A comparison of Mycobacterium tuberculosis complex isolates from seals (pinnipeds) in Australia, Argentina, Uruguay, Great Britain and New Zealand was undertaken to determine their relationships to each other and their taxonomic position within the complex. Isolates from 30 cases of tuberculosis in six species of pinniped and seven related isolates were compared to representative and standard strains of the M. tuberculosis complex. The seal isolates could be distinguished from other members of the M. tuberculosis complex, including the recently defined 'Mycobacterium canettii ' and 'Mycobacterium caprae', on the basis of host preference and phenotypic and genetic tests. Pinnipeds appear to be the natural host for this 'seal bacillus', although the organism is also pathogenic in guinea pigs, rabbits, humans, Brazilian tapir (Tapirus terrestris) and, possibly, cattle. Infection caused by the seal bacillus is predominantly associated with granulomatous lesions in the peripheral lymph nodes, lungs, pleura, spleen and peritoneum. Cases of disseminated disease have been found. As with other members of the M. tuberculosis complex, aerosols are the most likely route of transmission. The name Mycobacterium pinnipedii sp. nov. is proposed for this novel member of the M. tuberculosis complex (the type strain is 6482 T =ATCC BAA-688 T =NCTC 13288 T ).Abbreviations: BCG, Bacille Calmette-Gué rin; FAFLP, fluorescent amplified fragment length polymorphism; PZA, pyrazinamide; SS, seal spoligotype; TCH, thiophen-2-carboxylic acid hydrazide.

SummaryWith the hypothesis that genetic variability of Mycobacterium bovis could influence virulence and immunopathology, five M. bovis strains were selected from an epidemiological study in Argentina on the basis of their prevalence in cattle and occurrence in other species. We then determined the virulence and the immunopathology evoked by these strains in a well-characterized mouse model of progressive pulmonary tuberculosis. The reference strain AN5 was used as a control. BALB/c mice infected with this M. bovis reference strain showed 50% survival after 4 months of infection, with moderate bacillary counts in the lung. Two weeks after inoculation, it induced a strong inflammatory response with numerous granulomas and progressive pneumonia. In contrast, strain 04-303, isolated from a wild boar, was the most lethal and its most striking feature was sudden pneumonia with extensive necrosis. Strain 04-302, also isolated from wild boar but with a different spoligotype, induced similar pathology but to a lesser extent. In contrast, strains 534, V2 (both from cattle) and 02-2B (from human) were less virulent, permitting higher survival after 4 months of infection and limited tissue damage. Strain AN5 and the cattle and human isolates induced rapid, high and stable expression of interferon (IFN)-g and inducible nitric oxide synthase (iNOS). In contrast, the more virulent strains induced lower expression of IFN-g, tumour necrosis factor-a and iNOS. Interestingly, these more virulent strains induced very low expression of murine beta defensin 4 (mBD-4); whereas, the control strain AN5 induced progressive expression of this anti-microbial peptide, peaking at day 120. The less virulent strains induced high mBD-4 expression during early infection. Thus, as reported with clinical isolates of M. tuberculosis, M. bovis also showed variable virulence. This variability can be attributed to the induction of a different pattern of immune response.

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Abstract. The confirmatory diagnosis of Mycobacterium bovis (M. bovis) in animal samples is carried out by culture in Stonebrink media. However, culture is very slow because of the extremely long duplication time of the bacillus and difficult because of the scarcity of bacilli in diagnostic samples. This study describes the development of a single-tube touch-down polymerase chain reaction (PCR) protocol for the detection of M. bovis using primers that target the IS6110 element. Spiked water and milk as well as routine diagnostic samples (milk and nasal swabs) from M. bovis-positive cattle were tested. This protocol allows the rapid and sensitive detection of M. bovis in bovine samples by enhancing the sensitivity of standard PCR amplification.

The first case of tuberculosis is described in a wild subantarctic fur seal (Arctocephalus tropicalis) found on the Argentine coast. There was hydrothorax and white firm granulomatous lesions of 40-50 mm in diameter in the lungs. Lesions consisted of a central area of caseous necrosis, an intermediate zone of epithelioid and lymphocytic mononuclear cells, and a peripheral zone of connective tissue. Biochemical and drug sensitivity tests and inoculation of Guinea pigs confirmed the identification as Mycobacterium tuberculosis complex. Arctocephalus tropicalis is the fifth pinniped species in which the M. tuberculosis complex has been detected. Since subantarctic fur seals are widely distributed in the Southern Hemisphere, it is possible that the tuberculosis cases may have a common origin and could spread to other austral regions and species.

