Bacteriological culture and direct PCR for detecting the Mycobacterium tuberculosis complex in the Italian eradication campaign: a decade of experience at the National Reference Laboratory.

AIMS: Our study evaluates the capacity of direct real-time PCR for detecting Mycobacterium tuberculosis complex (MTBC), with a focus on diagnostic performances and the feasibility of implementing this protocol in an eradication campaign. Specifically, we compare the effectiveness of the direct PCR method to various culture systems used by the Italian National Reference Laboratory over the last decade to detect MTBC. METHODS AND RESULTS: Bovine tissue samples were routinely tested and analyzed for bovine tuberculosis (bTB) confirmation using microbiological culture (solid and liquid media), histopathological analysis, and a direct PCR assay targeting IS6110, an insertion sequence specific to the MTBC that is widely used for tuberculosis diagnosis. The direct real-time PCR demonstrated a high concordance (K = 0.871) with microbiological culture, as well as good sensitivity (91.84%) and specificity (95.24%). In contrast, histopathology demonstrated lower concordance (K = 0.746) and performance levels (sensitivity 91.41%, specificity 82.88%). Liquid media promoted faster and more efficient growth of MTBC than solid media. M. bovis and M. caprae had the comparable ability to respond to the direct real-time PCR test and grow on the microbiological medium. CONCLUSIONS: This study confirms that direct real-time PCR can detect MTBC with high diagnostic accuracy within a few days. This study found no significant differences in performance between culture media and direct PCR for M. bovis and M. caprae.

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.

Geo-epidemiology of animal tuberculosis and Mycobacterium bovis genotypes in livestock in a small, high-incidence area in Sicily, Italy.

Introduction: The persistence of animal tuberculosis (TB) in livestock is a major concern in Sicily, Italy. The objective of this study was to elucidate the transmission dynamics of M. bovis infection in a highly circumscribed, and at the same time geographically diverse, high-risk area of the island through an in-depth geo-epidemiological investigation of TB in cattle and black pigs raised in small-scale extensive farms across the district of Caronia. Methods: We used genotype analysis coupled with geographic information system (GIS) technology and phylogenetic inference to characterize the spatial distribution of TB and M. bovis genotypes in livestock and the genetic relationships between M. bovis isolates. A total of 589 M. bovis isolates collected from slaughtered cattle (n = 527) and Sicilian black pigs (n = 62) over a 5-year period (2014-2018) were included in the study. Results: TB was widespread throughout the district and was most frequent in the north-central area of the district, especially along one of the district's streams. We identified a total of 62 M. bovis genotypes. Identical genetic profiles were isolated from both neighboring and non-neighburing herds. The 10 most frequent genotypes, accounting for 82% of M. bovis isolates, showed geographic specificities in that they tended to cluster in specific spatial niches. The landscape structure of these niches-i.e. steep slopes, rocky ridges, meadows and streams-is likely to have had a significant influence on the distribution of TB among livestock in Caronia. Higher concentrations of TB were observed along streams and in open meadows, while rocky ridges and slopes appeared to have hampered the spread of TB. Discussion: The geographical distribution of TB cases among livestock in Caronia is consistent with several epidemiological scenarios (e.g., high density of infected herds along the streams or in hilly plateau where livestock share pastures). Landscape structure is likely to play an important role in the transmission and persistence of M. bovis infection across the district. Additional potential risk factors, such as livestock trading and extensive breeding methods, are also discussed. Our results will contribute to the improvement of surveillance, control and eradication activities of TB in Sicily by the implementation of ad hoc TB control measures, especially in farms located along streams, sharing common pastures or with mixed animal species.

Development and evaluation of a multi-antigen serological assay for the intra-vitam diagnosis of Tuberculosis caused by Mycobacterium bovis in pigs.

