Isolation of Nontuberculous Mycobacteria from Bovine Raw Lungs Bought in Butchers' Shops.

Nontuberculous Mycobacteria (NTM) can cause opportunistic disease in animals and humans, causing mycobacteriosis. In this study, bovine lungs were collected from butchers' shops and slaughterhouses after food official's inspection from the metropolitan area of Buenos Aires. All samples were cultured and then identified by molecular methods. Twelve isolates of NTM were identified being the most prevalent Mycolicibacterium insubricum. This demonstrates that viable Mycobacteria can pass food inspection and contaminate surfaces and food, making manipulation of raw organs and feeding of animals with raw lungs a potential source of infection for pets and owners.

Tuberculosis in wild South American sea lions Otaria flavescens stranded in Chubut, Argentina.

Pinniped tuberculosis, commonly caused by Mycobacterium pinnipedii, is a zoonotic disease reported in free-living and captive otariid species of the southern hemisphere. Currently, data concerning pinniped tuberculosis in South America are scarce, reinforcing the need for further studies of the disease in free-ranging pinnipeds. In this study, we investigated the presence of tuberculosis in South American sea lions Otaria flavescens (SASLs) stranded along the Chubut coastline (Argentina). Necropsies were performed in 9 SASLs, and tissue samples were collected for histopathology, bacteriology, and molecular diagnosis. Four SASLs showed enlarged tracheobronchial lymph nodes (TBLNs) with multifocal to coalescing granulomas. In these animals, a direct IS6110-PCR amplification confirmed the presence of a Mycobacterium tuberculosis complex member in TBLNs (n = 4) and lungs (n = 2), but the agent could not be further identified. In one SASL, Mycobacterium murale was isolated from lungs without lesions. This study confirms the presence of tuberculosis in SASLs from Chubut, where tourist activities promote close interaction with the animals, generating a potential risk to human health. Further research is currently focusing on addressing the prevalence of tuberculosis in wild SASLs, to assess the risk for public health and develop management strategies to avoid human infection.

Detection of Viable Mycobacterium bovis in Lungs and Livers Sold in Butchers' Shops in Buenos Aires, Argentina.

Although Mycobacterium bovis is the major etiological agent of tuberculosis in bovines, it can infect other mammalians. Previously reported cases of tuberculosis caused by M. bovis in cats from the Autonomous City of Buenos Aires (CABA) led to the conclusion that the main source of infection for these felines was the ingestion of raw bovine lungs. Thus, for this study, we collected samples of bovine viscera from butchers' shops of the Greater Buenos Aires (GBA) and the CABA to assess presence and viability of these mycobacteria in bovine lungs (including the lymph nodes) and livers. We analyzed 216 different samples and obtained 5 isolates of M. bovis (4 from lungs and 1 from liver) by culture analysis. We also confirmed the presence of different isolates by polymerase chain reaction, spoligotyping, and MIRU-VNTR assays. The results obtained in this work emphasizes the need of social education for food hygiene, and to change the habit of feeding pets with raw viscera, which carries the risk of epizootic and zoonotic transmission. Moreover, control and eradication programs of bovine tuberculosis should be strengthened and improved.

[Interlaboratory test: Isolation of Mycobacterium bovis from granulomatous lesions in bovine]. / Ensayo interlaboratorio: aislamiento de Mycobacterium bovis a partir de lesiones granulomatosas en bovinos.

Mycobacterium bovis is the causative agent of bovine tuberculosis. The diagnostic laboratory confirmation is made through bacterial isolation. The aim of interlaboratory tests is to assess the performance of each participant in comparison with other of similar capacities. The test objective was to determine the efficiency of isolation of M. bovis. Four laboratories were part of the test and processed 25 blind tissue samples from granulomatous lesions and with previous M. bovis isolation. The laboratory that had the highest proportion of isolates was A (68%), followed by C (60%) and then B and D (both with 52%). The greatest concordance was observed between B-D and B-C laboratories (68%). The differences could be due to specific factors in each laboratory procedures. This type of interlaboratory tests highlights errors in the bacteriology and identifies critical points in the process to detect M. bovis accurately.

Detection of non-tuberculous mycobacteria in native wildlife species at conservation risk of Argentina.

