Teresa Albuquerque

Implications and challenges of tuberculosis in wildlife ungulates in Portugal: A molecular epidemiology perspective

Mycobacterium bovis and, more rarely, Mycobacterium caprae, may cause zoonotic bovine tuberculosis (bTB) in an extensive range of animal species. In Portugal, during 2009, a remarkable raise of bTB incidence was registered in cattle along with an increase of new cases in wildlife. In this work, we reassess and update the molecular epidemiology of bTB in wild ungulates by including 83 new M. bovis and M. caprae isolates from wild boar and red deer obtained during 2008-2009. Spoligotyping identified 27 patterns in wild ungulates, including 11 patterns exclusive from deer and five from wild boar. The genetic relatedness of wildlife and livestock isolates is confirmed. However, the relative prevalence of the predominant genotypes is different between the two groups. Contrasting with the disease in livestock, which is widespread in the territory, the isolation of bTB in wildlife is, apparently, geographically localized and genotypic similarities of strains are observed at the Iberian level.

Enhanced Detection of Tuberculous Mycobacteria in Animal Tissues Using a Semi-Nested Probe-Based Real-Time PCR

Bovine tuberculosis has been tackled for decades by costly eradication programs in most developed countries, involving the laboratory testing of tissue samples from allegedly infected animals for detection of Mycobacterium tuberculosis complex (MTC) members, namely Mycobacterium bovis. Definitive diagnosis is usually achieved by bacteriological culture, which may take up to 6–12 weeks, during which the suspect animal carcass and herd are under sanitary arrest. In this work, a user-friendly DNA extraction protocol adapted for tissues was coupled with an IS6110-targeted semi-nested duplex real-time PCR assay to enhance the direct detection of MTC bacteria in animal specimens, reducing the time to achieve a diagnosis and, thus, potentially limiting the herd restriction period. The duplex use of a novel β-actin gene targeted probe, with complementary targets in most mammals, allowed the assessment of amplification inhibitors in the tissue samples. The assay was evaluated with a group of 128 fresh tissue specimens collected from bovines, wild boars, deer and foxes. Mycobacterium bovis was cultured from 57 of these samples. Overall, the full test performance corresponds to a diagnostic sensitivity and specificity of 98.2% (CIP95% 89.4–99.9%) and 88.7% (CIP95% 78.5–94.7%), respectively. An observed kappa coefficient was estimated in 0.859 (CIP95% 0.771–0.948) for the overall agreement between the semi-nested PCR assay and the bacteriological culture. Considering only bovine samples (n = 69), the diagnostic sensitivity and specificity were estimated in 100% (CIP95% 84.0–100%) and 97.7% (CIP95% 86.2–99.9%), respectively. Eight negative culture samples exhibiting TB-like lesions were detected by the semi-nested real-time PCR, thus emphasizing the increased potential of this molecular approach to detect MTC-infected animal tissues. This novel IS6110-targeted assay allows the fast detection of tuberculous mycobacteria in animal specimens with very high sensitivity and specificity, being amenable and cost effective for use in the routine veterinary diagnostic laboratory with further automation possibilities.

Multihost tuberculosis: insights from the portuguese control program.

This paper describes the current situation of animal tuberculosis in Portugal, reviewing the accomplishments and constraints of the 2001–2009 period. Notwithstanding the substantial progress achieved with the implementation of a comprehensive test and cull scheme, notification, postmortem inspection and surveillance at slaughterhouses, herd and animal prevalence have unexpectedly increased in 2009. In parallel, the recent awareness of tuberculosis in local free-ranging wildlife species causes concern regarding the final steps towards eradication, demanding new approaches to the existing disease control policies.

Snapshot of Mycobacterium bovis and Mycobacterium caprae Infections in Livestock in an Area with a Low Incidence of Bovine Tuberculosis

