José Antonio Infantes-Lorenzo

Background: Caprine tuberculosis (cTB), caused by members of the Mycobacterium tuberculosis complex (MTBC) represents a significant concern for animal and public health. Antibody-based diagnostic tests have emerged as promising tools to complement ante-mortem official diagnostic techniques in ruminants, which mainly use serum, although alternative, easier-to-collect samples, such as milk, oral fluid, or feces, could also be used. The objective of this exploratory study was to evaluate the reactivity of an in-house P22 enzyme-linked immunosorbent assay (ELISA) in fecal samples from goats belonging to cTB-infected herds, using serum P22 ELISA and official diagnostic tests as comparators. Methods: Serum and fecal samples were collected from 51 and 42 adult female goats from a high-prevalence herd (n = 149) and a low-prevalence herd (n = 220), respectively, which defecated during sampling. These animals were subjected to official diagnostic techniques: single and comparative intradermal tuberculin tests and the interferon-gamma release assay. As no TB-free herd was available, a preliminary fecal cut-off was established by receiver operating characteristic analysis using serum P22 ELISA as the reference classification. Results: The percentage of positive animals in fecal samples from the high-prevalence herd was 68.6%, while no positive results were observed in the low-prevalence herd. Serum samples showed a higher number of positives in both herds compared to fecal samples (p < 0.05). The agreement between official diagnostic techniques and P22 ELISA (using serum or feces) was slight in general terms. When a combined classification based on all official tests was used as the reference, fecal P22 ELISA detected 58.0% of test-positive goats, none of them in the low-prevalence herd. Conclusions: Under the conditions of this field study (no TB-free controls or post-mortem confirmation of infection were available), fecal P22 ELISA showed limited reactivity compared with serum, particularly in the low-prevalence herd, suggesting limited usefulness for antibody detection in TB infected goats.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects various mammalian species, with farmed minks experiencing the highest number of outbreaks. In Spain, we analyzed 67 whole genome sequences and eight spike sequences from 18 outbreaks, identifying four distinct lineages: B.1, B.1.177, B.1.1.7, and AY.98.1. The potential risk of transmission to humans raises crucial questions about mutation accumulation and its impact on viral fitness. Sequencing revealed numerous not-lineage-defining mutations, suggesting a cumulative mutation process during the outbreaks. We observed that the outbreaks were predominantly associated with different groups of mutations rather than specific lineages. This clustering pattern by the outbreaks could be attributed to the rapid accumulation of mutations, particularly in the ORF1a polyprotein and in the spike protein. Notably, the mutations G37E in NSP9, a potential host marker, and S486L in NSP13 were detected. Spike protein mutations may enhance SARS-CoV-2 adaptability by influencing trimer stability and binding to mink receptors. These findings provide valuable insights into mink coronavirus genetics, highlighting both host markers and viral transmission dynamics within communities.

Background: The European bison (Bison bonasus), a symbol of Polish nature, is a protected species that requires active health monitoring. However, conservation efforts are made difficult by the zoonotic diseases such as brucellosis and tuberculosis.Objective: The aim of this study was to screen the Polish European bison population for exposure to the Mycobacterium tuberculosis complex (MTC) and Brucella spp.Methods: A total of 323 free‐living and captive European bison from 13 localities were tested serologically for antibodies against the M. bovis P22 multi‐protein complex (in‐house ELISA) and against Brucella spp. (commercial ELISA).Results: Antibodies against the MTC (P22) were detected in 7% (22/323) of the tested European bison. Anti‐MTC antibody positivity was not significantly different by sex, age, and captive/free range status. Anti‐MTC antibodies were found in six of 13 populations sampled, always in populations with larger sample sizes including the four free‐living ones. Antibodies against Brucella spp. were detected in 36% (116/323) of the tested bison. While Brucella spp. antibody prevalence was not different by sex, it was significantly different by age (lower in adults) and captive/free‐living status. Brucella spp. seroprevalence decreased with sample size and seropositive bison were found in 12 of 13 sampling populations.Conclusions: Our findings identify potential emerging threats to the European bison population and confirm the first serological response to P22 in European bison. As Poland is currently officially free of brucellosis and bovine tuberculosis, our results require careful interpretation. Further studies are needed to establish the presence of cross‐reactions with atypical mycobacteria in the case of MTC and other bacteria (e.g. Yersinia enterocolitica O:9) in the case of Brucella spp.

