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Bovine tuberculosis: epidemiological surveillance in slaughterhouses of Santa Fe (Argentina) and evaluation of the immune response in granulomatous lesions of infected animals

Ana Maria Canal Didier defended the PhD Thesis at the Faculty of Veterinary Medicine of the Complutense University of Madrid

June 22nd, 2012

Bovine tuberculosis remains an important disease in the context of animal health and public health. The worldwide increase in food need highlights the importance of control and eradication of zoonotic diseases in the region, benefiting the economy of these countries, several producers and exporters of meat and dairy products, and health of their populations. The Province of Santa Fe is a cattle farming area par excellence, with nearly 7,000,000 cattle, 32,167 RENSPA (National Register of Agricultural Producers Health) corresponding to 60% for breeding, 22.7% for wintering, 13.8% for dairy cattle (tambo), 1% for Feed Lot and 2.3% for cabins and/or mixed with a productive and participative health system whose aim is to deepen coordination and optimize the necessary actions, respecting the roles of different actors, to achieve better health status in cattle herds and increase quality food production. In this framework, the implementation of an Epidemiological Surveillance System of Bovine Tuberculosis through the Faena (post-mortem surveillance) charges a superlative importance because it is a diagnosable disease macroscopically in the routine work of inspection services to the health of refrigerators. The Tuberculosis Surveillance System through the slaughter (SISVIT) was implemented in 2006 as a joint effort between the Ministry of Production, Food Safety Agency and Santa Fe SENASA, in order to record and analyze the information from animals slaughtered in cold storage and other factors of the Provinces, to supervise free certified herds, to notify producers who had sent to slaughter animals with gross lesions consistent with Bovine tuberculosis and to provide information to establish control and eradication plans. On the other hand and in order to provide knowledge of the pathogenesis and typing of strains of Mycobacterium bovis acting in the province, bacteriological analysis and molecular typing by spoligotyping together with evaluation by histopathology of cytokines IFN-γ, TNFα, IL-1β, IL-10 and TGF immunohistochemistry of samples obtained in slaughterhouses. Software was installed (SISVIT) in the 36 refrigerators for data loading regarding slaughtered cattle with lesions compatible with tuberculosis bovina. The reporting period covers the years 2008 and 2009. The Province of Santa Fe has an annual participation of estimated cattle slaughter of 17% (2,500,000) of the national slaughter cattle, which ranges approximately in 14,000,000. In the two years, the Epidemiological Surveillance System of Tuberculosis through slaughter (SISVIT) was able to record and monitor 3,254,937 cattle that passed through the slaughterhouse, of which 2,200,514 came directly from producers and 546,905 of Shots Fair. The remainder relates to cattle from other provinces, and there were also 46,205 cattle slaughtered in slaughterhouses of Santa Fe located off. In total, 29,062 cattle were detected with lesions compatible with bovine tuberculosis, 50% of them corresponded to direct sales from producer to slaughter by which it has complete information about the source of 15,637 affected cattle. From this information, notices were distributed to 6666 farmers. Establishments with affected cattle were detected in all departments and different production systems, with the highest percentage in dairy herds. Cows and bulls were the categories most committed (2.3% and 3% respectively), but lesions were observed in calves, steers, bulls and heifers also. Of a total of 4365 dairy farms, 51.4% had free certification. In the course of two years, 86 free establishments that sent troops to slaughter including cattle affected were detected, identifying 203 animals in this situation. Samples were taken from 57 cattle from 24 establishments, of which 18 had more than one organ with granulomatous lesions; granulomas with caseous center of typical grade 3 and 4 (Wangoo y col., 2005) predominate in retropharyngeal lymph nodes, mediastinal and lung. Ziehl-Neelsen staining revealed acid-fast bacilli, showing homogeneity between the granulomas number of grade 3 and 4. The isolation of M. bovis was performed in 83% of the cattle. Immunoreaction was observed for the five cytokines analyzed. IFN-γ was expressed strongly in necrotic centers in macrophages and epithelioid cells and less Langhans giant cells. TNF-α was not expressed in necrotic centers, however, was evident in macrophages of granulomas grade 4 rather than grade 3. IL-1β was the one with mild immune response in macrophages and epithelioid cells, coinciding in some granulomas grade 3 with low percentage of TNF-α. The anti-inflammatory cytokine IL-10 was expressed in macrophages and epithelioid cell granulomas grade 4, but three with generalized injury in TGF grade 3 and expressed in areas of necrosis in granulomas grade 4 and mainly in macrophages. Despite the chronicity, only eight had more than 30% of immunostaining. From the 47 isolates of M. bovis a total of two different spoligotypes were detected. Three were unique to Santa Fe, not found elsewhere ([spol-117 (SB1790), spol-132 (SB2166) and spol-133 (SB2117)]); both 132 and 133 were orphan types (with a single isolate each). Two spoligotypes were prevalent, spol-34 (SB0140) in 50% (n=24) of the isolates and present in 15 establishments in different regions, and the spb-21 (SB0130) in 15% (n=7) of the isolates, but all the animals came from the same establishment. Five types were also observed to form the 24% [spol-3 (SB0153), spol-17 (SB0131), spol-29 (SB0484), spol-75 (SB090) and spol-117 (SB1790)]; and five (11%) were unique [spol-4 (SB0145), spol-41 (SB1033), spol-123 (SB2165), spol-132 (SB2166) and spol-133 (SB2117)]. The information generated allowed the characterization of the geographical distribution of disease and spoligotypes. It gives a basis for future studies about the presence of bovine tuberculosis in the province, as well as allows its use as a tool for control strategies and eradication of this disease. It also provides official information to the producer about the health of their animals, the possible origin or source of infection, which aims to prompt actions to clean up the herd from consultation with the veterinarian. Also, it has revealed the presence of this disease in other provinces. Taking into account that the main cause of infection of bovine tuberculosis in the human population are the presence of bovine tuberculosis in cattle and the cattle and human contact (especially in the rural population, refrigerators and the meat industry staff, veterinarians, laboratory workers, among others), it is important to strengthen the control and eradication programs in the province, thereby promoting the early control of the disease. The study of lesions and the local immune response has revealed that the expression of cytokines in granulomas grade 3 and 4 was significant, with predominant immunoreactivity to IFN-γ. This fact suggests a strong immune response mediated by macrophages and epithelioid cells that remains over time, and a lower reactivity to IL-10 which would mean deficient anti-inflammatory cytokines in the medium. This work creates a model that can be used in other provinces, and could be the basis of a national network of epidemiological surveillance of bovine tuberculosis through the task, identifying the origin of each of the more than 14,000,000 million cattle that is slaughtered cattle annually in our country for the purpose of establishing their health status in view of future eradication of the disease

Bovine tuberculosis: epidemiological surveillance in slaughterhouses of Santa Fe (Argentina) and evaluation of the immune response in granulomatous lesions of infected animals

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Ana Maria Canal Didier

TITLE: Tuberculosis bovina: vigilancia epidemiológica en mataderos de la Provincia de Santa Fe (Argentina) y evaluación de la respuesta inmune en lesiones granulomatosas de animales infectados

TYPE: PhD Thesis

AUTHOR: Ana Maria Canal Didier

DIRECTORS: Aranaz A., Dominguez L. and Rodriguez-Bertos A.

DATE: June 22nd, 2012