Transmissibility of Mycobacterium pinnipedii in a murine model.

The causative agent of tuberculosis in pinnipeds is Mycobacterium pinnipedii, a member of the Mycobacterium tuberculosis complex (MTC). The natural hosts are pinnipeds; however, other non-marine mammals, including humans, can also be infected. The transmissibility of a pathogen is related to its virulence. The transmissibility of a M. pinnipedii strain (i.e., 1856) was investigated in a murine model and compared with that of two Mycobacterium bovis strains (i.e., 534 and 04-303) with different reported virulence. Non-inoculated mice (sentinels) were co-housed with intratracheally inoculated mice. Detailed inspection of mice to search for visible tuberculosis lesions in the lungs and spleen was performed, and bacillus viability at 30, 60, and 90 days post-inoculation (dpi) was assayed. A transmissibility of 100% was recorded at 30 dpi in sentinel mice co-housed with the inoculated mice from the M. pinnipedii and M. bovis 04-303 groups, as evidenced by the recovery of viable M. pinnipedii and M. bovis from the lungs of sentinel mice. Mice inoculated with M. pinnipedii (1856) and M. bovis (534) survived until euthanized, whereas five of the M. bovis 04-303-inoculated mice died at 17 dpi. This study constitutes the first report of the transmissibility of a M. pinnipedii strain in mice and confirms the utility of this experimental model to study virulence features such as the transmission of poorly characterized MTC species.

Assessing the impact of various tuberculin PPD brands on bovine tuberculosis diagnosis.

Although several brands of tuberculin purified protein derivatives (PPDs) are available for diagnosing bovine tuberculosis (bTB), comparative studies to determine their diagnostic accuracy are infrequent. In Ecuador we compared two different PPD brands for bTB diagnosis using skin testing and measuring skin thickness increase. Additionally, we evaluated four PPD brands, including those used for skin testing, in the Bovine Tuberculosis Interferon Gamma Test (IFN-γ test) measuring IFN-γ induction in whole blood. The study included 17 naturally tuberculosis-infected PPD and IFN-γ test positive bovines. Both the field and laboratory results showed significant differences in classifying the 17 bovines as bTB positive or negative. We hypothesize that several factors, such as the genetic background of the cows, sensitization to environmental mycobacteria, M. bovis strains involved in the bTB infection, and the manufacturing procedures of the PPDs, could have influenced the immune reaction toward the different tuberculin PPD brands. Our study emphasizes the necessity for comparative studies aimed at determining the diagnostic accuracy of PPD brands for bTB diagnosis as well as the development of standardized methods for PPD production and potency determination.

First Case Reports of Nontuberculous Mycobacterial (NTM) Lung Disease in Ecuador: Important Lessons to Learn.

Nontuberculous mycobacteria (NTM) lung infections are often misdiagnosed as tuberculosis, which can lead to ineffective antibiotic treatments. In this report, we present three cases of NTM lung infections in Ecuador that were initially diagnosed and treated as tuberculosis based on the results of sputum smear microscopy. The patients, all male, included two immunocompetent individuals and one HIV-positive subject. Unfortunately, sputum culture was not initiated until late in the course of the disease and the cause of the lung infection, Mycobacterium avium complex (MAC), was only identified after the patients had either passed away or were lost to follow-up. These cases are the first documented cases of NTM lung infections in the English medical literature from Ecuador. We emphasize the importance of accurate diagnosis of NTM infections by culture and identification to species level. Sputum smear staining alone cannot differentiate between mycobacterial species, which can lead to misidentification and ineffective treatments. Additionally, reporting NTM pulmonary disease as a notifiable disease to national TB control programs is recommended to obtain accurate prevalence data. These data are critical in determining the importance of this public health problem and the necessary actions needed to address it.

Spatial distribution of Mycobacterium bovis spoligotypes in cattle from the state of Minas Gerais, Brazil.