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Summary An ELISA which detects bovine circulating IgG mycobacterial antibodies using M. bovis PPD as antigen was assessed. PPDs prepared from unheated cultures of two M. bovis strains were compared with autoclaved bovine PPD; the latter was found to be a more reliable antigen. Ninety per cent of bacteriologically confirmed tuberculous cattle were detected by this method (18/20), whereas 89.8% of negative reactions (44/49) were observed in healthy cattle from a tuberculosis‐free area. Antibody levels of tuberculin negative cattle from an endemic area did not differ significantly from antibody levels of cattle from a tuberculosis‐free area. An almost complete qualitative correlation was observed between instrumental and visual readings. In the present trial, the sensitivity and specificity of the ELISA were similar to those of the tuberculin skin test at its best. In addition, operative advantages and low cost of the ELISA make of it a valuable tool for the diagnosis of bovine tuberculosis. Zusammenfassung Bestimmung der Sensitivität und Spezifität eines enzymgebundenen Immunadsorptionstests (ELISA) zum Nachweis von mykobakteriellen Antikörpern bei boviner Tuberkulose Ein ELISA zum Nachweis von zirkulierenden, bovinen IgG‐Antikörpern mit M. bovis als Antigen wurde überprüft. Autoklaviertes bovines PPD erwies sich, verglichen mit PPDs aus nicht erhitzten Kulturen zweier M. bovis‐Stämme, als geeignetes Antigen. Mit Hilfe dieser Methode konnten bei Rindern mit bakteriologisch abgesicherter Tuberkulose in 90% der Fälle (18/20) Antikörper nachgewiesen werden. Negative Befunde wurden dagegen zu 89,9% bei gesunden Tieren aus einem tuberkulosefreiem Gebiet ermittelt. Zwischen den Antikörpergehalten tuberkulinnegativer Rinder aus einem endemischen Gebiet und Tieren aus dem tuberkulosefreien Gebiet konnte keine signifikante Abweichung festgestellt werden. Eine nahezu vollständige Korrelation bestand zwischen instrumenteller und visueller Ablesung. In der vorliegenden Untersuchung waren die Ergebnisse des ELISA bezüglich Sensitivität und Spezifität ähnlich denen des Tuberkulin‐Hauttestes. Darüber hinaus machen die technischen Vorteile und geringen Kosten den ELISA zu einem nützlichen Hilfsmittel für die Diagnostik der bovinen Tuberkulose.

Summary The purpose of this study was to evaluate the sensitivity of meat inspection at the slaughterhouse for the detection of tuberculous lesions in bovines. Ten out of 719 cattle (1.4%) from 18 herds were condemned for tuberculosis upon inspection. A sample of 178 animals from the 709 not condemned was selected at random, and bacteriological examination for mycobacteria was performed on lymph node pools specimens taken from each of them. M. bovis was cultured from five of these 178 specimens (2.8%). This means that the number of TB infected bovines in these herds is at least 10/15: 33% higher than those detected by the inspection. This rather low sensitivity of meat inspection could be attributed to the high rate of TB transmission in this area resulting in a relatively high proportion of small incipient lesions in bovines. These results confirm the importance of improving the control measures of the National Program of Bovine TB Control in Argentina. Zusammenfassung Zweck dieser Studie war es, die Genauigkeit der Fleischbeschau im Schlachthaus für den Nachweis tuberkulöser Veränderungen beim Rind zu überprüfen. Von 719 Rindern aus 18 Herden wurden zehn (1,4%) nach Untersuchung auf Tuberkulose beschlagnahmt. Von den restlichen 709 nicht beschlagnahmten Tieren wurden 178 zufällig ausgewählt, um die bakteriologische Untersuchung der Lymphknoten eines jeden Tieres auf Mykobakterien durchzuführen. Mycobacterium bovis wurde aus fünf von diesen 178 Tieren geprüften Tieren (2,8%) isoliert. Daraus ist ersichtlich, daß die Anzahl der mit Tuberkulose infizierten Rinder in diesen Herden mindestens um 33% höher ist als die bei der Fleischbeschau ermittelte. Diese geringe Nachweisempfindlichkeit der Fleischbeschau für Tuberkulose könnte der hohen Übertragungsrate von Tuberkulose in diesem Gebiet und dem daraus resultierenden relativ hohen Anteil kleiner, sich im Anfangsstadium befindlicher Läsionen zugeschrieben werden. Diese Ergebnisse bekräftigen die Wichtigkeit die Kontrollmaßnahmen des Nationalen Programms der Tuberkulosekontrolle beim Rind in Argentinien zu verbessern.