Cássia Yumi Ikuta

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.

Evaluation of DNA extraction protocols for Brucella abortus pcr detection in aborted fetuses or calves born from cows experimentally infected with strain 2308

The objective of the present study was to improve the detection of B. abortus by PCR in organs of aborted fetuses from infected cows, an important mechanism to find infected herds on the eradication phase of the program. So, different DNA extraction protocols were compared, focusing the PCR detection of B. abortus in clinical samples collected from aborted fetuses or calves born from cows challenged with the 2308 B. abortus strain. Therefore, two gold standard groups were built based on classical bacteriology, formed from: 32 lungs (17 positives), 26 spleens (11 positives), 23 livers (8 positives) and 22 bronchial lymph nodes (7 positives). All samples were submitted to three DNA extraction protocols, followed by the same amplification process with the primers B4 and B5. From the accumulated results for organ, the proportion of positives for the lungs was higher than the livers (p=0.04) or bronchial lymph nodes (p=0.004) and equal to the spleens (p=0.18). From the accumulated results for DNA extraction protocol, the proportion of positives for the Boom protocol was bigger than the PK (p<0.0001) and GT (p=0.0004). There was no difference between the PK and GT protocols (p=0.5). Some positive samples from the classical bacteriology were negative to the PCR and viceversa. Therefore, the best strategy for B. abortus detection in the organs of aborted fetuses or calves born from infected cows is the use, in parallel, of isolation by classical bacteriology and the PCR, with the DNA extraction performed by the Boom protocol.

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.

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.

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.

ABSTRACT 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.

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.

Evaluation of Four DNA Extraction Protocols for Brucella abortus Detection by PCR in Tissues from Experimentally Infected Cows with the 2308 Strain

This study compared 4 protocols for DNA extraction from homogenates of 6 different organs of cows infected with the Brucella abortus 2308 strain. The extraction protocols compared were as follows: GT (guanidine isothiocyanate lysis), Boom (GT lysis with the carrying suspension diatomaceous earth), PK (proteinase K lysis), and Santos (lysis by boiling and freezing with liquid nitrogen). Positive and negative gold standard reference groups were generated by classical bacteriological methods. All samples were processed with the 4 DNA extraction protocols and amplified with the B4 and B5 primers. The number of positive samples in the placental cotyledons was higher than that in the other organs. The cumulated results showed that the Santos protocol was more sensitive than the Boom (p=0.003) and GT (p=0.0506) methods and was similar to the PK method (p=0.2969). All of the DNA extraction protocols resulted in false-negative results for PCR. In conclusion, despite the disadvantages of classical bacteriological methods, the best approach for direct diagnosis of B. abortus in organs of infected cows includes the isolation associated with PCR of DNA extracted from the cotyledon by the Santos or PK methods.

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

BackgroundTuberculosis 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 presentationAn 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.ConclusionsThe 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.