Zoonotic bovine tuberculosis (bTB), caused by Mycobacterium bovis and, more rarely, by Mycobacterium caprae, is an important disease of livestock and of public health concern (1). A low incidence of bTB has been observed until now in Portugal, where a comprehensive eradication scheme has been implemented. Epidemiological surveys carried out in several countries suggest that test-and-slaughter policies may reduce strain diversity, favoring clonal expansion as a result of a bovine population bottleneck (2, 6). A remarkable exception to this observation was the first epidemiological analysis carried out in Portugal (3), which highlighted the apparent high genotype diversity of Mycobacterium bovis, possibly due to the low prevalence rates and the absence of major epidemics favoring ongoing transmission of the same strain(s). In the present work, we reassess and update the molecular epidemiology of bTB in Portugal. The previous analysis, concerning the period 2002 to 2007 (3), was extended to 2009 by including an additional 183 M. bovis and 10 Mycobacterium caprae isolates from cattle, goats, and sheep. Spoligotyping, based on the direct repeat (DR) region (5), identified 30 M. bovis patterns, including 11 profiles previously unrecognized in Portugal and seven new profiles that were deposited in the international database (www.mbovis.org). Spoligotype SB0157 remains the single M. caprae pattern acknowledged in the territory. The epidemiological significance of the most relevant genotypes in relation to the temporal and geographical distribution was investigated globally for the period 2002 to 2009 (results from this study and from reference 3). Consistent with previous findings, the high prevalence of SB0121 (25%) and SB0119 (13%) is confirmed. Remarkably, SB0119 and seven other profiles identified in Portugal differ from SB0121 in the absence of a single spacer (Table ​(Table1).1). Altogether, their cumulative prevalence in cattle is superior to 53%. Furthermore, they have also been detected in wildlife (3). Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis based on eight previously validated loci (4) distinguished 90 SB0121-related strains into 50 closely associated allelic profiles (data not shown), further suggesting their genetic relatedness. Taking this into consideration and the trend of the DR region to evolve primarily by the loss of single or multiple contiguous direct variable repeats (DVRs) (7), our observations suggest the presence of a clonal complex. Supporting this hypothesis is also the recent detection of SB0121-like strains in the national territory in comparison to SB0121, the sole genotype identified until 2004. To our knowledge, three SB0121-related spoligotypes, SB1090, SB1093, and SB1095, have been reported only in Portugal. Geographical distribution analysis also shows that SB0121 and SB0119 have remarkably expanded throughout the territory, being isolated in roughly all bTB-affected districts, while most SB0121-related genotypes are geographically localized (Fig. ​(Fig.11). FIG. 1. Geographical distribution across the districts of the six bTB-affected regions of mainland Portugal during the period 2002 to 2009 (joint results from this study and from reference 3) of M. bovis spoligotype SB0121 (•) and of strains with spoligotyping ... TABLE 1. Patterns, hosts, prevalence, and geographical distribution of M. bovis and M. caprae spoligotypes isolated in Portugal from 2007 to 2009 (this study) and 2002 to 2009 (joint results from this study and from reference 3) Our results support the notion that a country tends to have a dominant and locally evolved clone. Moreover, clonally related strains may emerge, exhibiting biogeographical specificities, possibly in response to barriers for expansion or adaptation to new ecological niches and new host species. Further studies are needed to improve our understanding of the epidemiological significance of geographically important M. bovis strains which may define specific lineages with increased virulence and the ability to escape tuberculin test and, consequently, slaughter of their hosts.

Pre-Multidrug-Resistant Mycobacterium tuberculosis Beijing Strain Associated with Disseminated Tuberculosis in a Pet Dog

ABSTRACT Resistance to isoniazid, ethambutol, and streptomycin was detected in a Mycobacterium tuberculosis strain, belonging to the Beijing family lineage, isolated from two nodule exudates of a Yorkshire terrier with generalized tuberculosis. This report alerts medical practitioners to the risk of dissemination of pre-multidrug-resistant tuberculosis (preMDR-TB) through exposure to M. tuberculosis-shedding pets.

Tuberculosis in mandrills at the Lisbon zoo.

SIR, — We would like to report two tuberculosis cases in a group of 10 mandrill baboons ( Mandrillus sphinx ) at the Lisbon zoo. In early 2006, a 16-year-old female, brought in from France in 1998, started to show signs of apathy, coughing and weight loss. Identical clinical signs were observed

New insights into resistance to colistin and third-generation cephalosporins of Escherichia coli in poultry, Portugal: Novel blaCTX-M-166 and blaESAC genes

The increasing incidence of intestinal colonization with extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and Gram negative organisms that has been observed in food animals such as poultry, cattle and pigs, are suggestive that animals, food and environment are potential sources of ESBL-producing bacteria. Hence, the aim of this study was to characterized commensal E. coli obtained from healthy broiler and turkey flocks at slaughter for the presence of penicillinases-, ESBL-, extended-spectrum AmpC (ESAC)-, plasmid-mediated quinolone resistance- and MCR-encoding genes. Study of clonal relatedness showed genetic diversity among CTX-M-type, SHV-12 and TEM-52 producing isolates with human isolates of the same type, was also assessed. We detected that eleven (5.4%, 11/202) and forty-five (2.2%, 45/185) E. coli isolates from broilers and turkeys, respectively, carried blaESBL or blaESAC genes and two isolates from turkeys carried mcr-1 gene. A new variant blaCTX-M-166 was reported in a multidrug resistant isolate from a broiler flock. Overall, we detected a diversity of resistance mechanisms among E. coli from food-producing animals, all of them with high importance at a public health level.