Introduction: Non-tuberculous Mycobacteria (NTM) are mainly environmental but can cause opportunistic infections and diseases in humans and animals. Livestock and wild animals can be infected with NTM. In Argentina, there are native wild species facing conservation risks, and they are the focus of protection and reintroduction projects designed to preserve biodiversity in various ecoregions. The aim of this study was to report the presence of NTM in samples collected from four endangered native wild species from nine Argentine provinces, as part of their pre-release health assessment. Methods: A total of 165 samples from giant anteater, peccary, tapir and pampas deer were obtained, these included either bronchoalveolar or endotracheal lavages, or oropharyngeal, nasopharyngeal or tracheal swabs. Bacteriological culture followed by molecular identification and sequencing were performed. Results: A total of 27 NTM were detected, including Mycobacterium avium subsp. hominissuis, M. intracellulare, M. terrae, M. gordonense, M. kumamotonense, M. fortuitum, M. saskatchewanense, and M. genavense. Results revealed a 16,36% NTM recovery rate, with the giant anteater showing the highest prevalence among the mammals under study. Discussion: In Argentina, due to extensive production systems, the interaction between domestic and wild species sharing the same environment is frequent, increasing the exposure of all the species to these NTM. In this way, the transmission of infectious agents from one to another is feasible. Moreover, NTMs might interfere with the diagnosis of bovine tuberculosis and paratuberculosis. These findings emphasize the importance of active health surveillance in conservation programs. It highlights the need to address NTM epidemiology in wildlife and its impact on conservation and public health.

Tuberculosis in Wild Pigs from Argentina.

Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTC) and non-tuberculous Mycobacteria (NTM), may infect wild and domestic mammals, including humans. Although cattle are the main hosts and spreaders of M. bovis, many wildlife hosts play an important role worldwide. In Argentina, wild boar and domestic pigs are considered important links in mammalian tuberculosis (mTB) transmission. The aim of this work was to investigate the presence of M. bovis in wild pigs from different regions of Argentina, to characterize isolates of M. bovis obtained, and to compare those with other previously found in vertebrate hosts. A total of 311 samples from wild pigs were obtained, and bacteriological culture, molecular identification and genotyping were performed, obtaining 63 isolates (34 MTC and 29 NTM). Twelve M. bovis spoligotypes were detected. Our findings suggest that wild pigs have a prominent role as reservoirs of mTB in Argentina, based on an estimated prevalence of 11.2 ± 1.8% (95% CI 8.0-14.8) for MTC and the frequency distribution of spoligotypes shared by cattle (75%), domestic pigs (58%) and wildlife (50%). Argentina has a typical scenario where cattle and pigs are farm-raised extensively, sharing the environment with wildlife, creating conditions for effective transmission of mTB in the wildlife-livestock-human interface.

Molecular detection of Mycobacterium tuberculosis complex in a captive aguará popé (Procyon cancrivorus) with macroscopic tuberculosis like-lesions.

Tuberculosis is a chronic and contagious infectious disease caused by multi-host species of the genus Mycobacterium grouped within the Mycobacterium tuberculosis complex. These pathogenic bacteria mainly affect mammals, including humans. The most recognized species is Mycobacterium bovis, the causative agent of bovine tuberculosis in livestock. Although livestock is the main host of M. bovis, this species is frequently isolated from wild animals. Wild native mammals from Central and South America, as the crab-eating raccoon or "aguará popé" (Procyon cancrivorus), may act as a source of tuberculosis and may represent a human health risk, especially in captive scenarios, due to closer animal-human interaction. However, the only presence of infection in wild animals is not enough to determine their epidemiological role in the disease. Here we identify tuberculosis in a captive aguará popé with clinical signs and lung macroscopic tuberculosis-like lesions during necropsy. We detected tuberculosis by polymerase chain reaction assay. DNA was extracted directly from lung tissue and the amplification target was the insertion sequence 6110. This study contributes to investigate the presence of the disease in wild native animals of Argentina and supports the knowledge that wild mammals may act as a source of TB for humans and domestic animals.

Mycobacterium Avium in Miniature Schnauzer From Argentina: A Series of Cases.

