Canine Leproid Granuloma (CLG) Caused by Mycobacterial Species Closely Related to Members of Mycobacterium Simiae Complex in a Dog in Brazil.

This report describes the clinical features and molecular diagnosis of a case of canine leproid granuloma (CLG) caused by mycobacterial strains of the Mycobacterium simiae complex in Brazil. A 12-year-old non-neutered male Labrador Retriever dog was presented with a 2-week history of progressive painless cutaneous lesions. Ulcerated nodules with hematic crusts were observed on the dorsal surface of the right and left pinna and on the metacarpal, metatarsal, and digits. Complete blood count, serum biochemistry, aspiration cytology of cutaneous lesions, biopsy for histopathological evaluation, culture for aerobic and anaerobic bacteria, polymerase chain reaction and DNA sequencing to identify mycobacterial species were performed. According to the clinical and histopathological findings, a diagnosis of CLG was established. Despite the negative result of the bacterial culture, mycobacterial identification was made by sequencing the hsp65 gene. Our findings highlight that mycobacterial species closely related to members of the M simiae clade can be causative agents of CLG.

High discrimination of Mycobacterium bovis isolates in Brazilian herds by spoligotyping.

Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis (M. bovis), that leads to economic losses in infected herds and it is also considered an important zoonosis. The molecular typing methods of M. bovis isolates are fundamental for the bovine tuberculosis surveillance system, and spoligotyping is the standard genotyping technique for this species. Thus, the aim of the present study is to analyze the spatial and cluster distribution of M. bovis strains from several regions of Brazil through molecular typing. Spoligotyping technique was applied on 422 isolates identified as M. bovis, and Ripley's K function was used to perform the spatial and cluster analysis of each identified profile. Forty-three (43) different profiles were identified and spoligotype SB0121 was the most frequent and showed a uniform pattern in the spatial distribution while spoligotypes SB0295, SB1380 and SB1050 formed clusters. In addition, three novel spoligotype profiles (SB2361, SB2362, SB2364) were identified in different herds. In this perspective, it is believed that molecular identification and typing can significantly improve the performance of surveillance systems for bovine tuberculosis in Brazil.

Evolutionary analysis of Mycobacterium bovis genotypes across Africa suggests co-evolution with livestock and humans.

Mycobacterium bovis is the pathogenic agent responsible for bovine tuberculosis (bTB), a zoonotic disease affecting mostly cattle, but also transmittable to humans and wildlife. Genetic studies on M. bovis allow to detect possible routes of bTB transmission and the identification of genetic reservoirs that may provide an essential framework for public health action. We used a database with 1235 M. bovis genotypes collected from different regions in Africa with 45 new Mozambican samples. Our analyses, based on phylogeographic and population genetics' approaches, allowed to identify two clear trends. First, the genetic diversity of M. bovis is geographically clustered across the continent, with the only incidences of long-distance sharing of genotypes, between South Africa and Algeria, likely due to recent European introductions. Second, there is a broad gradient of diversity from Northern to Southern Africa with a diversity focus on the proximity to the Near East, where M. bovis likely emerged with animal domestication in the last 10,000 years. Diversity indices are higher in Eastern Africa, followed successively by Northern, Central, Southern and Western Africa, roughly correlating with the regional archaeological records of introduction of animal domesticates. Given this scenario M. bovis in Africa was probably established millennia ago following a concomitant spread with cattle, sheep and goat. Such scenario could translate into long-term locally adapted lineages across Africa. This work describes a novel scenario for the spread of M. bovis in Africa using the available genetic data, opening the field to further studies using higher resolution genomic data.

Mycobacteria in Minas cheese commercialized in open fairs in São Paulo, Brazil / Mycobacteria em queijos tipo Minas comercializados em feiras-livres de São Paulo, Brasil