Rapid identification of veterinary-relevant Mycobacterium tuberculosis complex species using 16S rDNA, IS6110 and Regions of Difference-targeted dual-labelled hydrolysis probes

Members of the Mycobacterium tuberculosis complex (MTC) are causative agents of tuberculosis (TB) in both humans and animals. MTC species are genetically very similar but may differ in their epidemiology, namely geographic distribution and host preferences, virulence traits and antimicrobial susceptibility patterns. However, the conventional laboratory diagnosis does not routinely differentiate between the species of the MTC. In this work we describe a rapid and robust two-step five-target probe-based real-time PCR identification algorithm, based on genomic deletion analysis, to identify the MTC species most commonly associated with TB in livestock and other animals. The first step allows the confirmation of the cultures as MTC members, by targeting their IS6110 element, or as a mycobacterial species, if only a 16S rDNA product is detected in the duplex amplification reaction. If a MTC member is identified, the second amplification step allows the assessment of the presence or absence of the RD1, RD4 and RD9 genomic regions. The correspondent pattern allows us to infer the species of the isolate as M. tuberculosis (if all RDs are present), Mycobacterium caprae (if only RD1 and RD4 are present) and Mycobacterium bovis (if only RD1 is present). The identification algorithm developed presented an almost perfect agreement with the results of the routine bacteriological analysis, with a kappa coefficient of 0.970 (CI(P95%) 0.929-1.000). The assay is able to be adaptable to automation and implementation in the routine diagnostic framework of veterinary diagnostic laboratories, with a particular focus for reference laboratories.

Implementation of a zebrafish health program in a research facility : A 4-year retrospective study

Abstract In the past two decades, zebrafish (Danio rerio)-based research has contributed to significant scientific advances. Still, husbandry and health programs did not evolve at the same pace, as evidenced by the absence of general guidelines. Health monitoring is essential to animal welfare, to permit animal exchanges across facilities, to contribute to robust experimental results, and for data reproducibility. In this study, we report a health program implemented in a zebrafish research facility to prevent, monitor, and control pathogen, and disease dissemination. This program includes quarantine, routine health screening of sentinels, and nonroutine screenings of retired animals and sick/moribund individuals. An extensive list of clinical signs, lesions, and pathogens was monitored based on: daily observation of fish, necropsy, histology, and bacterial culture. The results indicate that the combined analysis of sentinels with the evaluation of sick/moribund animals enables a comprehensive description not only of pathogen prevalence but also of clinical and histopathologic lesions of resident animals. The establishment of a quarantine program revealed to be effective in the reduction of Pseudoloma neurophilia frequency in the main aquaria room. Finally, characterization of the colony health status based on this multiapproach program shows a low prevalence of lesions and pathogens in the facility.

Zoonotic potential of multidrug resistant Escherichia coli clonal groups in Portugal

The efflux pump QepA confers decreased susceptibility to hydrophilic fluoroquinolones (e.g., norfloxacin, ciprofloxacin, and enrofloxacin). In this study, we characterized the third variant, named qepA3, collected from an Escherichia coli isolate in Portugal. INSRA6015 was isolated in 2005 from the urine of a 77-year-old female patient hospitalized at the Hospital Fernando Fonseca, Portugal. Susceptibility testing was performed by disk diffusion and MIC methods, (SFM and EUCAST guidelines, respectively). PCR and sequencing were used to screen and identify bla (bla TEM , bla SHV , bla OXA , bla CTX-M and plasmid-mediated ampC ) genes, as well as plasmid-mediated quinolone resistance ( qnrA , qnrB , qnrC , qnrD , qnrS , qepA and aac(6’)Ib-cr ), and the quinolone resistance-determining regions (QRDR: gyrA , gyrB , parC , and parE ) genes. PCR-mapping was used to characterize the genetic environment of the new qepA3 gene. Transfer of resistance of the QepA3 determinant, was performed through electroporation, using the E. coli TOP10 as recipient. Plasmid content was characterized by PCR-based replicon typing. Molecular characterization of INSRA6015 showed the presence of bla TEM-1, bla CMY-2 and a new variant of qepA possessing two nucleotide substitutions, leading to Phe85Leu and Val134Ile changes. This variant, named QepA3, conferred a similar phenotype to that of the QepA1 and QepA2 determinants. Sequencing of the QDRD detected substitutions Ser83Leu and Asp87Asn in the GyrA subunit and Glu84Lys in the ParC subunit, which are consistent with the high resistance to ciprofloxacin observed in the MICs. Sequence analysis of qepA3 genetic environment revealed that the gene was located inside a genetic structure identical to that of previously described for qepA1 and qepA2 . It is noteworthy that qepA3 gene, as qepA2 , was not associated with the rmtB gene encoding an aminoglycoside ribosomal methylase, contrarily to qepA1. PCR-based replicon typing indicated the presence of the IncF plasmid. We have identified and characterized a new variant of the plasmid-mediated efflux pump QepA, which is responsible for the increased levels of resistance to several clinically important quinolones, such as ciprofloxacin, and norfloxacin. This is, at our knowledge, the first description of the co-production of QepA and CMY-2. The study highlights the need of surveillance of this resistance mechanism and reinforces a more careful use of quinolones.