Comparative Pathology of the Natural infections by Mycobacterium bovis and by Mycobacterium caprae in Wild Boar (Sus scrofa)

The potential role of wild animals in the maintenance and spread of tuberculosis (TB) infection in domestic livestock is of particular importance in countries where eradication programs have substantially reduced the incidence of bovine tuberculosis but sporadic outbreaks still occur. Mycobacterium bovis is the agent mainly isolated in wildlife in Spain, but recently, infections by Mycobacterium caprae have increased substantially. In this study, we have analysed 43 mandibular lymph nodes samples containing TB‐like lesions from 43 hunted wild boar from Madrid and Extremadura (central and south‐western regions of Spain). After isolation, identification and typing of Mycobacterium tuberculosis complex isolates, we found that 23 mandibular lymph nodes involved M. caprae infections and 20 M. bovis. The lesions were compared for histopathology (different granuloma stage and number of multinucleated giant cells (MNGCs)), and acid‐fast bacilli (AFBs) were quantified in the Ziehl‐Neelsen‐stained slides. Granulomas produced by M. caprae showed more stage IV granulomas, more MNGCs and higher AFBs counts than those induced by M. bovis. In conclusion, lesions caused by M. caprae would be more prone to the excretion of bacilli, and infected animals result as a high‐risk source of infection for other animals.     

Factors that Influence Mycobacterium bovis Infection in Red Deer and Wild Boar in an Epidemiological Risk Area for Tuberculosis of Game Species in Portugal

Bovine tuberculosis (bTB) is a worldwide zoonotic disease of domestic and wild animals. Eradication has proved elusive in those countries with intensive national programmes but with ongoing transmission between wildlife and cattle. In Portugal, a high‐risk area for bTB was defined and specific measures implemented to assess and minimize the risk from wildlife. Data from the 2011 to 2014 hunting seasons for red deer (Cervus elaphus) and wild boar (Sus scrofa) were analysed with bovine demographic and bTB information to assess factors that determined the occurrence and distribution of bTB in both species. The likelihood of bTB‐like lesions in wild boar was positively associated with density of red deer, wild boar and cattle, while for red deer, only their density and age were significant factors. The likelihood of Mycobacterium bovis isolation in wild boar was associated with density of cattle and red deer and also with the anatomical location of lesions, while for red deer, none of the variables tested were statistically significant. Our results suggest that, in the study area, the role of red deer and wild boar may be different from the one previously suggested by other authors for the Iberian Peninsula, as red deer may be the driving force behind M. bovis transmission to wild boar. These findings may assist the official services and game managing bodies for the management of hunting zones, what could also impact the success of the bTB eradication programme.     

Seroprevalence of Mycobacterium bovis infection in warthogs (Phacochoerus africanus) in bovine tuberculosis‐endemic regions of South Africa

Bovine tuberculosis (bTB ), caused by Mycobacterium bovis (M. bovis), has been reported in many species including suids. Wild boar are important maintenance hosts of the infection with other suids, that is domestic and feral pigs, being important spillover hosts in the Eurasian ecosystem and in South Africa, warthogs (Phacochoerus africanus ) may play a similar role in M. bovis‐ endemic areas. However, novel diagnostic tests for warthogs are required to investigate the epidemiology of bTB in this species. Recent studies have demonstrated that serological assays are capable of discriminating between M. bovis ‐infected and uninfected warthogs (Roos et al., 2016 ). In this study, an indirect ELISA utilizing M. bovis purified protein derivative (PPD ) as a test antigen was used to measure the prevalence and investigate risk factors associated with infection in warthogs from uM hkuze Nature Reserve and the southern region of the Greater Kruger National Park (GKNP ). There was a high overall seroprevalence of 38%, with adult warthogs having a higher risk of infection (46%). Seroprevalence also varied by geographic location with warthogs from Marloth Park in the GKNP having the greatest percentage of positive animals (63%). This study indicates that warthogs in M. bovis ‐endemic areas are at high risk of becoming infected with mycobacteria. Warthogs might present an under‐recognized disease threat in multi‐species systems. They might also serve as convenient sentinels for M. bovis in endemic areas. These findings highlight the importance of epidemiological studies in wildlife to understand the role each species plays in disease ecology.     