Mycobacterium bovis is the causative agent of bovine tuberculosis, a disease that affects dairy herds throughout the Brazilian territory, constituting a neglected zoonosis transmitted by raw milk and its derivatives. In this study, we evaluated the presence of M. bovis and other mycobacteria in Minas cheese obtained from open fairs in the city of São Paulo between 2012 and 2013. Samples (n = 133) were decontaminated using hexa-cetylpyridinium chloride and seeded on Stonebrink­Leslie medium. The isolates were submitted to molecular identification by TB Multiplex PCR targeting the 16S rRNA gene and amplicon nucleotide sequencing. From 16 cheese samples (12%), we obtained 26 putative colonies of Mycobacterium spp, none of which belonged to any of the Mycobacterium tuberculosis, Mycobacterium avium, or Mycobacterium intracellulare complexes. Phylogenetic analysis showed that sample sequences were grouped in a clade that includes only non-tuberculous mycobacteria with proximity to sequences obtained from Mycobacterium novocastrense (3 sequences), Mycobacterium holsaticum (1 sequence), and Mycobacterium elephantis (2 sequences). Although no epidemiological evidence was found regarding the importance of oral transmission of mycobacteria in healthy people, their importance in the immunosuppressed population remains uncertain.(AU)

Mycobacterium bovis é o agente da tuberculose bovina, doença que acomete o rebanho em todo território brasileiro e é uma negligenciada zoonose transmitida pelo leite e seus derivados. Este trabalho avaliou a presença de M. bovise outras micobactérias, em queijo minas meia-cura, obtidos em feiras-livres na cidade de São Paulo, entre os anos de 2012 e 2013. As amostras (n = 133) foram descontaminadas pelo método HPC (hexa-cetyl-pyridinium chloride) e semeadas em meio Stonebrink Leslie. Os isolados foram submetidos à identificação molecular por PCR TB multiplex, pesquisando-se o gene 16S rRNA, e ao sequenciamento nucleotídico. Dezesseis amostras (12%) possuiam 26 colônias sugestivas de Mycobacterium spp, mas nenhuma delas pertencia aos complexos Mycobacterium tuberculosis, Mycobacterium avium e Mycobacterium intracellulare. A análise filogenética mostrou que todas as amostras estavam agrupadas em clados que incluem apenas micobactérias não tuberculosas (MNT), sendo que algumas possuiam proximidade com sequências obtidas de Mycobacterium novocastrense (3 sequências), Mycobacterium hosaticum(1 sequência) e Mycobacterium elephantis (2 sequências). Embora no momento não haja evidência epidemiológica da importância da transmissão oral das micobactérias pra indivíduos saudáveis, sua importância na população imunossuprimida ainda é incerta.(AU)

Tuberculosis caused by Mycobacterium bovis infection in a captive-bred American bullfrog (Lithobates catesbeiana).

BACKGROUND: Tuberculosis is widely known as a progressive disease that affects endothermic animals, leading to death and/or economical losses, while mycobacterial infections in amphibians are commonly due to nontuberculous mycobacteria. To the authors' knowledge, this report describes the first case of bovine tuberculosis in a poikilothermic animal. CASE PRESENTATION: An adult female captive American bullfrog (Lithobates catesbeianus Shaw, 1802) died in a Brazilian aquarium. Multiple granulomas with acid-fast bacilli were observed in several organs. Identification of Mycobacterium bovis was accomplished by culture and PCR methods. The other animals from the same enclosure were euthanized, but no evidence of mycobacterial infection was observed. CONCLUSIONS: The American bullfrog was introduced in several countries around the world as an alternative husbandry, and its production is purposed for zoological and aquarium collections, biomedical research, education, human consumption and pet market. The present report warns about an episode of bovine tuberculosis in an amphibian, therefore further studies are necessary to define this frog species' role in the epidemiology of M. bovis.

Identification of Mycobacterium species and Rhodococcus equi in peccary lymph nodes.

