Evaluation of the Effect of a Recent Comparative Intradermal Tuberculin Test on the Humoral Diagnosis of Paratuberculosis Using Serum and Milk Samples from Goats.

Paratuberculosis (PTB) and tuberculosis (TB) are two mycobacterial diseases with a severe economic and health impact on domestic ruminants. The ante mortem diagnosis of PTB is hampered, among other factors, by the limited sensitivity of all the available diagnostic techniques. Since TB-infected goats subjected to the comparative intradermal tuberculin test (CITT) may experience a booster effect on their antibody titer and a potential enhancement to the sensitivity of humoral techniques for tuberculosis, in the present study we aimed to evaluate this diagnostic strategy on the humoral diagnosis of PTB in serum and milk samples collected from a caprine herd that was TB free and PTB infected. The results from 120 goats indicated a significant increase (p < 0.001) in the quantitative response detected using an ELISA technique, conducted using serum and milk samples taken 15 and 30 days after performing a CITT (day 0 of the study); although, it did not translate into a significant increase in the number of reactors during any of the testing events (0, 3,15, 30 and 60 days post-CITT). Additionally, the number of ELISA-positive animals was higher for the serum versus the milk samples at both 15 and 30 days post-CITT. The increase in the quantitative ELISA result suggested a diagnostic strategy that maximizes ELISA sensitivity, mainly using serum samples, in PTB-infected herds; although, it may depend on individual differences and the interpretation criteria.

Effect of a recent intradermal test on the specificity of P22 ELISA for the diagnosis of caprine tuberculosis.

Caprine tuberculosis (TB) is a zoonotic disease caused by members of the Mycobacterium tuberculosis complex. TB eradication programs in goats are based on the single and comparative intradermal tuberculin tests (SITT and CITT, respectively). Antibody-based diagnostic techniques have emerged as potential diagnostic tools for TB. P22 ELISA has been previously evaluated using samples collected after the intradermal tuberculin tests to maximize the sensitivity, a phenomenon known as booster effect. However, there is no information available on whether the use of this diagnostic strategy could lead to a decrease of its specificity (Sp). The aim of the present study was to elucidate the interference effect of a recent CITT on the Sp of the P22 ELISA in serum and milk samples collected at different times after the CITT from a TB-free herd (n = 113). The number of reactors to P22 ELISA was significantly higher (p < 0.01) on serum samples collected 15 days post-CITT compared to day 0, showing a decrease in Sp from 99.1% (95% CI; 95.2-99.8%) to 88.5% (95% CI; 81.3-93.2%). The number of reactors and the quantitative values of P22 ELISA were significantly higher (p < 0.01) in serum samples compared to milk. No significant (p > 0.05) changes in the Sp of the P22 ELISA were observed throughout the different time samplings using milk No significant (p > 0.05) changes were observed on days 30 and 60 post-CITT. In conclusion, the booster effect strategy may significantly decrease the Sp of P22 ELISA in TB-free herds when serum samples are used but not when milk is tested.

Non-tuberculous mycobacteria: occurrence in skin test cattle reactors from official tuberculosis-free herds.

Non-tuberculous mycobacteria (NTM) are considered a relevant cause of non-specific reactions to the most widely applied bovine tuberculosis (bTB) test, the intradermal tuberculin test. In order to establish which NTM species might act as a potential source of such diagnostic interference, a collection of 373 isolates obtained from skin test positive cows from 359 officially tuberculosis-free (OTF) herds, culled in the framework of the bTB eradication campaign in Spain, were identified at the species level through PCR and Sanger sequencing of the 16S rDNA, hsp65 and rpoB genes. Of the 308 isolates for which a reliable identification was achieved, 32 different mycobacterial species were identified, with certain species being most represented: among M. avium complex members (n = 142, 46.1%), M. avium subsp. hominissuis (98; 69.0%) was the most abundant followed by M. avium subsp. avium (33, 23.2%), and M. intracellulare (7, 4.9%). Among non-MAC members (n = 166, 53.9%), M. nonchromogenicum (85; 27.6%) and M. bourgelatii (11; 5.6%) were the predominant species. In addition, mixed results were obtained in 53 isolates presenting up to 30 different genotypes, which could be indicative of new mycobacterial species. Our results represent a first step toward characterizing the diversity of NTM species that could interfere with official diagnostic tests for bTB eradication in Spain.