Genotyping methods have led to a better understanding of the epidemiology of Mycobacterium bovis (M. bovis) infection, and its transmission dynamics, as well as the possible phylogenetic relationships between Mycobacterium strains, thus making bovine tuberculosis control programs more efficient. The goal of this study was to characterize the main spoligotypes of M. bovis isolated from cattle in the State of Minas Gerais, Brazil. It was carried out in 28 municipalities of "Triângulo Mineiro" and "Alto Paranaíba" regions of the state. Viscera samples were obtained from 58 bovines positive for tuberculosis according to comparative cervical tests, and from another 100 bovines with lesions suggestive of tuberculosis, which were donated by the National Agricultural Laboratory of Pedro Leopoldo, Minas Gerais. Microbiological isolation was performed in Stonebrink medium, and molecular identification of mycobacteria was performed by PCR. Genotyping was performed using the spoligotyping method at the Agrobiotechnology and Molecular Biology Institute of National Agricultural Technology Institute-National Scientific and Technical Research Council, Buenos Aires, Argentina. Among the 158 viscera samples, we obtained 40 (25%) isolates of M. bovis, and detected 11 spoligotype patterns, with a predominance of SB1142 (37.5%), SB0121 (25.0%), and SB1145 (10.0%). Other standards, SB0295, SB1050, SB0881, SB1144, SB1802, SB0140, SB0120, and SB0849, varied from 2.5 to 7.5%, heterogeneously distributed among the municipalities. The presence of spoligotypes shared with other Brazilian states and different countries indicates their possible exchange through epidemiological relationships, such as the transit of live animals and/or genetic similarity between strains that share a common ancestor.

Replication and transmission features of two experimental vaccine candidates against bovine tuberculosis subcutaneously administrated in a murine model.

Cattle vaccination is an attractive approach in compliance with control and eradication programs against Bovine Tuberculosis (bTB). Today, there is no anti bTB vaccine licensed. Two vaccine candidates, MbΔmce2 and MbΔmce2-phoP previously designed were evaluated in BALB/c mice, including the parental M. bovis NCTC10772 and a M. bovis hypervirulent Mb04-303 strains as controls. Sentinel mice (non-inoculated) cohoused with subcutaneous inoculated mice. Persistence, visible tuberculosis lesions (VTL) in lungs and spleens and bacillary load were investigated subcutaneously delivered at 60 and 90 days after inoculation (dpi) as well as their potential transmission to naïve mice. While a 100% survival was observed at 90 dpi without VTL in all groups, transmission was not evidenced in the sentinels mice. Vaccine candidates and control strains were isolated from the spleen of all inoculated mice, while Mb04-303 was isolated from the lungs of one inoculated mouse. Vaccine candidate's attenuation considering survival, lung bacillary load and VTL was confirmed, administrated by the subcutaneous route. Future experiments are necessary to demonstrate whether the persistence of both mutants in the spleen, with low CFU, remains over time to increase the potential increasing risk of dissemination to organs and subsequent transmission to other animals by airborne or other routes.

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.

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.

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.

Prevalence of paratuberculosis in dairy cattle in ecuador.

Background: Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis, a chronic infectious contagious disease of the intestinal tract of ruminants that are also associated with Crohn's disease in humans. The existence of paratuberculosis in Ecuador is virtually unknown; hence, the present study was performed to gain insight into the prevalence of this disease. Methods: Three dairy cattle farms in different geographic regions in Ecuador were investigated for the infection with MAP, and 600 blood samples, 200 of each cattle herd, were processed with an indirect enzyme-linked immunosorbent assay. Fecal samples of the seropositive cows were processed for culture on modified Löwenstein-Jensen medium. Results: One hundred and fifty bovines (25%) resulted seropositive and we confirmed with culture the presence of MAP in 4.7% (7/150) of the seropositive cows. Approximately 20% of the fecal samples of seropositive cows yielded nontuberculous mycobacteria (NTM) species including M. avium subsp. avium, a NTM species closely related to MAP. Conclusions: The seroprevalence of paratuberculosis in this first study for Ecuador is high (25%). We discuss a possible interference of NTM species, isolated from fecal samples, with the diagnosis of paratuberculosis. With this report, a baseline study, we confirm for the first time the presence of paratuberculosis in Ecuador, and we provide the necessary information for future studies and control of this disease.

Molecular characterization of Mycobacterium bovis infection in cattle and buffalo in Amazon Region, Brazil.