Development of a Gene Expression Assay for the Diagnosis of Mycobacterium bovis Infection in African Lions (Panthera leo)

Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis‐exposed, M. bovis‐unexposed, M. tuberculosis‐exposed and M. bovis‐infected lions was incubated in QuantiFERON^®‐TB Gold (QFT) tubes containing either saline or ESAT‐6/CFP‐10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen‐dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT‐6/CFP‐10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut‐off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis‐infected and M. bovis‐uninfected lions.     

Non‐tuberculous Mycobacteria in Wild Boar (Sus scrofa) from Southern Spain: Epidemiological,Clinical and Diagnostic Concerns

Non‐tuberculous mycobacteria (NTM) are widely distributed in the environment, particularly in wet soil, marshland, rivers or streams, but also are causative agents of a wide variety of infections in animals and humans. Little information is available regarding the NTM prevalence in wildlife and their effects or significance in the bovine tuberculosis (bTB) epidemiology and diagnosis. This research shows the most frequently NTM isolated in lymph nodes of wild boar (Sus scrofa) from southern Spain, relating the NTM presence with the individual characteristics, the management of animals and the possible misdiagnosis of Mycobacterium bovis in concurrent infections. A total of 219 NTM isolates were obtained from 1249 wild boar mandibular lymph nodes sampled between 2007 and 2011. All but 75 isolates were identified by the PCR‐restriction analysis‐hsp65, and a partial sequencing of the 16S rDNA was carried out to identify the rest of the isolates. Results showed that Mycobacterium chelonae was the most frequently isolated NTM specie (133 isolates, 60.7%), followed by Mycobacterium avium (24 isolates, 11%). No relation was found regarding sex, body condition and management, but M. chelonae was more frequently detected in adults, whereas M. avium was more prevalent in subadults. The high NTM prevalence observed in the studied wild boar populations could make difficult the bTB diagnostic.     

Upregulation of IL‐17A,CXCL9 and CXCL10 in Early‐Stage Granulomas Induced by Mycobacterium bovis in Cattle

To gain further insight into the immunopathogenesis of bovine tuberculosis (bTB), the cytokine and chemokine expression of cattle experimentally infected with Mycobacterium bovis was analysed in TB granulomas, using immunohistochemistry (IHC) and laser capture microdissection (LCM) followed by qPCR. Immunohistochemistry was conducted for cell types using labelling for CD68, CD3, CD4, CD8, WC1 and CD79a and for the cytokines IFN‐γ, TNF‐α and TGF‐β as well as inducible form of nitric oxide synthase (iNOS). qPCR was conducted for mRNA expression of IFN‐γ, TNF‐α, TGF‐β, IL‐17A, IL‐22, IL‐2, granzyme A and the chemokines CXCL9 and CXCL10. Early stages of granuloma were primarily comprised of epithelioid MΦs expressing high levels of IFN‐γ and iNOS, with significantly upregulated expression of CXCL9 and CXCL10 when compared with control tissue. These chemokines displayed a trend of decreasing mRNA expression as lesion progressed, suggesting a higher level of importance during the early stages of the immune response to mycobacterial infection. IL‐22 levels showed a strong trend of decrease through granuloma development, and IL‐17A was shown to be upregulated, supporting its investigation as a potential biomarker of bTB. The use of LCM and qPCR may prove especially useful for the study of IL‐17A as previous attempts to analyse its expression using IHC and in situ hybridization proved unsuccessful.     

Spatiotemporal and Ecological Patterns of Mycobacterium microti Infection in Wild Boar (Sus scrofa)

Mycobacterium microti has recently been described as the causative agent of tuberculosis‐like lesions in wild boar (Sus scrofa), a reservoir specie of Mycobacterium tuberculosis complex (MTBC) in some European Mediterranean ecosystem. Through a five‐year survey on tuberculosis in free‐living wild boars, the epidemiological trend of M. microti infections and the host and population risk factors linked with its occurrence were described. Retropharyngeal and mandibular lymph nodes of 3041 hunted wild boars from six different districts were macroscopically inspected. The sex and age of each animal were registered, as well as the animal abundance in each district. Lesions compatible with tuberculosis (190) were collected and analysed using a gyrB PCR–RFLP assay. M. microti was identified directly in 99 tissue samples (Prev = 3.26%; 95% CI: 2.67–3.97%), while neither Mycobacterium bovis, nor other members of the MTBC were detected. The probability of being M. microti positive showed spatio‐temporal variability, with 26% of increase of risk of being infected for each year. Moreover, a positive effect of wild boar abundance and age on the prevalence was detected. The generalized increase in the European wild boar population, coupled with its sensitivity to M. microti infection, poses a future concern for the identification and management of MTBC members in wild boar.     

Disease Control in Wildlife: Evaluating a Test and Cull Programme for Bovine Tuberculosis in African Buffalo

Providing an evidence base for wildlife population management is difficult, due to limited opportunities for experimentation and study replication at the population level. We utilized an opportunity to assess the outcome of a test and cull programme aimed at limiting the spread of Mycobacterium bovis in African buffalo. Buffalo act as reservoirs of M. bovis, the causative agent of bovine tuberculosis (BTB), which can have major economic, ecological and public health impacts through the risk of infection to other wildlife species, livestock and surrounding communities. BTB prevalence data were collected in conjunction with disease control operations in Hluhluwe–iMfolozi Park, South Africa, from 1999 to 2006. A total of 4733 buffalo (250–950 per year) were tested for BTB using the single comparative intradermal tuberculin (SCIT) test, with BTB‐positive animals culled, and negative animals released. BTB prevalence was spatially and temporally variable, ranging from 2.3% to 54.7%. Geographic area was a strong predictor of BTB transmission in HiP, owing to relatively stable herds and home ranges. Herds experiencing more intensive and frequent captures showed reduced per capita disease transmission risk and less increase in herd prevalence over time. Disease hot spots did not expand spatially over time, and BTB prevalence in all but the hot spot areas was maintained between 10% and 15% throughout the study period. Our data suggest that HiP's test and cull programme was effective at reducing BTB transmission in buffalo, with capture effort and interval found to be the crucial components of the programme. The programme was thus successful with respect to the original goals; however, there are additional factors that should be considered in future cost/benefit analyses and decision‐making. These findings may be utilized and expanded in future collaborative work between wildlife managers, veterinarians and scientists, to optimize wildlife disease control programmes and mitigate conflict at the interface of conservation, agricultural and urban areas.     

Liver fluke (Fasciola hepatica) co‐infection with bovine tuberculosis (bTB) in cattle: A retrospective animal‐level assessment of bTB risk in dairy and beef cattle

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, remains a persistent problem for cattle industries in endemic countries. The frequency, quality, and performance of tests, and the presence of wildlife reservoirs, have been identified as impediments to eradication. Recently, exposure to helminth infection (Fasciola hepatica) has been associated negatively with the disclosure of bTB. Here, for the first time, we assess impact of concurrent infections of Fasciola hepatica and the disclosure of bTB at the animal‐level using large surveillance datasets. We utilized a dataset of 138,566 animal records from an abattoir from Northern Ireland (2011–2013). The presence of F. hepatica infection was assessed from macroscopic tissue inspection at abattoir. Multivariable models were developed to assess co‐infection associations with bTB status based on: Single Intradermal Comparative Tuberculin Test (SICTT), lesion, bacteriological confirmation, including either all animals, or only skin‐test negative animals (lesions at routine slaughter; LRS; confirmed nonreactors at routine slaughter; cNRs) or positive (reactors) animals alone, respectively. The relationship between skin tuberculin reaction sizes and fluke status was also explored for a subset of animals with field recordings (n = 24,680). Controlling for known risk factors (e.g., climatic, herd, and individual level characteristics), we did not find significant associations between the SICTT (standard or severe interpretation), lesion, nor confirmation status of animals and their liver fluke status. The only exception was a negative association between liver fluke positivity, and LRS or cNRs, respectively; though effect‐sizes were small (e.g., LRS Odds‐Ratio: 0.87; 95% CI: 0.76–1.00). There was limited evidence of a relationship between tuberculin reaction sizes during SICTT testing and liver fluke infection status. These data do not support the contention that the detection of bTB using skin‐tests or reactor postmortem follow‐up may be compromised by co‐infection at a population level, but the relationship with lesion formation (pathogenesis) may indicate an impact for postmortem surveillance.     