Bovine tuberculosis in Spain, is it really the final countdown?

Bovine tuberculosis (bTB) is a severe zoonotic disease that has major impacts on both health and the economy, and which has been subjected to specific eradication programmes in many countries for decades. This manuscript highlights the relevance of this disease in the context of the European Union (EU) and summarizes the epidemiological situation and the main tools (e.g. antemortem diagnostic tests, slaughterhouse surveillance, laboratories, comprehensive databases, etc.) used to control and eradicate bTB in the various EU countries with a focus on the situation in Spain. A comprehensive description of the specific bTB epidemiological situation in Spain is provided, together with an assessment of the evolution of different epidemiological indicators throughout the last decades. Moreover, the main features of the Spanish bTB eradication programme and its control tools are described, along with the studies carried out in Spain that have allowed the updating of and improvement to the programme over the years with the aim of eradication, which has been established for 2030.

Evaluation of the performance of the IFN-γ release assay in bovine tuberculosis free herds from five European countries.

The diagnostic methods for granting and maintenance of the official tuberculosis-free (OTF) status and for intra-Community movement of cattle are the tuberculin skin tests (single or comparative) and the interferon-γ (IFN-γ) release assay (IGRA). However, until now, IGRAs have been primarily applied in infected farms in parallel to the skin test to maximize the number of infected animals detected. Therefore, an evaluation of the performance of IGRAs in OTF herds to assess whether if their specificity is equal to or higher than that of the skin tests is needed. For this, a panel of 4365 plasma samples coming from 84 OTF herds in six European regions (five countries) was assembled and analysed using two IGRA kits, the ID Screen® Ruminant IFN-g (IDvet) and the Bovigam™ TB Kit (Bovigam). Results were evaluated using different cut-offs, and the impact of herd and animal-level factors on the probability of positivity was assessed using hierarchical Bayesian multivariable logistic regression models. The percentage of reactors ranged from 1.7 to 21.0% (IDvet: S/P ≥ 35%), and 2.1-26.3% (Bovigam: ODbovis-ODPBS ≥ 0.1 and ODbovis-ODavium ≥ 0.1) depending on the region, with Bovigam disclosing more reactors in all regions. The results suggest that specificity of IGRAs can be influenced by the production type, age and region of origin of the animals. Changes in the cut-offs could lead to specificity values above 98-99% in certain OTF populations, but no single cut-off yielding a sufficiently high specificity (equal or higher than that of skin tests) in all populations was identified. Therefore, an exploratory analysis of the baseline IFN-γ reactivity in OTF populations could help to assess the usefulness of this technique when applied for the purpose of maintaining OTF status.

Once bitten twice shy: Risk factors associated with bovine tuberculosis recurrence in Castilla y Leon, Spain.

Persistence of bovine tuberculosis (bTB) in cattle herd remains a major challenge in disease elimination due to the ineffectual removal of all infected animals in a bTB breakdown. Characterization of herds with a higher probability of experiencing further bTB breakdowns can help to implement specific risk-based policies for disease control and eradication. Here, our aim was to identify herd- and breakdown-level risk factors in bTB infected herds in Castilla y Leon, Spain, associated with a decreased time to recurrence and an increased risk of recurrence using a mixed effects Cox proportional hazards model and a multivariable logistic regression model, respectively. Results revealed that location (province), herd size and number of incoming animals/contacts were good predictors of a decreased time to bTB recurrence and an increased risk of becoming a recurrent herd. Additionally, the duration of the previous outbreak and the number of IFN-γ herd-tests applied in it were associated with increased odds of (an early) recurrence. Risk factors identified here can be used for early identification of herds in which bTB eradication may be more challenging and that should thus be subjected to increased control efforts. The characterization of high-risk herds may help to minimize the risk of reinfection and emphasize early detection and removal of bTB positive animals in the herd.

Differences in skin test reactions to official and defined antigens in guinea pigs exposed to non-tuberculous and tuberculous bacteria.