The aim of this study was to characterize Mycobacterium bovis from cattle and buffalo tissue samples, from two Brazilian states, and to analyse their genetic diversity by spoligotyping. Tissue samples from tuberculosis suspect animals, 57 in Amazonas State (12 cattle and 45 buffaloes) and six from Pará State (5 cattle and one buffalo) from slaughterhouses under State Veterinary Inspection, were isolated in culture medium Stonebrink. The positive cultures were confirmed by PCR and analysed by the spoligotyping technique and the patterns (spoligotypes) were identified and compared at the Mycobacterium bovis Spoligotype Database (http://www.mbovis.org/). There was bacterial growth in 44 (69.8%) of the tissues of the 63 animals, of which PCR for region of differentiation 4 identified 35/44 (79.5%) as Mycobacterium bovis. Six different spoligotypes were identified among the 35 Mycobacterium bovis isolates, of which SB0295, SB1869, SB0121 and SB1800 had already been described in Brazil, and SB0822 and SB1608 had not been described. The most frequent spoligotype in this study (SB0822) had already been described in buffaloes in Colombia, a neighbouring country of Amazonas state. The other identified spoligotypes were also described in other South American countries, such as Argentina and Venezuela, and described in the Brazilian states of Rio Grande do Sul, Santa Catarina, São Paulo, Minas Gerais, Mato Grosso do Sul, Mato Grosso and Goiás, indicating an active movement of Mycobacterium bovis strains within Brazil.

Analysing nonsynonymous mutations between two Mycobacterium bovis strains with contrasting pathogenic profiles.

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis, a chronic infectious disease that can affect cattle, other domesticated species, wild animals and humans. This disease produces important economic losses worldwide. Two M. bovis strains (04-303 and 534) have been isolated in Argentina. Whereas the 04-303 strain was isolated from a wild boar, the 534 strain was obtained from cattle. In a previous study, six weeks after infection, the 04-303 strain induced 100% mortality in mice. By contrast, mice infected with the 534 strain survived, with limited tissue damage, after four months. In this study we compared all predictive proteins encoded in both M. bovis genomes. The comparative analysis revealed 141 polymorphic proteins between both strains. From these proteins, nine virulence proteins showed polymorphisms in 04-303, whereas five did it in the 534 strain. Remarkably, both strains contained a high level of polymorphism in proteins related to phthiocerol dimycocerosate (PDIM) synthesis or transport. Further experimental evidence indicated that only mutations in the 534 strain have an impact on PDIM synthesis. The observed reduction in PDIM content in the 534 strain, together with its low capacity to induce phagosome arrest, may be associated with the reported deficiency of this strain to replicate and survive inside bovine macrophages. The findings of this study could contribute to a better understanding of pathogenicity and virulence aspects of M. bovis, which is essential for further studies aiming at developing new vaccines and diagnostic techniques for bovines.

Surveillance and characterization of drug-resistant Mycobacterium tuberculosis isolated in a reference hospital from Argentina during 8 years' period.

Background: Argentina is considered a country with a middle tuberculosis (TB) incidence. However, according to the last national epidemiological report released in 2018, since 2013, the trends are steadily increasing. The aims of this study were to determine the drug-resistance (DR), multi-DR and extensively DR (MDR/XDR-TB), and rifampicin resistance (RIF-R) burden as a part of the local TB diagnosis (June 2010-August 2018); to detect the mutations associated to isoniazid (INH) and RIF-R and their geographical distribution; and to analyze the lineage relationship among the genetic patterns of the isolates circulating in the community. Methods: Respiratory and extrapulmonary specimens were processed by Ziehl-Neelsen stain and cultured on specific media. Drug-susceptibility testing of isolates was performed by the MGIT 960 and a colorimetric micro-method. Mutations conferring DR were detected by Genotype and DNA sequencing. Results: The study showed a DR-TB prevalence of approximately 20% of the isolated strains, while M/XDR-TB-and particularly RIF-R-affected more than 5.0% of the total amount of cases. DR geographical distribution revealed isolates carrying mutations in the inhA gene promoter region only constrained to three districts where it was also registered two same family relatives' cases with the infrequent rpoB S522 L/Q mutation. The fact that most DR/MDR-TB isolates were not grouped in genetic clusters suggested that these cases may mostly have occurred due to endogenous reactivation rather than recently transmission. Conclusion: According to the obtained results, it would be convenient, in highly MDR-TB suspected individuals, to confirm phenotypically, the INH and RIF susceptibility detected by molecular tests.