Persistence of Mycobacterium bovis bacillus Calmette–Guérin (BCG) Danish In White‐tailed Deer (Odocoileus virginianus) Vaccinated with a Lipid‐Formulated Oral Vaccine

Mycobacterium bovis, the causative agent of tuberculosis in animals, has a broad host range, including humans. Historically, public health concerns prompted programs to eradicate tuberculosis from cattle in many nations. Eradication efforts decreased the prevalence of bovine tuberculosis; nevertheless, some countries encountered significant obstacles, not least of which was a wildlife reservoir of M. bovis. Efforts to decrease the size of the affected wildlife populations have neither eliminated disease nor eliminated transmission to cattle. Consequently, the use of a vaccine for wildlife is being explored. The vaccine most studied is M. bovis BCG, an attenuated live vaccine, first developed 100 years ago. The most efficient and effective means of vaccinating wildlife will be an oral vaccine. White‐tailed deer in Michigan, USA, constitute a reservoir of M. bovis. White‐tailed deer are a popular game species, and as such, represent a food animal to many hunters. BCG persistence in deer tissues could result in human exposure to BCG. Although non‐pathogenic, BCG exposure could induce false‐positive skin test results, confounding the central component of public health surveillance for TB. The objective of the present study in white‐tailed deer was to evaluate persistence of lipid‐encapsulated BCG and a liquid suspension of BCG after oral administration at two different dosages. Vaccine was not recovered at any time after oral consumption of a bait containing a single dose (1 × 10⁸ CFU) of lipid‐encapsulated BCG. However, persistence was consistent in deer consuming 10 lipid‐encapsulated baits (1 × 10⁹ CFU), with BCG recovered from at least one deer at 1, 3, 6, 9 and 12 months after consumption. Persistence of up to 9 months was seen in deer vaccinated with orally with a liquid suspension. Persistence of BCG was limited to lymphoid tissue and never found in samples of muscle collected at each time point. Although the risk of exposure to hunters is low, BCG persistence should be considered prior to field use in white‐tailed deer.     

First detection of Mycobacterium bovis infection in Giraffe (Giraffa camelopardalis) in the Greater Kruger National Park Complex: Role and implications

Bovine tuberculosis (bovine TB) caused by Mycobacterium bovis has become endemic in some wildlife populations in South Africa. The disease has been reported in 21 wildlife species in the country. In this study, we report M. bovis infection in two female giraffes (Giraffa camelopardalis) from two different nature reserves within the Greater Kruger National Park Complex (GKNPC). Mycobacterium bovis was isolated from tissue lesions consistent with macroscopic appearance of tuberculosis (TB) and confirmed by polymerase chain reactions (PCRs), targeting the RD4 region of difference on the genome of the isolates. Spoligotyping and variable number of tandem repeat (VNTR) typing revealed infection of one giraffe with a strain (SB0294) previously not detected in South Africa, while a resident M. bovis strain (SB0121) was detected from the other giraffe. Our work is first to report M. bovis infection in free‐ranging giraffes in South Africa. We have further demonstrated the existence of at least three genetically unrelated strains currently infecting wildlife species within the GKNPC. This finding suggests that the epidemiological situation of M. bovis within the GKNPC is not only driven by internal sources from its established endemic presence, but can be additionally fuelled by strains introduced from external sources. It further emphasizes that regular wildlife disease surveillance is an essential prerequisite for the timely identification of new pathogens or strains in ecospheres of high conservation value.     

