Limited usefulness of the IS6110 touchdown-PCR in blood for tuberculin skin test false-negative cattle with serological response to Mycobacterium bovis.

Ante-mortem diagnosis of bovine tuberculosis (bTB) is based mainly on the tuberculin skin test (TST) and the ɣ-IFN release assay (IGRA). Some infected animals escape screening tests, thus, limit herd sanitation. Previous reports have suggested a predominant pattern of multi-organ lesions attributable to Mycobacterium bovis (the causative agent of bTB) bacteraemia. A case-control study was conducted to investigate blood PCR as an alternative tool for improving ante-mortem detection of TST false-negative bovines. Cases comprised 70 TST false-negative bovines (cases), which were serology positive, and controls included 81 TST positive bovines; all of them confirmed as infected with M. bovis. Detection of the IS6110 target through touchdown blood-PCR (IS6110 TD-PCR) was performed. The positivity of the blood-PCR was 27.2% in the control group. This performance was similar to the 15% obtained among cases (p = 0.134). Most cases identified by the IS6110 TD-PCR exhibited focalized lesions (p = 0.002). Results demonstrated that blood-PCR could detect TST false-negative cattle, even if they are negative for IGRA. Considering that cases exhibited humoral response to M. bovis, further studies conducted in a pre-serological stage could provide evidence about the real contribution of the technique in herds.

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.

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.

Production of Mycobacterium bovis Antigens Included in Recombinant Occlusion Bodies of Baculovirus.

Bovine tuberculosis (bTB) is a disease produced by Mycobacterium bovis that affects livestock, wild animals, and humans. The classical diagnostic method to detect bTB is measuring the response induced with the intradermal injection of purified protein derivative of M. bovis (PPDb). Another ancillary bTB test detects IFN-γ produced in whole blood upon stimulation with PPDb, protein/peptide cocktails, or individual antigens. Among the most used M. bovis antigens in IFN-γ assays are the secreted proteins ESAT-6 and CFP-10, which together with antigen Rv3615c improve the sensitivity of the test in comparison to PPDb. Protein reagents for immune stimulation are generally obtained from Escherichia coli, because this bacterium produces a high level of recombinant proteins. However, E. coli recombinant antigens are in general contaminated with lipopolysaccharides and other components that produce non-specific IFN-γ secretion in in vitro assays. In this work, we produced the relevant ESAT-6, CFP-10, and Rv3615c M. bovis antigens as fusions to the polyhedrin protein from the baculovirus AcMNPV. We obtained chimeric proteins effectively incorporated to the occlusion bodies and easily purified the recombinant polyhedra with no reactive contaminants. In an IFN-γ assay, these fusion proteins showed equivalent sensibility but better specificity than the same M. bovis proteins produced in E. coli.

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.

Hepatitis B virus and hepatitis D virus in blood donors from Argentina: circulation of HBsAg and reverse transcriptase mutants.

In Argentina, current procedures to ensure the safety of the blood supply for transfusion include the serologic detection of specific blood-borne infections. The aim of this study was to evaluate the prevalence and the genetic diversity of hepatitis B virus (HBV) and hepatitis D virus (HDV) in blood donor populations from two distantly located Argentine regions. Data from 56,983 blood donations from the Favaloro Foundation, in the city of Buenos Aires (Central Region), and the Central Blood Bank of Misiones Province (Northeast Region) were analyzed. Samples that were reactive for HBsAg were analyzed for HBV-DNA characterization and HDV serological and molecular analysis. The HBV prevalence was 0.12 % for HBsAg and 1.68 % for anti-HBc antibodies in Buenos Aires, and 0.73 % and 8.55 %, respectively, in Misiones. Seventy-seven HBsAg-reactive samples were analyzed by polymerase chain reaction for HBV-DNA. Subgenotypes A2, B2, C2, F1b and F4 (Buenos Aires) and F1b and D3 (Misiones) were detected. Several mutations within the major hydrophilic region of HBsAg, the reverse transcriptase, the basal core promoter, and the precore/core were detected. HDV genotype 1 was identified in Buenos Aires. This study confirms the circulation of several HBV subgenotypes, as well as known and newly identified variants, and the presence of HDV1 in this population. A thorough investigation has to be carried out to evaluate the clinical importance of some of the documented mutations as well as those detected in the HDV1 case.

Pautas para el diagnóstico, notificación y asesoramiento de la infección por virus linfotrópico T humano tipo 1 y 2 (HTLV-1/2) en Argentina / Guidelines for the diagnosis, notification and advising for human T-cell lymphotropic virus type 1 and 2 infection in Argentina

El virus linfotrópico T-humano tipo 1 (HTLV-1), primer oncoretrovirus humano descubierto, es el agente etiológico de la Leucemia de Células T del Adulto (ATL) y de la Mielopatía Asociada al HTLV-1 o Paraparesia Espástica Tropical (HAM/TSP). El HTLV-2, no tiene un rol etiológico definido, si bien se lo ha asociado con síndromes neurológicos similares a la HAM/TSP. En Argentina, la detección de anticuerpos para HTLV-1/2 en donantes de sangre es obligatoria desde noviembre de 2005 (resolución 865/2006 del Ministerio de Salud y Ambiente), si bien fue recomendada por la Asociación Argentina de Hemoterapia e Inmunohematología desde el año 1997. Uno de los problemas que se presenta en nuestro país, es la notificación de resultados de esta infección y las dificultades que debe afrontar el médico para brindar la información correcta. En este trabajo se presenta una visión general sobre estos retrovirus, y en especial se brinda información sobre diagnóstico, patogenia y las conductas a seguir por los profesionales de la salud ante la necesi­dad de informar resultados basados únicamente en pruebas de tamizaje o con serología positiva para HTLV­1/2. Para el mismo, nos hemos basado en las recomendaciones y lineamientos elaborados por los Centros de Control de Enfermedades (CDC) y Prevención y el grupo de trabajo del Servicio de Salud Pública de Estados Unidos (USPHS Working Group) dirigidas a las personas infectadas y a trabajadores de la salud e instituciones oficiales de salud pública.

Human T-celllymphotropic virus type 1 (HTLV-1), the first human oncoretrovirus discovered, is the ethiologic agent of Adult T-cell Leukimia (ATL) and HTLV-1 Associated Mielopathy o Tropical Spastic Paraparesis (HAM/TSP). HTLV-2, has not a defined ethiopathology, although it has been associated to neurologic syndroms similar to HAM/TSP Screening for HTLV-1/2 antibodies in blood donors is mandatory since 2005 (Resolution 865/2006 of the Ministry of Health, Argentina) although it has been recomended by the Hemotherapy and Immunehemathology Association since 1997. One of the problems in our country is the notification of the results and the difficulties encountered by the medical doctor in order to provide the appropriate information. In this study, we provide an outlook of these retroviruses, and especially we give information about diagnosis, pathogenesis and guidelines for health professionals when HTLV-1/2 positive serology needs to be notified. We based these recommendations on the guidelines elaborated by the Centers for Disease Control and Prevention and the working group of the US Public Health Service (USPHS Working Group) directed to infected people and to health workers and official institutions of public health.