Diversidad de especies de micobacterias no tuberculosas aisladas en ambientes acuáticos de la ciudad de General Pico, La Pampa, Argentina / Species diversity of non-tuberculous mycobacteria isolated from aquatic environments of General Pico city, Province of La Pampa (Argentina)

Las micobacterias no tuberculosas (MNT) no solo se estudian por su importancia como patógenos oportunistas, sino también por sus aplicaciones en biotecnología y biorremediación. Nuestro objetivo fue determinar la presencia de micobacterias en los distintos hábitats acuáticos de la ciudad de General Pico (provincia de La Pampa), así como su diversidad. Los porcentajes de muestras positivas a micobacterias fueron los siguientes: 37,5% en el sistema de distribución de agua de red, 32,6% en el acuífero que abastece dicho sistema, 36,8% en el agua proveniente de las precipitaciones, 53,1% en los humedales del área de influencia, 80% en los natatorios cubiertos y 33,3% en las fuentes decorativas ubicadas en plazas públicas. De los 90 aislamientos de MNT obtenidos el 8,9% no logró ser identificado a nivel de especie con los métodos utilizados, que incluyeron pruebas fenotípicas y métodos moleculares. Las especies más frecuentemente aisladas fueron Mycobacterium fortuitum y Mycobacterium gordonae. Algunas especies identificadas han sido reportadas en casos de micobacteriosis en nuestro país, entre ellas M. fortuitum, M. gordonae, M. intracellulare, M. vaccae, M. lentiflavum y M. nonchromogenicum. No se aislaron MNT en muestras de agua de red con concentraciones de cloro activo residual mayores de 0,8mg/l, mientras que en los natatorios la presencia de hasta 1,5mg/l de cloro activo residual no fue una limitante para la proliferación de estos microorganismos. Se puede considerar que la incidencia de micobacterias en los ambientes acuáticos de General Pico es cercana al 35%, y que la presencia de estos microorganismos y su diversidad se ve afectada por el contacto con el hombre y sus actividades, como así también por la existencia de vida animal.

Non-tuberculous mycobacteria (NTM) are studied not only for their importance as emerging opportunistic pathogens but also for their applications in biotechnology and bioremediation. Our aim was to determine the occurrence and diversity of mycobacteria in different aquatic habitats of General Pico city, Province of La Pampa. The percentage of samples with positive cultures for mycobacteria were the following: 37.5% recovered from the water supply distribution system; 32.6% from the aquifer that supplies water to the distribution system; 36.8% from rain water; 53.1% from the two wetlands in the area of influence; 80% from indoor swimming pools; and 33.3% from water fountains in downtown public squares. Of the 90 NTM isolates, 8.9% could not be identified at the species level with any of the used methods, phenotypic tests and molecular methods. Mycobacterium fortuitum and Mycobacterium gordonae were the most frequently isolated species. Some of the identified species such as, M. fortuitum, M. gordonae, M. intracellulare, M. vaccae, M. lentiflavum and M. nonchromogenicum, have been reported in cases of mycobacteriosis in Argentina. Mycobacteria with values higher than 0.8mg/ml of residual active chlorine were not recovered from the drinking water supply network, whereas in the swimming pools the presence of up to 1.5 mg/l was not a constraint. Based on our results, the presence of mycobacteria in aquatic environments is close to 35% and their occurrence and diversity is affected both by contact with man and his activities as well as by the existence of animal life.

Matrix Assisted Laser Desorption Ionization-Time-of-Flight mass spectrometry identification of Mycobacterium bovis in Bovinae.

In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future.

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.

Prevalence and species diversity of nontuberculous mycobacteria in drinking water supply system of Bahía Blanca City, Argentina.