Isolation and Potential for Transmission of Mycobacterium bovis at Human–livestock–wildlife Interface of the Serengeti Ecosystem,Northern Tanzania

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), is a multihost pathogen of public health and veterinary importance. We characterized the M. bovis isolated at the human–livestock–wildlife interface of the Serengeti ecosystem to determine the epidemiology and risk of cross‐species transmission between interacting hosts species. DNA was extracted from mycobacterial cultures obtained from sputum samples of 472 tuberculosis (TB) suspected patients and tissue samples from 606 livestock and wild animal species. M. bovis isolates were characterized using spoligotyping and Mycobacterial Interspersed Repetitive Units‐Variable Tandem Repeats (MIRU‐VNTR) on 24 loci. Only 5 M. bovis were isolated from the cultured samples. Spoligotyping results revealed that three M. bovis isolates from two buffaloes (Syncerus caffer) and 1 African civet (Civettictis civetta) belonged to SB0133 spoligotype. The two novel strains (AR1 and AR2) assigned as spoligotype SB2290 and SB2289, respectively, were identified from indigenous cattle (Bos indicus). No M. bovis was detected from patients with clinical signs consistent with TB. Of the 606 animal tissue specimens and sputa of 472 TB‐suspected patients 43 (7.09%) and 12 (2.9%), respectively, yielded non‐tuberculous mycobacteria (NTM), of which 20 isolates were M. intracellulare. No M. avium was identified. M. bovis isolates from wildlife had 45.2% and 96.8% spoligotype pattern agreement with AR1 and AR2 strains, respectively. This finding indicates that bTB infections in wild animals and cattle were epidemiologically related. Of the 24 MIRU‐VNTR loci, QUB 11b showed the highest discrimination among the M. bovis strains. The novel strains obtained in this study have not been previously reported in the area, but no clear evidence for recent cross‐species transmission of M. bovis was found between human, livestock and wild animals.     

Molecular epidemiology of Mycobacterium tuberculosis complex strains isolated from livestock and wild animals in Italy suggests the need for a different eradication strategy for bovine tuberculosis

Bovine tuberculosis (bTB ) is an important zoonosis, which has been re‐emerging in different ecological scenarios. In Sicily, Italy, from 2004 to 2014, an anatomopathological survey for tuberculosis‐like lesions both in farmed and wild animals was performed. The isolates were genotyped using spoligotyping and Mycobacterial Interspersed Repetitive Units‐Variable Number of Tandem Repeats (MIRU ‐VNTR ) techniques. High prevalence of lesions was observed for cattle (4%), pigs (4.9%) and wild boars (6.8%), and a total of 625 Mycobacterium bovis isolates were identified. Genotyping analysis showed the presence of 37 different spoligotypes including fifteen spoligotypes not present in other Italian regions and 266 MIRU ‐VNTR profiles. Spoligotype SB 0120 exhibited the highest prevalence in cattle (50%) and pigs (56%) and the highest genetic variety with 126 different MIRU ‐VNTR profiles. The isolation of M. bovis in a farmer underlines the importance of M. bovis identification during the human TB diagnostic processes. This study supported the use of the genotyping analysis as a valuable tool for the evaluation of the epidemiological role of pigs and other domestic reservoirs such as goats and the role of wildlife in the maintenance of bTB infection.     

Transboundary tuberculosis: Importation of alpacas infected with Mycobacterium bovis from the United Kingdom to Poland and potential for serodiagnostic assays in detecting tuberculin skin test false‐negative animals

The present study highlights the transboundary nature of tuberculosis (TB) in alpacas and the failure of current antemortem testing protocols to identify TB‐free alpaca herds and individuals for exportation. The tuberculin skin test (TST) failed to identify Mycobacterium bovis‐infected animals prior to movement from the United Kingdom (UK) to Poland. This study describes the use of four serological assays [Enferplex Camelid TB, dual‐path platform (DPP) VetTB and BovidTB assays, and multi‐antigen print immunoassays (MAPIAs)] to detect TB in an alpaca herd with negative TST results. The breeding in Poland purchased alpacas for several years from the UK with the last group arriving in May 2018. In July 2018, two sick alpacas from the centre were hospitalized in a veterinary clinic and both died of TB a few weeks later. In November 2018, 20 alpacas remaining in this M. bovis‐affected herd were euthanized and samples were collected. The study population included 20 M. bovis‐infected and 20 uninfected alpacas, but only 15 infected animals were tested by all serology tests. The DPP VetTB and DPP BovidTB assays detected antibodies in 14 of the 20 infected alpacas, with results confirmed by MAPIA, and in none (MAPIA and DPP BovidTB) or one (DPP VetTB) of the 20 uninfected animals. None of the infected alpacas tested positive using the Enferplex assay. In addition, the group included three orphans and two cria–dam pairs, which provided an opportunity to analyse immune aspects of cria–mother relationships in this herd. The results suggest high susceptibility of this host species to M. bovis infection and rapid progression to disease. The serological tests used in this study offer useful tools for the detection of M. bovis infection in TST and Enferplex test non‐reactive alpacas. These tests should be further evaluated for implementation into TB management and control strategies for camelid species.     

Animal tuberculosis: Impact of disease heterogeneity in transmission,diagnosis and control

Animal tuberculosis (TB) in terrestrial mammals is mainly caused by Mycobacterium bovis. This pathogen is adapted to a wide range of host species, representing a threat to livestock, wildlife and human health. Disease heterogeneity is a hallmark of multi‐host TB and a challenge for control. Drivers of animal TB heterogeneity are very diverse and may act at the level of the causative agent, the host species, the interface between mycobacteria and the host, community of hosts, the environment and even policy behind control programmes. In this paper, we examine the drivers that seem to contribute to this phenomenon. We begin by reviewing evidence accumulated to date supporting the consensus that a complex range of genetic, biological and socio‐environmental factors contribute to the establishment and maintenance of animal TB, setting the grounds for heterogeneity. We then highlight the complex interplay between individual, species‐specific and community protective factors with risk/maintenance variables that include animal movements and densities, co‐infection and super‐shedders. We finally consider how current interventions should seek to consider and explore heterogeneity in order to tackle potential limitations for diagnosis and control programmes, simultaneously increasing their efficacy.     

Tuberculosis in Southern Brazilian wild boars (Sus scrofa): First epidemiological findings

Bovine tuberculosis (bTB ) is a zoonosis caused mainly by Mycobacterium bovis that affects domestic and wild animals. In Brazil, there are no epidemiological studies on tuberculosis in wild animal populations and their possible role in the disease maintenance in cattle herds; thus, the aim of this study was to evaluate the occurrence of tuberculosis in wild boars in Rio Grande do Sul, southern Brazil. Tissue samples of animals hunted under government consent were submitted to histopathology and M. bovis polymerase chain reaction (PCR ) as screening tests; the positive samples were subsequently submitted to bacterial isolation, the gold standard diagnosis. Eighty animals were evaluated, of which 27.9% and 31.3% showed histopathological changes and M. bovis genome presence, respectively. Moreover, 23.8% of the animals had at least one organ with isolates classified as Mycobacterium tuberculosis complex (MTC ). Three hunting points were risk factors for positive results on screening tests. This study shows the occurrence of tuberculosis in a wild boars’ population, and raise the possibility of these animals to play a role as disease reservoirs in southern Brazil. These results may help to improve the Brazilian tuberculosis control programme, as well as elucidate the circulation of mycobacteria in this country.     

Distribution of Mycobacterium avium subsp. paratuberculosis in a Subclinical Naturally Infected German Fleckvieh Bull