Bovine Tuberculosis (bTB) is a chronic disease caused by Mycobacterium bovis, affecting cattle and other mammalian species, such as pigs. In the present work, we developed a novel multi-antigen assay (The TB-Luminex multiplex test) to diagnose bTB in pig sera. Moreover, we investigated the seroreactivity to the different antigens employed (MPB83, MPB70, CFP10 and ESAT6) and the possible correlation with bTB lesions distribution in the positive pigs. The serum samples were collected from 59 bTB positive pigs and 186 pigs reared in an officially Tuberculosis free area. Sera were processed according to an optimized protocol for the detection of antibodies by a multiantigen assay using Luminex technology. The positive group showed visible lesions with localized (54.2%) or generalized (45.8%) distribution. Culture confirmed the infection in 62.7% of the cases, and histopathology and intra-vitam assays were used as additional confirmatory tests. Within the set of antigens tested, the immunodominant was MPB83 (positive in 94.9% of the affected pigs), followed by CFP10, MPB70 and ESAT6 (positivity shown in 81.3%, 67.8% and 25.4% of the positive pigs tested, respectively). The best antigens combination was MPB83/CFP10, with a 96.6% sensitivity and 96.8% specificity. Overall, the test showed high sensitivity (98.3% and 86.4%) and specificity (96.2% and 97.8%), if sera were considered positive according to the positivity to a single antigen or at least two antigens, respectively. The TB-Luminex multiplex test results did not give significantly different outcomes according to lesions distribution. Given the present study results, the TB-Luminex multiplex test is a reliable test capable of detecting bTB in most infected pigs with good Se and Sp, regardless of the stage of the disease. In conclusion, multi-antigen tests can be used as individual tests and screening tools for domestic and wild suids within bTB eradication programs.

Influence of Anthropic Environmental-Related Factors on Erysipelas in Wild Boar.

Erysipelothrix rhusiopathiae (ER) is an old but still emerging zoonotic infection that is not yet completely understood. ER infects a wide range of species and wild boar is of significant interest because of their similarities to pigs, a known ER reservoir. Moreover, the increase of its densities and the limited data available about ER in this species should be considered. The need is to investigate whether wild boar could represent a risk of erysipelas at the wildlife-domestic-human interface. Here, 1067 sera and 149 tonsils of wild boar from five hunting districts in Northwest Italy were tested using ELISA and bacteriological culture, respectively. Using generalized linear models, we evaluated host and environmental factors influencing ER spread and dynamics. We found an ER seroprevalence of 69.4% among wild boar. Increased human density and pig farm density lead to an increase of ER seropositivity highlighting its association with anthropic environmental-related factors. The high ER percentage of isolation (34.2%) found in healthy wild boar suggests that this species can serve as a healthy carrier. This fact, together with the high seroprevalence, supports a role of wild boar as an ER reservoir. Potential zoonotic and economic risks should be considered in light of these data.

Evaluation of the Presence and Viability of Mycobacterium bovis in Wild Boar Meat and Meat-Based Preparations.

The aim of the present study is to provide information about the ability of Mycobacterium bovis to survive within wild boar (Sus scrofae) meat and meat-based preparations and the duration of this survival, and to consider the preservation of its infectious potential toward humans and animals. Meat samples were artificially contaminated with an M. bovis field strain and then stored at -20 °C, while two sausages batches were contaminated with the same field strain at two different concentrations, 105 CFU/g and 103 CFU/g, before storing them in proper conditions to allow for their ripening. A third sausage batch was contaminated by adding 2 g of wild boar lymph nodal tissue with active tuberculous lesions to the meat mixture. Bacteriological and biomolecular (PCR) methods were used to test the meat and sausage samples every 60 days and every 7-10 days, respectively. M. bovis was detected as still alive and viable on the frozen meat for the last test on the 342nd day, while from the sausage samples, M. bovis was isolated until 23 days after contamination. Our results indicate that M. bovis can stay alive and be viable for 23 days within sausages prepared with contaminated meat from infected wild boars. These products are usually eaten as fresh food after grilling, often cooking at a temperature that does not ensure complete inactivation of the pathogenic microorganisms present, which can pose a risk for humans to develop zoonotic tuberculosis.

Mycobacterium microti at the Environment and Wildlife Interface.