Environmental mycobacteria such as those from the Mycobacterium avium-intacellulare complex may cause disseminated and severe disease in dogs with genetic predisposition. A series of cases of 4 miniature schnauzers with nonspecific clinical signs and the diagnostic tests are described. Complementary means of diagnosis including complete blood count, biochemical serum analyses and fine needle aspiration cytology staining were performed. The bacteriological culture followed by PCR amplification of 1245 and 901 insertion sequences, allowed the identification of Mycobacterium avium subsp. hominissuis. This environmental Mycobacteria normally do not cause severe disease in dogs or other species, but when CARD-9 gene presents mutations, dogs may become extremely susceptible and disease is fast, disseminated, and fatal. Antibiotic therapy can be applied under veterinary consideration in specific situations, as treatment is usually applied for a long period of time. Although zoonotic risk is low as the Mycobacterium is environmental, contamination of the location may be high, and immunosuppressed animals and humans can develop infection as well. This report may aid clinical veterinarians in the diagnosis, treatment, and prognosis in similar cases of this breed and others with the genetic predisposition.

Mycobacterium tuberculosis infection in a free-ranging urban dog from Argentina.

Mycobacterium bovis and Mycobacterium avium are the most important Mycobacteria isolated from diseased dogs, but Mycobacterium tuberculosis can be isolated as well, especially when close contact with humans exists. Free ranging street dogs may carry zoonotic diseases, being a potential health risk to new owners after adoption. In this study, the clinical case of a dog affected by Mycobacterium tuberculosis is described. A six-year-old bitch that had been living with a homeless man was rescued and put for adoption in dog shelter. After being adopted, her health declined, and abdominal ultrasound and exploratory laparoscopy were performed. A tuberculosis-like lesion in the liver was biopsied and histopathological, bacteriological, and molecular analyses were carried out. Then, the animal was euthanized and necropsied, and disseminated macroscopic tuberculosis-like lesions were observed in abdominal organs. Mycobacterium presence was confirmed by histopathological and bacteriological methods. Genotyping identified a SIT-1228 Mycobacterium tuberculosis strain. Notification to the sanitary authorities was performed and the couple that had contact with the dog were alerted and sent to the hospital for assessment, as Mycobacterium tuberculosis is a primary pathogenic agent for humans. The epidemiology in this clinical case is unclear, but the most likely source of infection might have been consumption of sputum contaminated food during the years that this bitch lived with the homeless man, because this person had died with chronic respiratory symptoms. Veterinarians must consider this disease and perform a complete diagnosis when dogs that used to live on the streets show nonspecific clinical signs.

Serological survey suggests circulation of coronavirus on wild Suina from Argentina, 2014-2017.

Swine coronaviruses affecting pigs have been studied sporadically in wildlife. In Argentina, epidemiological surveillance of TGEV/PRCV is conducted only in domestic pigs. The aim was to assess the prevalence of TGEV/PRCV in wild Suina. Antibodies against these diseases in wild boar and captive collared peccary were surveyed by ELISA. Antibodies against TGEV were found in three collared peccaries (n = 87). No TGEV/PRCV antibodies were detected in wild boar (n = 160). Preventive measures should be conducted in contact nodes where the transmission of agents may increase. Epidemiological surveillance in wildlife populations and in captive animals before their reintroduction should be attempted.

Epidemiology of Pig Tuberculosis in Argentina.