Although it has been known for years that Mycobacterium avium subsp. paratuberculosis (MAP) is detectable in the reproductive organs and semen of infected bulls, only few studies have been conducted on this topic worldwide. This study surveyed the MAP status of a bull, naturally infected due to close contact with its subclinically infected parents over a period of 4 years. From the age of 7 weeks to necropsy, faecal, blood and, after sexual maturity, semen samples were drawn repeatedly. Already at the first sampling day, MAP‐DNA was detected in faeces by semi‐nested PCR. True infection was confirmed by the detection of MAP‐DNA in blood at the age of 40 weeks. In total, MAP‐DNA was present in 25% faecal (34/139), 16% blood (23/140) and 5% semen (4/89) samples, including MAP‐free intervals of up to 9 weeks. MAP genome equivalents (MAP‐GE) of up to 6.3 × 10⁶/g faeces and 1.8 × 10⁵/ml blood were determined. Cultivation of MAP occurred only in three of 137 faecal and two of 109 blood, but never in semen samples. Over the whole period, the bull was a serological negative MAP shedder. During necropsy, 42 tissue samples were collected. Neither macroscopic nor histological lesions characteristic of a MAP infection were observed. Cultivation of MAP in tissue sections failed. However, MAP‐DNA was spread widely in the host, including in tissues of the lymphatic system (7/15), digestive tract (5/14) and the urogenital tract (5/9) with concentrations of up to 3.9 × 10⁶ MAP‐GE/g tissue. The study highlighted the detection of MAP in male reproductive organs and semen. It supports the hypothesis that bulls may probably transmit MAP, at least under natural mating conditions. In artificial insemination, this might not be relevant, due to antibiotics included currently in semen extenders. However, the survivability of MAP in this microenvironment should be investigated in detail.     

Environmental Presence of Mycobacterium tuberculosis Complex in Aggregation Points at the Wildlife/Livestock Interface

The members of the Mycobacterium tuberculosis complex (MTC) cause tuberculosis (TB). Infection is transmitted within and between livestock and wildlife populations, thus hampering TB control. Indirect transmission might be facilitated if MTC bacteria persist in the environment long enough to represent a risk of exposure to different species sharing the same habitat. We have, for the first time, addressed the relationship between environmental MTC persistence and the use of water resources in two TB endemic areas in southern Spain with the objective of identifying the presence of environmental MTC and its driving factors at ungulates’ water aggregation points. Camera‐trap monitoring and MTC diagnosis (using a new MTC complex‐specific PCR technique) were carried out at watering sites. Overall, 55.8% of the water points tested positive for MTC in mud samples on the shore, while 8.9% of them were positive in the case of water samples. A higher percentage of MTC‐positive samples was found at those waterholes where cachectic animals were identified using camera‐trap monitoring, and at the smallest waterholes. Our results help to understand the role of indirect routes of cross‐species TB transmission and highlight the importance of certain environmental features in maintaining infection in multihost systems. This will help to better target actions and implement control strategies for TB at the wildlife/livestock interface.     

Highly sensitive nested PCR and rapid immunochromatographic detection of Babesia bovis and Babesia bigemina infection in a cattle herd with acute clinical and fatal cases in Argentina

Bovine babesiosis is a tick‐transmitted haemoparasitic disease caused by Babesia bovis and B. bigemina affecting cattle of tropical and subtropical regions around the world. Pathogens are transmitted by the tick vector Rhipicephalus microplus displaying a widespread distribution in northeastern Argentina. The disease is characterized by significant animal morbidity and mortality resulting in considerable economic loss. In this study, B. bovis and B. bigemina infection was investigated in a cattle herd of 150 adult bovines of pure Braford breed raised in a tick‐hyperendemic field using molecular and serum antibody tests. A highly sensitive nested polymerase chain reaction (nPCR) assay targeting a species‐specific region of the apocytochrome b gene resulted in direct B. bovis and B. bigemina detection in 27.3% and 54.7% of bovines, respectively. A recently developed immunochromatographic strip test (ICT) based on recombinant forms of spherical body protein 4 and the C‐terminal region of rhoptry‐associated protein 1 showed that 71.3% and 89.3% of bovines were seropositive for B. bovis and B. bigemina, respectively. The mixed infection rate as observed by direct (19.3%) and indirect detection (65.3%) coincided with those expected, respectively. Importantly, four months after sampling, nine bovines of the studied herd showed clinical signs of bovine babesiosis of which six animals eventually died. Microscopic detection of infected erythrocytes in Giemsa‐stained blood smears confirmed B. bovis infection. Our study demonstrates that although animals showed a relatively high and very high rate of immunity against infection with B. bovis (71.3%) and B. bigemina (89.3%) parasites, respectively, clinical cases and fatalities due to the infection with B. bovis were observed. It is proposed that the most adequate control measure in the studied epidemiological situation is to vaccinate animals to prevent losses and/or an outbreak of bovine babesiosis.