An unexpected high presence of Mycobacterium microti in wild boar in Northern Italy (Garda Lake) has been reported since 2003, but the factors contributing to the maintenance of this pathogen are still unclear. In this study, we investigated the presence of M. microti in wild rodents and in water and soil samples collected at wild boar aggregation areas, such as watering holes, with the aim of clarifying their role in M. microti transmission. In total, 8 out of 120 captured animals tested positive for the Mycobacterium tuberculosis complex (MTBC) as assessed by real-time PCR, and six samples were confirmed to be M. microti. A strain with a genetic profile similar to those previously isolated in wild boars in the same area was isolated from one sample. Of the 20 water and 19 mud samples, 3 and 1, respectively, tested positive for the presence of MTBC, and spacer oligotype SB0118 (vole type) was detected in one sample. Our study suggests that wild rodents, in particular Apodemus sylvaticus, Microtus sp. and Apodemus flavicollis, play roles in the maintenance of M. microti infections in wild boar through ingestion or by contact with either infected excreta or a contaminated environment, such as at animal aggregation sites.

Mycobacterium microti Infection in Wild Boar (Sus scrofa): Histopathology Analysis Suggests Containment of the Infection.

The European wild boar (WB) (Sus scrofa) population has rapidly expanded over the years, raising public health concerns over the species reservoir of several pathogens, including Mycobacterium microti (Mm), a Mycobacterium tuberculosis complex member. In this study, we aimed to investigate the Mm natural infection in WB in Lombardy and Emilia Romagna Italian regions by statistically evaluating the granulomatous lesions' histological features and Mm microbiological isolation. We analyzed 103 WB retropharyngeal and submandibular lymph nodes (LNs) for Mm identified by gyrB PCR-restriction fragment length polymorphism, and were retrospectively selected and histologically assessed. For each sample, Hematoxylin-eosin and Ziehl-Neelsen stained slides were evaluated. Considered histological variables were: the number of granulomas, size and maturational stage of granulomas, granulomas completeness within the section, number of multinucleated giant macrophages (MGMs), and acid-fast (AF) bacilli per granuloma. Furthermore, Mm microbiological results were also considered. Mm microbiological isolation was negatively influenced by granulomas maturation and positively affected by AF bacilli's presence within the section. Granuloma maturation was positively influenced by granuloma size and granuloma incompleteness and negatively affected by the number of granulomas in the section and the number of MGMs within the granuloma. The results indicate that granuloma maturation should ensures an efficient containment of Mm infection in the WB, suggesting that the intra-species transmission of the disease might be an unlikely event.

A new nomenclature for the livestock-associated Mycobacterium tuberculosis complex based on phylogenomics.

Background:  The bacteria that compose the Mycobacterium tuberculosis complex (MTBC) cause tuberculosis (TB) in humans and in different animals, including livestock. Much progress has been made in understanding the population structure of the human-adapted members of the MTBC by combining phylogenetics with genomics. Accompanying the discovery of new genetic diversity, a body of operational nomenclature has evolved to assist comparative and molecular epidemiological studies of human TB. By contrast, for the livestock-associated MTBC members, Mycobacterium bovis, M. caprae and M. orygis, there has been a lack of comprehensive nomenclature to accommodate new genetic diversity uncovered by emerging phylogenomic studies. We propose to fill this gap by putting forward a new nomenclature covering the main phylogenetic groups within M. bovis, M. caprae and M. orygis. Methods:  We gathered a total of 8,736 whole-genome sequences (WGS) from public sources and 39 newly sequenced strains, and selected a subset of 829 WGS, representative of the worldwide diversity of M. bovis, M. caprae and M. orygis. We used phylogenetics and genetic diversity patterns inferred from WGS to define groups. Results:  We propose to divide M. bovis, M. caprae and M. orygis in three main phylogenetic lineages, which we named La1, La2 and La3, respectively. Within La1, we identified several monophyletic groups, which we propose to classify into eight sublineages (La1.1-La1.8). These sublineages differed in geographic distribution, with some being geographically restricted and others globally widespread, suggesting different expansion abilities. To ease molecular characterization of these MTBC groups by the community, we provide phylogenetically informed, single nucleotide polymorphisms that can be used as barcodes for genotyping. These markers were implemented in KvarQ and TB-Profiler, which are platform-independent, open-source tools. Conclusions:  Our results contribute to an improved classification of the genetic diversity within the livestock-associated MTBC, which will benefit future molecular epidemiological and evolutionary studies.