The single and comparative intradermal tuberculin tests (SITT and CITT) are official in vivo tests for bovine tuberculosis (TB) diagnosis using bovine and avian purified protein derivatives (PPD-B and PPD-A). Infection with bacteria other than Mycobacterium tuberculosis complex (MTC) can result in nonspecific reactions to these tests. We evaluated the performance of the skin test with PPDs and new defined antigens in the guinea pig model. A standard dose (SD) of Rhodococcus equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis, M. avium subsp. avium, M. avium subsp. hominissuis, M. scrofulaceum, M. persicum, M. microti, M. caprae and M. bovis, and a higher dose (HD) of M. nonchromogenicum, M. monacense, M. intracellulare, M. avium subsp. paratuberculosis were tested using PPD-B, PPD-A, P22, ESAT-6-CFP-10-Rv3615c peptide cocktail long (PCL) and fusion protein (FP). The SD of R. equi, Nocardia sp., M. nonchromogenicum, M. monacense, M. intracellulare and M. avium subsp. paratuberculosis did not cause any reactions. The HD of M. nonchromogenicum, M. monacense, M. intracellulare, and M. avium subsp. paratuberculosis and the SD of M. avium subsp. hominissuis, M. scrofulaceum and M. persicum, caused nonspecific reactions (SIT). A CITT interpretation would have considered M. avium complex and M. scrofulaceum groups negative, but not all individuals from M. nonchromogenicum HD, M. monacense HD and M. persicum SD groups. Only animals exposed to M. bovis and M. caprae reacted to PCL and FP. These results support the advantage of complementing or replacing PPD-B to improve specificity without losing sensitivity.

A proof-of-concept study to investigate the efficacy of heat-inactivated autovaccines in Mycobacterium caprae experimentally challenged goats.

This study aimed to assess the efficacy of a heat-inactivated Mycobacterium caprae (HIMC) vaccine in goats experimentally challenged with the same strain of M. caprae. Twenty-one goats were divided into three groups of seven: vaccinated with heat-inactivated Mycobacterium bovis (HIMB), with HIMC and unvaccinated. At 7 weeks post-vaccination all animals were endobronchially challenged with M. caprae. Blood samples were collected for immunological assays and clinical signs were recorded throughout the experiment. All goats were euthanized at 9 weeks post-challenge. Gross pathological examination, analysis of lung pathology using computed tomography, and bacterial load quantification in pulmonary lymph nodes (LN) by qPCR were carried out. Only HIMC vaccinated goats showed a significant reduction of lung lesions volume and mycobacterial DNA load in LN compared to unvaccinated controls. Both vaccinated groups showed also a significant reduction of the other pathological parameters, an improved clinical outcome and a higher proportion of IFN-γ-producing central memory T cells after vaccination. The results indicated that homologous vaccination of goats with HIMC induced enhanced protection against M. caprae challenge by reducing lung pathology and bacterial load compared to the heterologous vaccine (HIMB). Further large-scale trials are necessary to assess the efficacy of autovaccines under field conditions.

Effect of a recent parenteral dexamethasone and ketoprofen administration on the immunological diagnosis of tuberculosis in goats.

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. Different factors may affect the performance of the TB diagnostic tests used in caprine herds and, therefore, their ability to detect infected animals. The present study evaluates the effect of the fraudulent administration of two anti-inflammatory substances, dexamethasone and ketoprofen, on the performance of the TB diagnostic techniques used in goats, as well as the suitability of high performance liquid chromatography (HPLC) for their detection in hair samples. The animals (n = 90) were distributed in three groups: (1) a group treated with dexamethasone (n = 30); a second group treated with ketoprofen (n = 30); and a third non-treated control group (n = 30). Both dexamethasone and ketoprofen groups were subjected to intramuscular inoculation with the substances 48 h after the administration of bovine and avian purified protein derivatives (PPDs), that is, 24 h before the tests were interpreted. All the animals were subjected to the single and comparative intradermal tuberculin (SIT and CIT, respectively) tests, interferon-gamma release assay (IGRA) and P22 ELISA. The number of SIT test reactors was significantly lower in the dexamethasone (p = 0.001) and ketoprofen (p < 0.001) groups 72 h after the bovine PPD inoculation compared with the control group. A significantly higher number of positive reactors to IGRA was detected within the dexamethasone group (p = 0.016) 72 h after PPD administration compared to the control group. Dexamethasone and ketoprofen detection in either hair or serum samples was challenging when using HPLC since these substances were not detected in animals whose skin fold thickness (SFT) was reduced, what could be an issue if they are used for fraudulent purposes. In conclusion, the parenteral administration of dexamethasone or ketoprofen 48 h after the PPDs administration can significantly reduce the increase in SFT (mm) and subsequently the number of positive reactors to SIT test.