Mycobacterium species and the virulence-associated proteins (vapA, vapB, and vapN genes) of Rhodococcus equi isolated from 330 lymph nodes of collared peccaries (Tayassu tajacu) and white-lipped peccaries (Tayassu pecari) intended for human consumption were investigated. Thirty-six (10.9%) R. equi strains were isolated; 3.3% (n = 11/330) were from white-lipped peccary lymph nodes, and 7.6% (25/330) were from collared peccary lymph nodes. Among the 11 isolates of R. equi from the white-lipped peccaries, 90.9% (n = 10/11) were obtained from the mesenteric lymph nodes, and only 9.1% (n = 1/10) were obtained from the mediastinal lymph nodes. In the 25 isolates of R. equi obtained from the collared peccaries, 40.0% (n = 10/25) were recovered from the mesenteric lymph nodes, 36% (n = 9/25) from the submandibular lymph nodes, and 24.0% (n = 6/25) from the mediastinal lymph nodes. No vapA, vapB, or vapN genes (plasmidless) or three host-associated types (pVAPA, pVAPB, and pVAPN) were identified among the R. equi isolates. Mycobacterium species were isolated in 3.03% (n = 10/330) of all the lymph nodes analyzed. Among the 10 mycobacterial isolates, 60% (n = 6/10) were from the white-lipped peccary lymph nodes, and 40% (n = 4/10) were from the collared peccary lymph nodes. Ten Mycobacterium species were detected by PCR-PRA with a predominance of M. avium type 1. Sequencing of the hsp65 and rpob genes revealed mycobacteria that were saprophytic (M. sinense and M. kumamotonense) and potentially pathogenic (M. colombiense and M. intracellulare) to humans and animals. To our knowledge, this is the first description of R. equi and/or mycobacterial species identified in the lymph nodes of peccary specimens. R. equi (plasmidless) and the mycobacterial species described here have been reported as causes of pulmonary and extrapulmonary infections in both immunocompetent and immunocompromised humans.

Cutaneous mycobacteriosis in a captive Amazonian manatee Trichechus inunguis.

An adult male Amazonian manatee Trichechus inunguis under human care presented with 3 circular cutaneous lesions on the dorsal aspect of the rostrum and between the nostrils (plenum). Initially these lesions were superficial, hypopigmented, without warmth and non-painful. Microbiological cultures of skin swabs isolated Candida sp. and Pseudomonas aeruginosa, and topical treatment with antiseptic, antifungal, anti-inflammatory and antibiotic medication was instituted. This treatment strategy did not lead to any clinical improvement, and after 6 mo, the lesions progressed to a confluent abscess (5.0 × 3.0 cm) with increased temperature and obvious discomfort on palpation. An impression smear of a cutaneous biopsy was submitted for Ziehl-Neelsen staining and after detection of acid-fast bacilli, the cutaneous biopsy and a swab from the lesion were sent for histopathology, culture and sensitivity testing. After 5 d of incubation and through PCR-restriction analysis of the isolates, Mycobacterium fortuitum and M. abscessus were identified. Sensitivity testing indicated that the isolates were susceptible to ciprofloxacin and clarithromycin, and after draining of the lesion and administration of systemic antibiotic treatment, there was rapid clinical improvement. This report describes non-healing lesions in an aquatic animal and illustrates the importance of evaluating the presence of non-tuberculous mycobacteria, opportunistic pathogens which are ubiquitous in the aquatic environment, in protracted, non-responsive cases. We also highlight the importance of a correct diagnosis and treatment approach, and we review concerns that these bacteria are zoonotic agents and are frequently resistant to conventional antibiotics.

Nontuberculous mycobacteria in milk from positive cows in the intradermal comparative cervical tuberculin test: implications for human tuberculosis infections.

Although the tuberculin test represents the main in vivo diagnostic method used in the control and eradication of bovine tuberculosis, few studies have focused on the identification of mycobacteria in the milk from cows positive to the tuberculin test. The aim of this study was to identify Mycobacterium species in milk samples from cows positive to the comparative intradermal test. Milk samples from 142 cows positive to the comparative intradermal test carried out in 4,766 animals were aseptically collected, cultivated on Lowenstein-Jensen and Stonebrink media and incubated for up to 90 days. Colonies compatible with mycobacteria were stained by Ziehl-Neelsen to detect acid-fast bacilli, while to confirm the Mycobacterium genus, conventional PCR was performed. Fourteen mycobacterial strains were isolated from 12 cows (8.4%). The hsp65 gene sequencing identified M. engbaekii (n=5), M. arupense (n=4), M. nonchromogenicum (n=3), and M. heraklionense (n=2) species belong to the Mycobacterium terrae complex. Despite the absence of M. tuberculosis complex species in the milk samples, identification of these mycobacteria highlights the risk of pathogen transmission from bovines to humans throughout milk or dairy products, since many of mycobacterial species described here have been reported in pulmonary and extrapulmonary diseases both in immunocompetent and immunocompromised people.

Serosurvey of Smooth Brucella, Leptospira spp. and Toxoplasma gondii in Free-Ranging Jaguars (Panthera onca) and Domestic Animals from Brazil.

This study investigated the exposure of jaguar populations and domestic animals to smooth Brucella, Leptospira spp. and Toxoplasma gondii in the Cerrado, Pantanal and Amazon biomes of Brazil. Between February 2000 and January 2010, serum samples from 31 jaguars (Panthera onca), 1,245 cattle (Bos taurus), 168 domestic dogs (Canis lupus familiaris) and 29 domestic cats (Felis catus) were collected and analysed by rose bengal test for smooth Brucella, microscopic agglutination test for Leptospira spp. and modified agglutination test for T. gondii. Cattle populations from all sites (9.88%) were exposed to smooth Brucella, but only one jaguar from Cerrado was exposed to this agent. Jaguars captured in the Cerrado (60.0%) and in the Pantanal (45.5%) were seropositive for different serovars of Leptospira spp., cattle (72.18%) and domestic dogs (13.1%) from the three sites and one domestic cat from Pantanal were also seropositive for the agent. The most prevalent serotype of Leptospira spp. identified in jaguars from the Cerrado (Grippotyphosa) and the Pantanal (Pomona) biomes were distinct from those found in the domestic animals sampled. Jaguars (100%), domestic dogs (38.28%) and domestic cats (82.76%) from the three areas were exposed to T. gondii. Our results show that brucellosis and leptospirosis could have been transmitted to jaguars by domestic animals; and jaguars probably play an important role in the maintenance of T. gondii in nature.

Draft Genome Sequence of Mycobacterium bovis Strain SP38, a Pathogenic Bacterium Isolated from a Bovine in Brazil.

We report a draft genome sequence of Mycobacterium bovis strain SP38, isolated from the lungs of a cow in Brazil. The assembly of reads resulted in 36 contigs in a total of approximately 4.37 Mb. Comparison of M. bovis strains sequenced to date will aid in understanding bovine tuberculosis in Brazil.

Serosurvey for selected viral infections in free-ranging jaguars (Panthera onca) and domestic carnivores in Brazilian Cerrado, Pantanal, and Amazon.

We investigated the exposure of jaguar (Panthera onca) populations and domestic carnivores to selected viral infections in the Cerrado, Amazon, and Pantanal biomes of Brazil. Between February 2000 and January 2010, we collected serum samples from 31 jaguars, 174 dogs (Canis lupus familiaris), and 35 domestic cats (Felis catus). Serologic analyses for antibodies to rabies virus, canine distemper virus (CDV), feline immunodeficiency virus (FIV), and for feline leukemia virus (FeLV) antigen were conducted. The jaguars from Cerrado and Pantantal were exposed to rabies virus, while the jaguars from the Pantanal and the dogs from all three areas were exposed to CDV. Two cats from the Amazonian site were antigen-positive for FeLV, but no jaguars had FeLV antigen or FIV antibody. Canine distemper and rabies viruses should be carefully monitored and considered potential threats to these jaguar populations. Currently FIV and FeLV do not appear to represent a health threat for jaguar populations in this area. Domestic dogs and cats in these areas should be vaccinated, and the movement of domestic animals around protected areas should be restricted.

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.

Isolation of Mycobacterium tuberculosis from captive Ateles paniscus.

An adult female red-faced black spider monkey (Ateles paniscus), housed for 2 years in the Parque Estoril Zoo in São Paulo, Brazil, showed apathy. Clinical examination revealed discrete emaciation, swelling and induration of lymph nodes, and presence of a mass in the abdominal cavity. Therapies with enrofloxacin, azithromycin, and ceftiofur were ineffective. The animal died after 6 months. Necropsy and histopathology confirmed granulommas in lymph nodes, parietal and visceral pleura, lungs, liver, spleen, and kidneys. Acid-fast bacilli were isolated and identified as Mycobacterium tuberculosis by polymerase chain reaction restriction analysis and Spoligotyping techniques. The zoo personnel and other animals that had had contact with the infected primate were negative to tuberculosis diagnostic procedures, such as sputum exam (baciloscopy) and thorax radiography. It was impossible to determine whether the infection occurred before or after the arrival of the animal to the Parque Estoril Zoo. This is the first report of M. tuberculosis infection in Ateles paniscus, a neotropical primate.