)]. A higher cut-off of 150 improved Sp in Norwegian goats [97% (CI95% 91.6-99)], but still yielded a poor Sp of 56.1% (CI95% 47.3-64.6) in Spanish goats. In Norway at the 100 cut-off the Sp was 58.3 (CI95% 42.2-72.9) and 90.6% (CI95% 81-95.6) in MAP vaccinated and non-vaccinated goats, respectively, indicating interference due to MAP vaccination. Sp in sheep was between 94.4 (CI95% 91.7-96.3) and 100% (CI95% 96.3-100) depending on the cut-off and country, and no diagnostic interference due to infection with C. pseudotuberculosis was recorded. Sp in pigs was 100%, regardless the cut-off point applied, and no significant differences were observed between pigs from Norway and from Spain. CONCLUSIONS: Due to its excellent Sp in pigs and acceptable Sp in cattle and sheep, this ELISA may constitute a suitable option for TB screening at herd level, particularly in OTF-countries.

A new test to detect antibodies against Mycobacterium tuberculosis complex in red deer serum.

Red deer (Cervus elaphus) farming is a growing economic activity worldwide. However, the capacity of this species to act as reservoir of animal tuberculosis (TB) poses a threat to other wildlife and to livestock. Diagnostic assay accuracy in this species is therefore highly relevant for prevention and control measures. Our aim was to evaluate the diagnostic performance of the protein complex P22, obtained from Mycobacterium bovis derived purified protein derivative (bPPD), as a candidate antigen for the detection of antibodies against Mycobacterium tuberculosis complex (MTC). We assessed the performance of this new antigen in indirect enzyme-linked immunosorbent assays (ELISA) in TB-positive and TB-negative red deer, in comparison with a bPPD-based ELISA. The P22 ELISA achieved a higher specificity (Sp) and similar sensitivity (Se) in comparison with the bPPD ELISA at all the cut-off points considered. The P22 ELISA yielded optimal Sp (99.02%; 95% confidence intervals [CI95%]: 96.5-99.8) and appropriate Se (70.1%; CI95%: 63.6-76) at the selected cut-off point of 100%. These results suggest that P22 can be used as an alternative antigen in the immunodiagnosis of animal TB through the use of an ELISA-type detection of antibodies against MTC in red deer, thus contributing to the diagnosis of animal TB in this species as a measure for further disease prevention and control programs.

Management of hunting waste as control measure for tuberculosis in wild ungulates in south-central Spain.

In recent decades, habitat change and the intensive management of wild ungulates for hunting have led to an increase in their populations in south-central Spain. This implies a higher generation of hunting waste, which can favour the transmission of infectious diseases, including tuberculosis (TB). The aim of this study was to assess the usefulness of the proper disposal of hunting waste as TB control measure in wild boar (Sus scrofa) and red deer (Cervus elaphus) during the 2008/2009 to 2016/2017 hunting seasons. Blood samples from 664 wild boar and 934 red deer were obtained in 14 game estates in two provinces in Andalusia (Area 1), where the disposal of hunting waste was implemented since the 2012/2013 hunting season. Besides, six game estates in the province of Ciudad Real, in Castilla-La Mancha (Area 2), an adjacent region where this management measure was not implemented during the studied period, were used as controls, sampling 277 wild boar and 427 red deer sera. The Mycobacterium tuberculosis complex (MTC), seroprevalence detected in wild boar from Area 1, was significantly higher before the disposal of big game hunting by-products (82.8%; 2008/2009-2012/2013) compared to the second period (61.8%; 2013/2014-2016/2017) (p < .001), after this control measure became established. By contrast, no significant differences between periods were found in wild boar (41.3% versus 44.8%; p = .33) and red deer (14.9% versus 11.6%; p = .19) from Area 2 as well as in red deer (10.8% versus 10.5%; p = .48) from Area 1. The proper disposal of hunting waste contributed to achieve a 25% reduction in MTC seroprevalence in wild boar. These results are of particular relevance regarding wild boar in the current context of re-emerging and emerging diseases such as TB and African Swine Fever in Europe. Further studies are needed to assess the effect of this measure on the health status of livestock and other wildlife species.