Field Evaluation of the Interferon Gamma Assay for Diagnosis of Tuberculosis in Water Buffalo (Bubalus bubalis) Comparing Four Interpretative Criteria.

Bovine tuberculosis (bTB) is a worldwide zoonosis that affects many species of domestic and wild animals. Mycobaterium bovis is the main cause of infection in water buffalo (Bubalus bubalis) and bovines and is of great concern for human health and for buffalo producers in Italy. The bTB eradication programme is based on slaughterhouse surveillance and intradermal skin tests. Other in vivo diagnostic methods such as the interferon-gamma (IFN-γ) assay have been developed and are widely used in cattle to accelerate the elimination of bTB positive animals. The present study is the first to assess the use and performance of IFN-γ assays, which is used as an ancillary test for bTB diagnosis in water buffalo, and presents the results of a field-evaluation of the assay from 2012 to 2019 during the buffalo bTB eradication programme in Italy. The study involved 489 buffaloes with a positive result to the single intradermal tuberculin test (SITT). The IFN-γ assays and single intradermal comparative tuberculin test were used as confirmation tests. Then, a total of 458 buffaloes, reared on officially tuberculosis-free (OTF) herds, that were confirmed bTB-free for at least the last 6 years were subjected to IFN-γ testing. Furthermore, to evaluate the IFN-γ test in an OTF herd with Paratuberculosis (PTB) infection, 103 buffaloes were subjected to SITT and IFN-γ test simultaneously. Four interpretative criteria were used, and the IFN-γ test showed high levels of accuracy, with sensitivity levels between 75.3% (CI 95% 71.2-79.0%) and 98.4% (CI 95% 96.7-99.4%) and specificity levels between 94.3% (CI 95% 91.2-96.50%) and 98.5% (CI 95% 96.9-99.4%), depending on the criterion used. Finally, in the OTF herd with PTB infection, in buffalo, the IFN-γ test displayed high specificity values according to all 4 interpretative criteria, with specificity levels between 96.7% (CI 95% 88.4-99.5%) and 100% (CI 95% 96.2-100%), while SITT specificity proved unsatisfactory, with a level of 45.3% (CI 95% 35.0-55.7%). Our results showed that the IFN-γ test in the buffalo species could reach high Sensitivity and Specificity values, and that the level of Sensitivity and Specificity could be chosen based on the interpretative criterion and the antigens used depending on the health status of the herd and the epidemiological context of the territory. The IFN-γ test and the use of different interpretative criteria proved to be useful to implement bTB diagnostic strategies in buffalo herds, with the possibility of a flexible use of the assay.

Infection by Mycobacterium caprae in three cattle herds in Emilia-Romagna Region, Northern Italy.

Bovine tuberculosis (bTB) is a contagious chronic disease associated with progressive emaciation (starvation) and tubercles (granuloma) formation commonly caused by Mycobacterium bovis. In cattle, M. caprae may also be responsible for bTB. In EU, human tuberculosis due to M. bovis had a notification rate of 0.04 cases per 100,000 inhabitants in 2017, but data did not include M. caprae infections. From September 2018 to April 2019, bTB outbreaks were investigated in three neighbouring dairy cattle herds in Parma province, Northern Italy. Parma municipality belongs to an officially free of bovine tuberculosis (OTF) Italian region. Official testing on cattle herds, performed every three years as legally required, revealed no positive animals. Tubercular lesions were found during the post mortem (PM) examination of slaughtered cattle and M. caprae genotype SB0418/VNTR 4,3,5,3,4,5,2,2,4, 3,15,5 was isolated. This report confirms the crucial importance of PM veterinary inspection at slaughterhouse, despite the OTF status of cattle herds.

Acute lameness in a cat with disseminated mycobacteriosis.

Mycobacterium avium infection was diagnosed in an adult cat showing acute lameness of the right hind limb, enlargement of the right popliteal lymph node and two cutaneous nodular lesions of the right chest wall. Conventional radiography of the proximal tibia showed a proliferative osteolytic lesion. Cytological examination of the right popliteal lymph node and the nodular skin lesions fine needle aspiration smears, demonstrated granulomatous inflammation with many negative staining bacilli within macrophages or in smears background. The diagnosis was confirmed by Ziehl­Neelsen staining of the smears and the identification of mycobacteria was performed by microbiological and molecular methods. Histopathology performed after the necropsy revealed disseminated mycobacteriosis with granulomatous mesenteric lymphadenitis, granulomatous pneumonia, hepatitis and tibial osteomyelitis. M. avium is a well­known agent of gastro­enteric, respiratory or disseminated disease in immunocompromised cats but there are few cases reported in literature of bone involvement in systemic mycobacteriosis.

Genotype diversity and distribution of Mycobacterium bovis from livestock in a small, high-risk area in northeastern Sicily, Italy.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis is an important re-emerging disease affecting livestock, wildlife and humans. Epidemiological studies are crucial to identifying the source of bTB infection, and its transmission dynamics and host preference, and thus to the implementation of effective strategies to contain it. In this study, we typed M. bovis isolates from livestock, and investigated their genetic diversity and distribution. A total of 204 M. bovis isolates were collected from cattle (n = 164) and Sicilian black pigs (n = 40) reared in a limited area of the province of Messina, northeastern Sicily, an area that had previously been identified as having the highest incidence of bTB in livestock on the island. All M. bovis isolates were typed by both spoligotyping and 12-loci MIRU-VNTR analysis. Results from both methods were then combined in order to improve the discriminatory power of M. bovis typing. We identified 73 combined genetic profiles. Thirty-five point six percent of the profiles were common to at least two animals, whereas 64.4% of profiles occurred in only one animal. A number of genetic profiles were predominant in either cattle or black pigs. We identified common genetic patterns in M. bovis isolates originating not only from neighboring districts, but also from non-neighboring districts. Our findings suggest that bTB is widespread in our setting, and is caused by a large number of genetically diverse M. bovis strains. The ecology and farming practices characteristic of the area may explain the substantial M. bovis heterogeneity observed, and could represent obstacles to bTB eradication.

Systemic tuberculosis by MYCOBACTERIUM BOVIS in a free-ranging MARSICAN brown bear (URSUS ARCTOS MARSICANUS): a Case report.

BACKGROUND: Mycobacterium bovis is known to have a wide host range and has been isolated from numerous free-ranging wildlife species, carnivores included. In bears, M. bovis has been previously reported only from a culture of pooled lymph nodes of a black bear (Ursus americanus) in the absence of lesions. The aims of this study were to describe gross and microscopic pathological findings of M. bovis tuberculosis in a deceased Marsican brown bear (Ursus arctos marsicanus). CASE PRESENTATION: In March 2014, an adult female Marsican brown bear was found in the Abruzzo, Lazio and Molise National Park (Italy) showing severe non-specific clinical signs. The animal died soon after its discovery and the carcass was submitted to post-mortem examination to identify the cause of death. The bear was diagnosed with a severe Mycobacterium bovis infection, with both pathological and microbiological aspects suggesting ongoing generalization. A presumptive diagnosis of mycobacterial infection was initially made based on gross findings. Histopathology showed the presence of acid-fast bacilli in all sampled tissues along with poorly organized granulomatous lesions. Slow-growing Mycobacterium sp. was isolated from multiple organs (intestine, mesenteric lymph nodes, liver, spleen, lung and kidneys). The PCR and sequencing algorithm identified the Mycobacterium sp. isolate as M. bovis. Spoligotyping demonstrated that the M. bovis isolate belonged to spoligotype SB0120. CONCLUSIONS: This is the first report of lethal M. bovis tuberculosis infection in a free-ranging brown bear. This pathogen could have serious adverse effects in an endangered relic population such as the Marsican brown bear. Stricter application of health regulations in force, surveillance of M. bovis infections in wild ungulates and carnivore scavengers, along with dismissal of supplementary feeding points intended for cattle or wildlife, are warranted to control the presence of bovine tuberculosis in wild and domestic animals in protected areas.

Mycobacterium caprae in a dairy farm in Northeast Italy.

Veneto region, Northeast Italy, has been declared officially free from bovine tuberculosis since 2008, although the disease is sporadically detected in association with cattle trade. In September 2015, bovine tuberculosis was detected in a dairy cattle farm of the region, in a holding with 69 animals. The herd underwent single intradermal tuberculin testing as part of the regional surveillance plan, and 24 animals resulted positive. Mycobacterium caprae was evidenced in 22 samples, further genotyped by PCR-based assays, as Allgäu type. Epidemiological investigation reported that sixteen animals were introduced from an officially tuberculosis free Member State in previous years. Nevertheless, spoligotyping and multilocus variable tandem repeat analysis (MLVA) indicated that M. caprae was strictly related to the strain circulating in 2007-2009 in Trento province, although no at-risk contacts were described. M. caprae is a zoonotic pathogen and further analyses are warranted in order to control its spread and impact on public health and animal trade.

Mycobacterium genavense Infection in a Domestic Ferret (Mustela putorius furo).

Mycobacterium genavense infection was diagnosed in an adult ferret with ptosis of the left eye, a proliferative lesion of the conjunctiva of the nictitating membrane, conjunctival swelling, and tumefaction of the periorbital tissues with a watery ocular discharge and the presence of a retrobulbar mass. The diagnosis was based on characteristic cytology of the retrobulbar mass and left mandibular lymph node that revealed granulomatous inflammation. Ziehl-Neelsen staining showed the presence of positive acid-fast bacilli in the cytoplasm of the macrophages. The diagnosis was confirmed by sequence analysis of the 16S rRNA gene amplified by using a multiplex polymerase chain reaction from a fresh lymph node biopsy. Therapy with marbofloxacin, rifampicin, and clarithromycin was recommended for 6 months and after this period, the veterinarian who was treating the ferret reported the disappearance of clinical signs. Six months after the end of the antibiotic treatment, the symptoms described previously reoccurred. Confirmatory laboratory tests were not performed but a recurrence of M genavense infection was suspected and the veterinarian, in agreement with the owner, euthanized the ferret.

Development and evaluation of two multi-antigen serological assays for the diagnosis of bovine tuberculosis in cattle.

There is currently an increased interest in the use of serological approaches in combination with traditional cell-mediated immunity-based techniques to improve the detection of tuberculosis (TB)-infected animals. In the present study, we developed and validated two different serological TB-detection assays using four antigens, MPB70, MPB83, ESAT6 and CFP10, and the tuberculin PPDb. A conventional multi-antigen TB-ELISA method and a novel TB multiplex test, based on Luminex technology, were developed to detect antibodies to multiple antigen targets. The performance levels of the two tests were evaluated and compared using selected panels of samples having known TB states. The TB-ELISA test (containing five antigens, including PPDb) had a sensitivity (Se) of 74.2% and a specificity (Sp) of 94.9%, while the TB-Luminex test had higher Se (79.0%) and Sp (99.1%) rates even when only one reactive antigen was used to classify the test as positive. If a more restrictive criterion, requiring two positive antigens to classify the test as positive, was used, then the TB-ELISA's Sp rate increased to 99.8% but the Se decreased to 61.3%, while the TB-Luminex test's Sp rate increased to 100% but the Se decreased to 51.2%. TB-ELISA and TB-Luminex were applied to a panel of 257 sera collected from bTB-positive herds, as determined by a post-mortem inspection. They showed good performance levels, identifying 49 (80.3%) and 48 (78.7%), respectively, of 61 samples that had tested positive by the intradermal tuberculin (IDT) test and/or interferon-gamma assay. In addition, TB-ELISA and TB-Luminex were able to identify 60 and 42 samples as positive, respectively, out of the 196 samples that tested negative to IDT and interferon-gamma at the time of serum collection. Subsequent IDT tests performed after 1-2 months, confirmed the positivity of 18 samples, indicating the strategic value of having two serological assays to detect TB-infected herds that were not reactive to initial IDT testing, thereby allowing for the rapid control of outbreaks and eradication of the disease.

Comparison of semi-automated commercial rep-PCR fingerprinting, spoligotyping, 12-locus MIRU-VNTR typing and single nucleotide polymorphism analysis of the embB gene as molecular typing tools for Mycobacterium bovis.

Purpose. Highly discriminatory genotyping strategies are essential in molecular epidemiological studies of tuberculosis. In this study we evaluated, for the first time, the efficacy of the repetitive sequence-based PCR (rep-PCR) DiversiLab Mycobacterium typing kit over spoligotyping, 12-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and embB single nucleotide polymorphism (SNP) analysis for Mycobacterium bovis typing.Methodology. A total of 49 M. bovis animal isolates were used. DNA was extracted and genomic DNA was amplified using the DiversiLab Mycobacterium typing kit. The amplified fragments were separated and detected using a microfluidics chip with Agilent 2100. The resulting rep-PCR-based DNA fingerprints were uploaded to and analysed using web-based DiversiLab software through Pearson's correlation coefficient.Results. Rep-PCR DiversiLab grouped M. bovis isolates into ten different clusters. Most isolates sharing identical spoligotype, MIRU-VNTR profile or embB gene polymorphism were grouped into different rep-PCR clusters. Rep-PCR DiversiLab displayed greater discriminatory power than spoligotyping and embB SNP analysis but a lower resolution power than the 12-locus MIRU-VNTR analysis. MIRU-VNTR confirmed that it is superior to the other PCR-based methods tested here.Conclusion. In combination with spoligotyping and 12-locus MIRU-VNTR analysis, rep-PCR improved the discriminatory power for M. bovis typing.

An outbreak of bovine tuberculosis in a fallow deer herd (Dama dama) in Sicily.

Wild ruminants have an important role in the epidemiology of bovine tuberculosis (bTB). This study describes an outbreak of bovine tuberculosis occurring in a fallow deer herd in Sicily. In 2012 a Sicilian herd of 47 animals was referred for cachexia. Pathological examination of 2 dead animals revealed disseminated granulomas predominantly involving the skin and subcutaneous tissues. Tissue samples were submitted for histological analysis, bacteriological culture, and biomolecular assay. PCR analysis identified Mycobacterium strains. Genotyping by spoligotyping and MIRU-VNTR profiles identified Mycobacterium bovis spoligotype SB0120 in both animals. In 2014, bTB skin testing of 28 fallow deer from the same group was positive in 4 and inconclusive in another 4. All 8 positive/inconclusive reactors were euthanized. Disseminated granulomatous lesions were noted in 6 of these animals, 3 of which (2 positive and 1 negative to skin tests) also presented cutaneous lesions. M. bovis spoligotype SB0120 was identified from all animals in which tuberculous-like lesions were observed, including 2 negative reactors. Many of the animals involved in this outbreak presented diffuse skin lesions, a potential route of transmission of M. bovis infection. Given the epidemiological role wildlife play in the maintenance of bTB infection and its potential risk for humans, a comprehensive monitoring plan for this zoonosis in wildlife species in Sicily is needed.

Professional Acquisition of M. bovis in Calabria Region (Southern Italy): A Challenging Case of Osteomyelitis in a Migrant Patient from Bulgaria.

We report herein the first case of a coinfection with Brucella spp., M. bovis, and Enterobacter cloacae in a butcher who moved from Bulgaria to Italy. Molecular typing suggested professional acquisition of M. bovis in Italy. So, surveillance and preventive measures need to be implemented.