Extracellular matrix proteins (fibronectin, collagen III, and collagen I) immunoexpression in goat tuberculous granulomas (Mycobacterium caprae).

The lesion resulting from the interaction between Mycobacterium and the host immune response is the tuberculous granuloma. Tuberculous granulomas, except in incipient stages, are partially or totally encapsulated by connective tissue. The aim of this study was to assess the immunoexpression of the extracellular matrix proteins fibronectin, collagen III, and collagen I in granulomas caused by Mycobacterium caprae in goats (Capra aegagrus hircus) to understand capsule development at different granuloma stages. For this purpose, a retrospective study of 56 samples of tuberculous granulomas in lung (n = 30) and mediastinal lymph node (n = 26) from 17 goats naturally infected with M. caprae in stages I (n = 15), II (n = 14) and III (n = 27) was carried out. Fibronectin immunoreaction was extracellular, fibrillar-reticular in the center of stage I, II and III granulomas and peripheral in stages II and III granulomas. Collagen III immunoexpression was extracellular and fibrillar in the center of stages I, II and III tuberculous granulomas in lung and mediastinal lymph node, and progressive expression was observed in the periphery of stages II and III granulomas. Finally, collagen I immunoexpression was extracellular and fibrillar, showing a progressive loss of central expression and an increase in peripheral expression in stage III granulomas compared to stage I granulomas. Immunoexpression of these extracellular matrix proteins could help understand fibrogenesis and dating in tuberculous granuloma in both animal models and humans.

Effect of heat-inactivated Mycobacterium avium subspecies paratuberculosis (MAP) vaccine on the lesions and immunopathology developed in target tissues of naturally MAP-infected goats.

Paratuberculosis (PTB) is a disease caused by Mycobacterium avium subspecies paratuberculosis (MAP), which affects a broad range of hosts, including domestic and wild animals. PTB is a chronic granulomatous enteritis and lymphadenitis that compromises animal welfare and causes economic losses. The objective of this study was to evaluate the effect of a commercial heat-inactivated MAP vaccine on lesions and immunopathology developed in the target tissues of goats naturally infected with MAP. Lesions compatible with PTB in the intestine and regional lymph nodes (LNs), as well as local immune response to MAP, were evaluated and compared in Gudair®-vaccinated (n = 14) and unvaccinated (n = 11) goats from a MAP-infected farm. The percentage of animals with multifocal granulomatous lesions in the jejunal (p = 0.05) and ileocecal (p = 0.02) LNs was higher in unvaccinated animals, while a lesion score reduction of 50% was found in the LNs of vaccinated animals. Unvaccinated animals showed increased numbers and wider distribution of macrophages (MΦs, CD68 +) in histiocytic infiltrate (p = 0.0003), associated with increased numbers of mycobacteria. Increased inducible nitric oxide synthase (iNOS) expression was also reported in these animals, while M2 MΦs (CD163 +) were scarce in both groups. Vaccinated animals showed an increase in CD3 + lymphocytes, although differences in interferon gamma (IFNγ) were negligible. These results support the hypothesis that heat-inactivated MAP vaccination could reduce the severity of PTB lesions and mycobacterial load in target tissues in vaccinated adult goats.

Use of Whole-Genome Sequencing to Unravel the Genetic Diversity of a Prevalent Mycobacterium bovis Spoligotype in a Multi-Host Scenario in Spain.