The single and comparative intradermal tuberculin (SIT and CIT) tests are used for the ante-mortem diagnosis of caprine tuberculosis (TB). The tuberculin injection site has been associated with a different performance of the test in cattle. In contrast to that required in cattle in Europe (cervical injection), it can be carried out in the scapular region in goats. Nevertheless, there are no previous data concerning the effect of the injection site on the performance of the test in goats. The aim of the present study was to evaluate the effect of two different inoculation sites (cervical and scapular) on the performance of the SIT/CIT tests. This was done by intradermally inoculating 309 goats from two infected herds and one TB-free herd with both avian and bovine PPDs in the mid-cervical and scapular regions. None of the animals from the TB-free herd had positive reactions, and the number of reactors was not significantly higher, regardless of the inoculation site, in the high and low prevalence herds. However, significantly higher increases in skin fold thickness were observed on the cervical site when compared to the scapular site after the avian and bovine PPD inoculations in the TB-free herd (p < 0.001) and after the bovine PPD injection in the high prevalence herd (p = 0.003). The presence of clinical signs was also more evident on the cervical site when using avian and bovine PPDs in the high prevalence herd (p < 0.01). In contrast, increases in higher skin fold thickness were observed on the scapular site when compared to the cervical site after the bovine and avian PPD inoculations were employed in the low prevalence herd (p < 0.01). These results suggest that the cervical injection of PPDs may improve the sensitivity of the intradermal tuberculin test in high TB prevalence caprine herds, mainly owing to the increased presence of local clinical signs and a better performance of the CIT test. Moreover, specificity was not affected when using standard interpretations, although further analyses in a great number of herds are required in order to confirm these findings.

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 aMycobacterium capraeinfection 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 ofM. 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 theM. tuberculosiscomplex. 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 foundM. capraeinfection in OWC. This is the first report ofM. capraeinfection 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.

Current eradication strategies of tuberculosis (TB) in goats resemble bovine TB eradication programs and mainly relay on single and comparative intradermal tuberculin tests (SITT and CITT). TB vaccination has been proposed as a cost-effective option in high prevalent herds or countries were economic compensation for slaughter positive animals is not affordable. However, TB vaccination compromises the efficiency of TB standard diagnostic tests. In this study, the performance of a new diagnostic platform, based on the P22 antigenic complex, was assessed for skin test (ST), Interferon gamma release assay (IGRA) and serology under different TB scenarios. The sensitivity (Se) of diagnostic tests was assessed in TB infected goats from the same farm (herd A, N=77) and the specificity (Sp) was assessed in two TB negative farms, one TB unvaccinated (herd B, N=77) and another vaccinated with BCG (herd C, N= 68). The single (s) P22-IGRA showed the highest Se among IGRA tests (91%) and the comparative (c) P22-ST showed the highest Sp (100% in Herd B and 98% in Herd C). Combined interpretation of techniques enabled the best diagnostic performances. Combining the CITT + sP22-IGRA improved Se (97%) compared to CITT + tuberculin-based-IGRA (95%), with a reduction of Sp (95% and 100%, respectively). In addition, combination of P22-ELISA + cP22-ST or CITT elicited similar performance in the non-vaccination context (Se: 94% and 95%; Sp: 95% and 95%, respectively) but Sp was significantly higher in the combination with cP22-ST compared to CITT in vaccination context (95% and 79%, respectively). The combination of serological tests based on P22 and MPB83 showed higher complementarity and improved 13% the Se of P22-ELISA alone. These findings suggest that either cell-mediated or antibodies based diagnostic techniques, using the P22 antigen complex, can contribute to improve the immunodiagnostic of TB in goats under different TB control strategies.

Attaining and maintaining the Official Tuberculosis Free status continues to be a challenge when several domestic and wild hosts contribute to the maintenance of the Mycobacterium tuberculosis complex (MTC). Local tuberculosis hotspots are sometimes identified in cattle in low-prevalence regions. We have, therefore, studied one such hotspot in depth in order to produce an epidemiological diagnosis. Host population size and MTC prevalence were estimated in selected wildlife and in livestock, while on-cattle environmental DNA detection was additionally used as a proxy for risk of exposure at the farm (herd) level. Positive skin test reactors were found on16 of the 24 cattle farms studied in the period 2012–2016. Although all goats tested negative to the skin test during this period, MTC was confirmed in four sheep at slaughter, thus indicating an unknown prevalence of infection in this host species. With regard to wildlife, the prevalence of MTC infection based on culture was 8.8% in the case of wild boar (Sus scrofa), and the only road-killed badger (Meles meles) submitted for culture tested positive. Two criteria were employed to divide the cattle farms into higher or lower risk: tuberculosis testing results and environmental DNA detection. Environmental MTC DNA detection yielded significant differences regarding “use of regional pastures” and “proximity to woodland”. This study suggests that on-animal environmental DNA sampling may help when assessing contact risk as regards MTC in livestock at the herd level. This tool opens up new avenues of epidemiological research in complex multi-host settings.