Bovine tuberculosis (bTB) is a disease caused mainly by the Mycobacterium bovis and that is endemic to livestock populations in most Latin American countries. Traditionally, bTB control programs are costly and targeted to cattle, largely disregarding other species such as swine and wildlife. According to official services, in Argentina disease prevalence in pigs is comparable to that observed in cattle, suggesting the need for efficient control programs to manage the disease in both species. Additionally, extensive farming systems, which are commonly practiced in Argentina, allow the interaction between livestock and wildlife such as wild boar (Sus scrofa), which is considered a natural host of the disease. Here, we evaluated the bTB pigs- cattle interface, studying the dynamics of M. bovis isolates in the pig population and identifying farm-level epidemiological variables associated with the disease confirmation at slaughterhouses. Additionally, to assess the potential multi-host systems in the transmission of bTB, the molecular characterization of wild boar mycobacterial strains was included in the study, as this interaction has not been previously evaluated in this region. Multivariable logistic regression models were used to assess the association between farm-level epidemiological variables (location, farm size, and co-existence with cattle and goats) and bTB confirmation in pig tuberculosis-like lesions samples. Results showed that when cattle were present, the odds of bTB in pigs decreased 0.3 or 0.6% for every additional sow when cattle were present or absent in the farm, respectively. Pigs shared 60% (18/30) of the genotypes with cattle and wild boar, suggesting transmission at the interface between pigs and cattle and highlighting the potential role of wild boar in bTB maintenance. These results provide novel information about the molecular diversity of M. bovis strains in pigs in Argentina and proposes the potential relevance of a multi-host system in the epidemiology of bTB in the region. The statistical models presented here may be used in the design of a low cost, abattoir-based surveillance program for bTB in the pig industry in Argentina, with potential extension to other settings with similar epidemiological conditions.

Development of a lateral flow immunochromatography test for the rapid detection of bovine tuberculosis.

Detection of specific antibodies would be a useful test strategy for bovine tuberculosis (bTB) as a complement to the single skin test. We developed a lateral flow immunochromatography (LFIC) test for rapid bTB detection based on the use of a conjugate of gold nanoparticles with a recombinant G protein. After evaluating 3 Mycobacterium bovis (MB) antigens: ESAT-6, CFP-10 and MPB83 for the control line, we selected MPB83 given it was the most specific. The performance of the test was analyzed with 820 bovine sera, 40 sera corresponding to healthy animals, 5 sera from animals infected with Mycobacterium avium subsp. paratuberculosis (MAP) and 775 sera of animals from herds with bTB. All these sera were also submitted to a validated bTB-ELISA using whole-cell antigen from MB. From the 775 sera of animals from herds with bTB, 87 sera were positive by the bTB-ELISA, 45 were positive by LFIC and only 5 animals were positives by skin test (TST). To confirm bTB infection in the group of TST (-), bTB-ELISA (+) and LFIC (+) animals, we performed postmortem examination in 15 randomly selected animals. Macroscopically, these 15 animals had numerous small and large yellow-white granulomas, characteristic of bTB, and the infection was subsequently confirmed by PCR in these tissues with lesions (gold standard). No false positive test result was detected with the developed LFIC either with the sera from healthy animals or from animals infected with MAP demonstrating that it can be a useful technique for the rapid identification of animals infected with bTB.

Bayesian Assessment of the Accuracy of a PCR-Based Rapid Diagnostic Test for Bovine Tuberculosis in Swine.

Infection with the Mycobacterium bovis (M. bovis) causes a disease referred to as bovine tuberculosis (bTB), which affects a wide range of mammal hosts. Many countries have implemented control and eradication plans that have resulted in variable levels of efficacy and success. Although bTB is a notifiable disease in Argentina, and a control plan that targets cattle herds has been in place for decades, M. bovis is still prevalent in cattle, swine, and certain wild species. The aim of the paper here was to assess the sensitivity (Se), specificity (Sp), and positive and negative predictive values (PPV and NPV) of PCR from tissue, which is a test for rapid M. bovis detection in swine. Bacteriological culture was also performed for comparison purposes. A Bayesian approach was applied to estimate the accuracy of the diagnostic tests, PCR and bacteriological culture, in 266 swine samples with bTB-like lesions recovered during routine official inspections at slaughterhouses. A one-population model, assuming conditional dependence between test results, and incorporating prior information on the performance of the tests obtained from the literature, was used to estimate the tests Se and Sp. The accuracy of the combined (in parallel) application of both tests was also estimated. The Se of the PCR (82.9%) was higher than the Se of the bacteriological culture (79.9%), whereas the Sp of both tests was similar (88.5 and 89.0%, respectively). Furthermore, when both techniques were assessed in parallel, the Se of the diagnostic system increased substantially (Se = 96.6%) with a moderate Sp loss (Sp = 78.8%; PPV = 92.8%; NPV = 89%). Results suggest that the PCR, or the combined application of bacteriological culture and PCR, may serve as an accurate diagnostic tool to confirm bTB in swine samples. Results here will help the design and implementation of effective surveillance strategies for the disease in swine of Argentina and other settings in which the disease is prevalent.