Background: There is evidence that tap water is the vehicle through which nontuberculous mycobacteria (NTM) infect or colonize the human body. The objective of this study was to determine the presence and diversity of NTM in the water distribution system of Bahía Blanca city, Argentina (sites S2/S3) and in the dike that supplies water to it (S1). Methods: Culture-dependent method, biochemical tests, and molecular method (16S rRNA sequencing gene) were combined to detect and identify NTM. Results: NTM were isolated in 51.6% (64/124) of all the samples analyzed. Mycobacterium gordonae was the most frequently isolated organism (15/64) in all samples analyzed, followed by Mycobacterium peregrinum and Mycobacterium frederiksbergense. Significant differences were found in the residual chlorine values between sampling S2 and S3. In both sites, maximum counts were recorded but they did not correlate with low chlorine values. A concentration higher than 500 colony-forming unit/L of NTM was never found, which can be attributed to the negative effect caused by decontamination methods being a point to consider for the recovery of NTM. In 46.9% (30/64) of samples, both methods coincided in the identification, and the obtained sequences presented ≥99% identity. Identification at the species level was achieved in 50% (32/64) of the isolates. Nearly 17.2% (11/64) of the isolates showed a similarity <99%. Conclusions: It should be taken into account that sequencing of the 16S rRNA gene and biochemical tests are useful for the identification of several species, but it is necessary to incorporate other genes (hsp 65 and rpo B) to obtain accurate identification.

Epidemiological Study of Mycobacterium bovis Infection in Buffalo and Cattle in Amazonas, Brazil.

Bovine Tuberculosis (BTB) is an endemic disease in about one hundred countries, affecting the economy causing a decrease in productivity, condemnation of meat, and damaging the credibility on international trade. Additionally, Mycobacterium bovis the major causative agent for BTB can also infect humans causing a variety of clinical presentations. The aim of this study was to determine BTB prevalence and the main risk factors for the Mycobacterium bovis prevalence in cattle and buffalos in Amazonas State, Brazil. Tissue samples from 151 animals (45 buffalo and 106 cattle from five herds with buffalo only, 22 herds with cattle only, and 12 herds with buffalo and cattle) were obtained from slaughterhouses under State Veterinary Inspection. M. bovis were isolated on Stonebrink medium. The positive cultures were confirmed by polymerase chain reaction (PCR) testing. The apparent herd and animal prevalence rates were 56.4 and 5.40%, respectively. Regarding animal species, the apparent prevalence rates were 3% in cattle and 11.8% in buffalo. Generalized Linear Mixed Models (GLMM) with random effect were used to assess the association with risk factors on the prevalence. Species (buffalo), herds size (>100 animals) and the presence of both species (buffalo and cattle) in the herd were the major risk factors for the infection by Mycobacterium bovis in the region. The findings reveal an urgent need for evidence-based effective intervention to reduce BTB prevalence in cattle and buffalo and prevent its spread to the human population. Studies are needed to understand why buffalo are more likely to be infected by M. bovis than cattle in Amazon. Recommendations for zoning, use of data from the inspection services to generate information regarding BTB focus, adoption of epidemiological tools, and discouragement of practices that promote the mixing of cattle and buffalo, were made.

[Species diversity of non-tuberculous mycobacteria isolated from aquatic environments of General Pico city, Province of La Pampa (Argentina)]. / Diversidad de especies de micobacterias no tuberculosas aisladas en ambientes acuáticos de la ciudad de General Pico, La Pampa, Argentina.

Non-tuberculous mycobacteria (NTM) are studied not only for their importance as emerging opportunistic pathogens but also for their applications in biotechnology and bioremediation. Our aim was to determine the occurrence and diversity of mycobacteria in different aquatic habitats of General Pico city, Province of La Pampa. The percentage of samples with positive cultures for mycobacteria were the following: 37.5% recovered from the water supply distribution system; 32.6% from the aquifer that supplies water to the distribution system; 36.8% from rain water; 53.1% from the two wetlands in the area of influence; 80% from indoor swimming pools; and 33.3% from water fountains in downtown public squares. Of the 90 NTM isolates, 8.9% could not be identified at the species level with any of the used methods, phenotypic tests and molecular methods. Mycobacterium fortuitum and Mycobacterium gordonae were the most frequently isolated species. Some of the identified species such as, M. fortuitum, M. gordonae, M. intracellulare, M. vaccae, M. lentiflavum and M. nonchromogenicum, have been reported in cases of mycobacteriosis in Argentina. Mycobacteria with values higher than 0.8mg/ml of residual active chlorine were not recovered from the drinking water supply network, whereas in the swimming pools the presence of up to 1.5mg/l was not a constraint. Based on our results, the presence of mycobacteria in aquatic environments is close to 35% and their occurrence and diversity is affected both by contact with man and his activities as well as by the existence of animal life.

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.