Despite the efforts invested in the eradication of bovine tuberculosis in Spain, herd prevalence has remained constant in the country during the last 15 years (~1.5-1.9%) due to a combination of epidemiological factors impairing disease control, including between-species transmission. Here, our aim was to investigate the molecular diversity of Mycobacterium bovis isolates belonging to the highly prevalent SB0339 spoligotype in the cattle-wildlife interface in different regions of Spain using whole-genome sequencing (WGS). Genomic data of 136 M. bovis isolates recovered from different animal species (cattle, wild boar, fallow deer, and red deer) and locations between 2005 and 2018 were analyzed to investigate between- and within-species transmission, as well as within-herds. All sequenced isolates differed by 49-88 single nucleotide polymorphisms from their most recent common ancestor. Genetic heterogeneity was geographic rather than host species-specific, as isolates recovered from both cattle and wildlife from a given region were more closely related compared to isolates from the same species but geographically distant. In fact, a strong association between the geographic and the genetic distances separating pairs of M. bovis isolates was found, with a significantly stronger effect when cattle isolates were compared with wildlife or cattle-wildlife isolates in Spain. The same results were obtained in Madrid, the region with the largest number of sequenced isolates, but no differences depending on the host were observed. Within-herd genetic diversity was limited despite the considerable time elapsed between isolations. The detection of closely related strains in different hosts demonstrates the complex between-host transmission dynamics present in endemic areas in Spain. In conclusion, WGS results a valuable tool to track bTB infection at a high resolution and may contribute to achieve its eradication in Spain.

MALDI-TOF Mass Spectrometry as a Rapid Screening Alternative for Non-tuberculous Mycobacterial Species Identification in the Veterinary Laboratory.

Non-tuberculous mycobacteria (NTM) are difficult to identify by biochemical and genetic methods due to their microbiological properties and complex taxonomy. The development of more efficient and rapid methods for species identification in the veterinary microbiological laboratory is, therefore, of great importance. Although MALDI-TOF Mass Spectrometry (MS) has become a promising tool for the identification of NTM species in human clinical practise, information regarding its performance on veterinary isolates is scarce. This study assesses the capacity of MALDI-TOF MS to identify NTM isolates (n = 75) obtained from different animal species. MALDI-TOF MS identified 76.0% (n = 57) and 4% (n = 3) of the isolates with high and low confidence, respectively, in agreement with the identification achieved by Sanger sequencing of housekeeping genes (16S rRNA, hsp65, and rpoB). Thirteen isolates (17.3%) were identified by Sanger sequencing to the complex level, indicating that these may belong to uncharacterised species. MALDI-TOF MS approximated low confidence identifications toward closely related mycobacterial groups, such as the M. avium or M. terrae complexes. Two isolates were misidentified due to a high similarity between species or due to the lack of spectra in the database. Our results suggest that MALDI-TOF MS can be used as an effective alternative for rapid screening of mycobacterial isolates in the veterinary laboratory and potentially for the detection of new NTM species. In turn, Sanger sequencing could be implemented as an additional method to improve identifications in species for which MALDI-TOF MS identification is limited or for further characterisation of NTM species.

Benchtop nuclear magnetic resonance-based metabolomic approach for the diagnosis of bovine tuberculosis.

Even though enormous efforts and control strategies have been implemented, bovine tuberculosis (TB) remains a significant source of health and socioeconomic concern. The standard method used in TB eradication programs for in vivo detection is the tuberculin skin test. However, the specificity of the tuberculin skin test is affected by infection with non-tuberculous mycobacteria or by vaccination. Thus, some animals are not correctly diagnosed. This study aimed first to identify a plasma metabolic TB profile by high-field (HF) nuclear magnetic resonance (NMR) spectroscopy and second measure this characteristic TB metabolic profile using low-field benchtop (LF) NMR as an affordable molecular technology for TB diagnosis. Plasma samples from cattle diagnosed with TB (derivation set, n = 11), diagnosed with paratuberculosis (PTB, n = 10), PTB-vaccinated healthy control (n = 10) and healthy PTB-unvaccinated control (n = 10) were analyzed by NMR. Unsupervised Principal Component Analysis (PCA) was used to identify metabolic differences between groups. We identified 14 metabolites significantly different between TB and control animals. The second group of TB animals was used to validate the results (validation set, n = 14). Predictive models based on metabolic fingerprint acquired by both HF and LF NMR spectroscopy successfully identified TB versus control subjects (Area under the curve of Receiver Operating Characteristic over 0.92, in both models; Confidence Interval 0.77-1). In summary, plasma fingerprinting using HF and LF-NMR differentiated TB subjects from uninfected animals, and PTB and PTB-vaccinated subjects who may provide a TB-false positive, highlighting the use of LF-NMR-based metabolomics as a complementary or alternative diagnostic tool to the current diagnostic methods.

Effect of the Inoculation Site of Bovine and Avian Purified Protein Derivatives (PPDs) on the Performance of the Intradermal Tuberculin Test in Goats From Tuberculosis-Free and Infected Herds.

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.

Evaluation of P22 ELISA for the Detection of Mycobacterium bovis-Specific Antibody in the Oral Fluid of Goats.

The ante-mortem diagnosis of tuberculosis (TB) in ruminants is based mainly on the intradermal tuberculin test and the IFN-γ assay. Antibody (Ab)-based tests have emerged as potential tools for the detection of TB infected animals using serum, plasma, or even milk samples. Oral fluids have also been evaluated as alternative samples with which to detect specific Abs against Mycobacterium bovis in pigs or wild boars, but not in ruminants. The objective of this study was, therefore, to evaluate the performance of an in house-ELISA for TB diagnosis (P22 ELISA) in goats as an experimental model for the diagnosis of TB using oral fluid samples. Oral fluid samples from 64 goats from a TB-infected herd (n = 197) and all the animals from a TB-free herd (n = 113) were analyzed using the P22 ELISA. The estimated sensitivity (Se) and specificity (Sp) were 34.4% (95% CI: 22.4-45.6) and 100% (95% CI: 97.4-100), respectively. The optimal cut-off point was set at 100% according to the ROC analysis. Those animals with a higher level of Abs in their oral fluid attained a higher lesion score (p = 0.018). In fact, when taking into account only the setting of the animals with severe lesions (n = 16), the ELISA showed a Se of 75% (95% CI: 53.7-96.2). Results of the present study suggest that the P22 ELISA is highly specific but has a limited value detecting infected animals in oral fluid samples. Nevertheless, its performance is significantly higher in the presence of severe lesions.

Spoligotype-specific risk of finding lesions in tissues from cattle infected by Mycobacterium bovis.

BACKGROUND: Although the pathogenic effect of members of the Mycobacterium tuberculosis complex in susceptible hosts is well known, differences in clinical signs and pathological findings observed in infected animals have been reported, likely due to a combination of host and pathogen-related factors. Here, we investigated whether Mycobacterium bovis strains belonging to different spoligotypes were associated with a higher risk of occurrence of visible/more severe lesions in target organs (lungs and/or lymph nodes) from infected animals. A large collection of 8889 samples belonging to cattle were classified depending on the presence/absence of tuberculosis-like lesions and its degree of severity. All samples were subjected to culture irrespective of the presence of lesions, and isolates retrieved were identified and subjected to spoligotyping. The association between the presence/severity of the lesions and the isolation of strains from a given spoligotype was assessed using non-parametric tests and Bayesian mixed multivariable logistic regression models that accounted for origin (region and herd) effects. RESULTS: Results suggested a difference in severity in lesioned samples depending on the strain's spoligotype. An association between specific spoligotypes and presence of lesions was observed, with a higher risk of finding lesions in animals infected with strains with spoligotypes SB0120, SB0295 and SB1142 compared with SB0121, and in those coming from certain regions in Spain. CONCLUSIONS: Our results suggest that strains belonging to certain spoligotypes may be associated with a higher probability in the occurrence of gross/macroscopic lesions in infected cattle, although these observational findings should be confirmed in further studies that allow accounting for the effect of other possible confounders not considered here, and ultimately through experimental studies.

Evaluation of the performance of slaughterhouse surveillance for bovine tuberculosis detection in Castilla y Leon, Spain.

Post-mortem inspection (PMI) of routinely slaughtered cattle in abattoirs is an extremely valuable tool for detecting bovine tuberculosis (bTB) infected herds that can supplement active surveillance activities. However, its true performance is difficult to assess due to the multiple factors that may affect it. Here, we determined relative efficiencies in the detection of bTB-compatible lesions and probabilities of subsequent laboratory confirmation of abattoirs located in Castilla y Leon, one of the regions with the largest cattle population in Spain, between 2010 and 2017. The slaughtered animal population was split based on the results of the ante-mortem tests (reactors or non-reactors), and two generalized linear multivariable mixed models were fitted to each subpopulation to calculate the risk of lesion detection and laboratory confirmation per abattoir while accounting for the effect of potential confounding variables. Throughout the 8-year period, ∼30,000 reactors and >2.8 million non-reactor animals in the ante-mortem tests were culled in the abattoirs under study. Bovine TB compatible lesions were detected in 4,710 (16%) reactors and 828 (0.03%) non-reactor animals, of which >95% were confirmed as infected through bacteriology. The probability of disclosure of bTB-like lesions was associated with the animal subpopulation, type of source unit, the herd size, the year of slaughter, the breed and age of the animal, and/or the season of slaughter. The probabilities of detection of bTB-like lesions varied largely depending on the abattoir in both subpopulations, ranging from 603 to 3,070 per 10,000 animals for the reactors and 0.2-16.1 per 10,000 animals for the non-reactor animals. Results obtained here will help to quantify the performance of PMI in abattoirs in Castilla y Leon and the between-abattoir variability, and to identify animals at increased risk of having bTB-like lesions detected during PMI based on animal- and farm-related factors.

Efficacy of a Salmonella enterica serovar Abortusovis (S. Abortusovis) inactivated vaccine in experimentally infected gestating ewes.

Salmonella enterica serovar Abortusovis (S. Abortusovis) infection is one of the most important causes of infectious late-term abortion as well as birth of weak lambs in sheep in many countries throughout the world. Implementation of protocols based on the application of effective vaccines is one of the most effective approaches for controlling this disease, but variable efficacy has been reported, possibly related to factors associated with the host, the vaccine, the parameters used for determining efficacy and the challenge protocols. In this context, a new commercial inactivated vaccine (INMEVA; Laboratorios Hipra S.A., Spain) was evaluated in 20 control and 17 vaccinated gestating ewes, subcutaneously challenged at 90 days of gestation with 5 × 106 colony-forming units (cfu) of a wild strain of S. Abortusovis. Incidence of reproductive failures, bacterial vaginal excretion (by real time PCR), and lamb survival were evaluated as indicators of the vaccine's level of protection. Moreover, humoral response (by ELISA test in serum samples) was studied. Vaccination was showed to be safe under the study conditions. Vaccine efficacy was demonstrated in two different ways: i) it significantly decreased the percentage of abortions [29.4% (5/17) in the vaccinated group compared to the control group (65%; 13/20)] and ii) there was a significant reduction of the overall vaginal excretion in the sampling period (3.05 log cfu/mL ±â€¯0.84 in the vaccinated group vs. 5.68 ±â€¯0.67 in the control group). Given these results, the vaccine evaluated can be considered as an effective alternative for controlling S. Abortusovis infection in ovine flocks.

Evaluation of Risk Factors Associated With Herds With an Increased Duration of Bovine Tuberculosis Breakdowns in Castilla y Leon, Spain (2010-2017).

The persistence of bovine tuberculosis (bTB) in certain cattle herds is a major concern in countries pursuing disease eradication worldwide. The chronic nature of the disease, the lack of performance of diagnostic tools, and the presence of wildlife reservoirs may lead infected herds to require longer periods to achieve the officially tuberculosis-free (OTF) status. Here, we evaluated the impact of farm and breakdown characteristics on the probability of disease persistence in infected farms in Castilla y Leon, a bTB-endemic region of Spain, using survival and logistic regression models. Data from bTB breakdowns occurring in 3,550 bTB-positive herds detected in 2010-2017 were analyzed. A multivariable Cox proportional hazards model was fitted using time to recover OTF status as the response variable, and a multivariable logistic regression model using the chronic status (yes/no) for herds experiencing particularly long breakdowns as the outcome variable was also used. Both analyses revealed that county-level bTB herd prevalence, herd size, number of incoming animals in the previous 3 years, number of skin test reactors in the disclosing test, and number of days between the disclosing and follow-up tests were associated with increased breakdown duration. Production type was not consistently associated with chronic infection, suggesting that once infected, it is not a significant predictor of outbreak duration beyond the initial stages of the breakdown. Province-level location and number of animals that are bacteriology-positive also affected significantly the expected herd breakdown duration, but their effect became less significant over time. Risk factors identified in this study may help to identify herds more prone to suffer chronic bTB infection that may require additional control measures early on in a breakdown.