Mycobacterioses in dogs and cats from Buenos Aires, Argentina.

Mycobacterioses can produce nonspecific clinical signs in dogs and cats that make diagnosis difficult. Furthermore, the full characterization of mycobacterial agents is not always possible or practical. We characterized mycobacteria detected through cytology in 12 dogs and 7 cats with generalized clinical signs from the province of Buenos Aires in Argentina. In dogs, molecular testing confirmed the presence of Mycobacterium avium subsp. hominissuis (MAH) in 8 cases and M. fortuitum in 1 case. All dogs were Miniature Schnauzers, suggesting that this breed may be more susceptible to M. avium than other dog breeds. The cat isolates were 2 M. bovis, 1 M. fortuitum, and 1 MAH. Mycobacterial interspersed repetitive unit-variable-number tandem repeat patterns suggested possible links with cattle, swine, and humans studied previously in Argentina. The results show that pets may act as susceptible hosts with the potential risk of transmitting the infection to humans and other animals.

Mycobacterium bovis Δmce2 double deletion mutant protects cattle against challenge with virulent M. bovis.

A Mycobacterium bovis strain deleted in mce2A and mce2B genes (M. bovis Δmce2) was tested as an experimental vaccine in cattle challenged with a virulent M. bovis strain. Three-and-a-half-month old calves (n = 5 to 6 per group) were vaccinated and challenged with a virulent strain of M. bovis by the intratracheal route 9 weeks after vaccination. A non-vaccinated group and a group vaccinated with BCG were included as controls. Blood samples were collected to measure IFN-γ by an interferon-gamma release assay (IGRA), cytometry and cytokine responses of bovine purified protein derivative (PPD) restimulated peripheral blood mononuclear cells (PBMCs). The IGRA test showed IFN-γ values similar to pre-vaccination except for the animals vaccinated with M. bovis Δmce2, where a significant increase was observed at 30 days post-vaccination. The expression of IL-2R on CD4(+) cells in response to PPD from the animals vaccinated with Δmce2 increased at 15 days post-vaccination compared to cells from non-vaccinated group. Vaccination of cattle with M. bovis Δmce2 induced the highest (P < 0.05) expression of IFN-γ and IL-17 mRNA upon PPD stimulation of PBMCs compared to vaccination with BCG or that for the non-vaccinated group. There was a weak positive correlation between the production of these proinflammatory cytokines post-vaccination and reduced pathology scores post-challenge. The animals were euthanized and necropsied 100 days after challenge. The group vaccinated with M. bovis Δmce2 displayed a significantly lower histopathological score for lesions in lungs and pulmonary lymph nodes than for the other groups (P < 0.05). A marked positive reaction to tuberculin intradermal test was observed post-vaccination in animals vaccinated with M. bovis Δmce2 compared to those vaccinated with BCG or the non-vaccinated group. In contrast, after challenge, non-vaccinated animals had greater skin test responses than the vaccinated animals. In summary, M. bovis Δmce2 is a promising vaccine candidate to control M. bovis pathogenesis in cattle.

Mycobacterium bovis in Swine: Spoligotyping of Isolates from Argentina.

A total of 143 Mycobacterium bovis isolates of pigs, from the most productive swine area in Argentina, were typed by spoligotyping. Twenty-two different spoligotypes were identified, and 133 (93%) isolates were grouped into 12 clusters. One of them, designed SB0140, was the most frequent because it held 83 (58%) isolates. This spoligotype also grouped 362 (43%) out of 841 isolates from previously typed cattle and, thus, constitutes the most frequent in our country. In addition, 135 (94%) isolates revealed spoligotypes identical to those of cattle, showing an epidemiological link. On the other hand, there were seven novel spoligotypes, six of which were also unique since they had only one isolate each. This study aimed to identify the spoligotypes of M. bovis isolated from pigs to contribute to a better understanding of the distribution of bovine tuberculosis in the main productive area of Argentina.

European 1: a globally important clonal complex of Mycobacterium bovis.

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle.