Publicação
Epidemiological Importance of Rodents as Reservoirs of Leptospira in Maronesa Cattle Farms in Trás-os-Montes Region
| Resumo: | Leptospirosis is a zoonosis caused by pathogenic spirochetes of genus Leptospira, maintained in nature by numerous reservoirs, free-living and domestic animals, constituting a potential source of infection for humans, and other susceptible hosts (Turner, 1967; Little, 1986). Leptospirosis is considered by O.I.E. (2008) as a disease of importance to international trade, and it is included in the list of “Multiple species diseases.” Leptospirosis is also regarded as a re-emerging zoonosis (Higgins, 2004). The emergence of several zoonoses seems related to a combination of factors namely: the global warming, expanding human populations, globalisation of trade, intensification of wildlife farming, and microorganisms associated to wildlife incoming intensive livestock production systems (Bengis et al., 2004). Indeed, leptospirosis causes a decrease in the income of cattle farms, mainly due to its impact on the reproduction programs. Financial damages of considerable economic loss, through reproductive wastage due to chronic infection with Hardjo serovar (Sejroe serogroup), result mainly from infertility, abortion in the last months of gestation, premature births, death of calves of 15-60 days, death of adult animals, decrease of the milk and meat productions, and rejection of bulls for artificial insemination (Ellis, 1994). Rodents, the major renal carriers of Leptospira, assume a very important role on disease transmission to man and animals (Little, 1986). Previous studies on the role of wildlife leptospirosis in Portugal have been conducted in the south and central mainland Portugal and in the archipelago of Azores by Collares-Pereira et al. (2000a), but not in the north. However, the last serological survey, in cattle (n=1,433), carried out at a national level (Paiva-Cardoso et al., 2000) found the high seropositivity rates in the northern regions, namely Trás-os-Montes (66.3%), Beira Litoral (53.3%) and Entre-Douro e Minho (39,7%), though the important percentage observed in the south, in Alentejo (75.9%) and Ribatejo e Oeste (36.8%) regions. In addition, the occurrence of different agglutination patterns in the bovines studied when compared to cattle from other regions of Portugal would be reported in further studies (Paiva-Cardoso, 2000; Paiva-Cardoso et al., 2001). The main objective of the present research was to contribute to the knowledge of bovine leptospirosis in the north of Portugal, particularly in Trás-os-Montes, region where indigenous Maronesa cattle is bred under traditional management systems. Nowadays, the main income source from the production of this breed is the production of calves for beef consumption, due to the high quality of their meat; thus, the maintenance of high reproductive levels is essential, within a strategy of preservation of this indigenous breed, based on measures beyond the genetic improvement, that allow a viable economic production without loosing the high standard for meat quality. Given that, the present research attempted for the first time the isolation of pathogenic strains from local small mammals, in view of a more specific contribution to the pioneer research about the role of these renal Leptospira chronic carriers, in Maronesa breeding smallholdings. A total of 308 small mammals, mainly rodents, were trapped on cattle smallholdings of Trás-os-Montes region, with the following distribution: Mus musculus (n=255), M. spretus (n=24), Rattus norvegicus (n=19), R. rattus (n=3), Apodemus sylvaticus (n=4) and Crocidura russula (n=3) (Paiva-Cardoso et al., 2005a). Seventeen rodent-isolates were obtained by culture in selective media as follows: 13 strains from Mus musculus, three from Mus spretus and one from Rattus norvegicus. Of these, only 14 strains were forwarded for identification by microscopic agglutination test (MAT), monoclonal antibodies and restriction endonuclease analyses, being further used in the reference MAT under the small mammals and cattle serosurveys. Eleven strains have been identified as belonging to Ballum serogroup (serovar Ballum), one to Sejroe group (serovar Saxkoebing) and the remaining two strains to Pomona group (serovar Kunming?). This latest isolated-serovar is still unknown in Portugal (Paiva-Cardoso et al., 2005a) and showed reactivity profiles very different from those obtained with Mozdok and Tsaratsovo strains previously isolated in Portugal (Collares-Pereira et al., 1996, 1997a, 1997b, 2000a, 2000b; Rocha, 1990; Rocha, 2004). Once the most closely related serovar to these Pomona isolated-strains has only been found in China (Zhang et al., 1987; Brenner et al., 1999; Salaün et al., 2006), there is a distinct possibility that these isolates represent a new serovar, whose clinical meaning to cattle health needs further evaluation. Regarding Sejroe serogroup, the isolation of a serovar (Saxkoebing) from the house-mouse Mus musculus is a very important epidemiological finding, not only because it‟s the first report in Portugal, but also in the Iberian Peninsula (Paiva-Cardoso et al., 2005a). To overcome the reduced information gathered by culturing, for the renal carrier state evaluation of the small mammals analysed, a polymerase chain reaction (PCR) was performed on 278 (90.3%) kidney samples, using three pairs of primers: G1/G2 derived from the sequence of the recombinant plasmid pLIPs60; a primer set designed from LipL21 gene sequence and a Southern Blotting using a 257 bp probe; and a primer set designed from LipL32 gene sequence with a 423 bp probe in the Southern Blotting. Leptospiral DNA was detected in 54% (150+/278) of the small mammals examined, that is, near 10 times greater than the percentage of 5.5 % (17+/308) obtained by culture. These bacteriological data confirm the generalized occurrence of Leptospira infection in the rodents‟ population studied, and anticipate the increased importance of these vertebrates in the transmission of leptospires to cattle, especially concerning accidental infections (Paiva-Cardoso et al., 2005b). Two hundred and eighty two sera from small mammals (91.6%) were tested by the microscopic agglutination test. The seroreactivity rate (55%) was similar to that found in the molecular approach for the search of leptospiral DNA, meaning that a spirochete infection took place in a recent past. The use of local isolated field-strains, as MAT live-antigens, contributed to increase the diagnostic value of this laboratory approach in animals with a potentially reduced immune response due to their chronic renal carrier condition of these leptospires. In parallel, the study of anti-Leptospira seroreactivity in cattle, aiming at the indirect evaluation of the occurrence of accidental infections by murine transmitted leptospires, evidenced a high (61.0%) percentage of reactive animals at low titres, equal or higher than 1: 10 (geometric mean titre 1:17). As expected, the anti-Sejroe titres were the most common, representing 26.7% of the agglutinations detected. This finding may not only be due to the high number of bovines with an anti-Hardjo response, as an endemic cattle infection, but also to the contact with serovar Saxkoebing isolated from the house-mice Mus musculus present in the same ecosystem. The seroreactivity of 12.1% with Ballum serogroup was also important, suggesting that exposure of cattle to rodent maintained-serovars of this serogroup is high in this region. Serogroup Pomona registered a serological prevalence of 11.8%, which was compatible with the existence of incidental infections with its serovars, namely serovar Kunming (?) firstly isolated in Portugal. Similarly to rodents, the greatest immunogenicity of the antigens (isolates) used in MAT, certainly contributed to the important detection of specific agglutinins in the cattle surveyed. The intensive management systems for bovines have shown to be an increased risk for the greater contact with infected rodents, especially Mus musculus, the most trapped animal species and the one with a higher number of isolates obtained. However, Leptospira transmission risk by other species was also high, due to the strong seropositivity detected in Rattus rattus (100%), Apodemus sylvaticus (75%), Mus spretus (56.5%) and Rattus norvegicus (55.7%). These findings assess the need for an effective control of rodents in the smallholdings studied, due to the wide inexistence of cattle protective measures, in particular, in Bragança district, where the presence of anti-Leptospira agglutinins was significantly higher (p<0.0001) than the one for Vila Real district. Amongst the pointed reasons to justify this observation, genetic factors of the breed (Mirandesa) with a higher density in that geographical area are admitted, but other eco-epidemiological studies are needed to explain the differences observed. On the contrary, Maronesa showed to be less sensitive to leptospirosis than the remaining non-Maronesa breeds, being genetic factors together with the type of cattle management also suggested as potential reasons to explain this feature. In conclusion, Leptospira infection is widely spread in rodents of Trás-os-Montes region and the percentage of renal chronic carriers is sufficiently important to increase awareness regarding cattle production of indigenous breeds, including Maronesa, though its (apparent) higher resistance to disease. Thus, there is an urgent need towards the implementation of surveillance and control measures of leptospirosis, in order to reduce both the consequences for human and animal health, besides the inherent social and economic effects in this Northern Portuguese region. |
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| Autores principais: | Paiva-Cardoso, Maria das Neves |
| Assunto: | Bovinae Leptospirose Leptospira Região de Trás-os-Montes (Portugal) Raça maronesa Epidemiologia molecular e serologia |
| Ano: | 2009 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso restrito |
| Instituição associada: | Universidade de Trás-os-Montes e Alto Douro |
| Idioma: | inglês |
| Origem: | Repositório da UTAD |
| Resumo: | Leptospirosis is a zoonosis caused by pathogenic spirochetes of genus Leptospira, maintained in nature by numerous reservoirs, free-living and domestic animals, constituting a potential source of infection for humans, and other susceptible hosts (Turner, 1967; Little, 1986). Leptospirosis is considered by O.I.E. (2008) as a disease of importance to international trade, and it is included in the list of “Multiple species diseases.” Leptospirosis is also regarded as a re-emerging zoonosis (Higgins, 2004). The emergence of several zoonoses seems related to a combination of factors namely: the global warming, expanding human populations, globalisation of trade, intensification of wildlife farming, and microorganisms associated to wildlife incoming intensive livestock production systems (Bengis et al., 2004). Indeed, leptospirosis causes a decrease in the income of cattle farms, mainly due to its impact on the reproduction programs. Financial damages of considerable economic loss, through reproductive wastage due to chronic infection with Hardjo serovar (Sejroe serogroup), result mainly from infertility, abortion in the last months of gestation, premature births, death of calves of 15-60 days, death of adult animals, decrease of the milk and meat productions, and rejection of bulls for artificial insemination (Ellis, 1994). Rodents, the major renal carriers of Leptospira, assume a very important role on disease transmission to man and animals (Little, 1986). Previous studies on the role of wildlife leptospirosis in Portugal have been conducted in the south and central mainland Portugal and in the archipelago of Azores by Collares-Pereira et al. (2000a), but not in the north. However, the last serological survey, in cattle (n=1,433), carried out at a national level (Paiva-Cardoso et al., 2000) found the high seropositivity rates in the northern regions, namely Trás-os-Montes (66.3%), Beira Litoral (53.3%) and Entre-Douro e Minho (39,7%), though the important percentage observed in the south, in Alentejo (75.9%) and Ribatejo e Oeste (36.8%) regions. In addition, the occurrence of different agglutination patterns in the bovines studied when compared to cattle from other regions of Portugal would be reported in further studies (Paiva-Cardoso, 2000; Paiva-Cardoso et al., 2001). The main objective of the present research was to contribute to the knowledge of bovine leptospirosis in the north of Portugal, particularly in Trás-os-Montes, region where indigenous Maronesa cattle is bred under traditional management systems. Nowadays, the main income source from the production of this breed is the production of calves for beef consumption, due to the high quality of their meat; thus, the maintenance of high reproductive levels is essential, within a strategy of preservation of this indigenous breed, based on measures beyond the genetic improvement, that allow a viable economic production without loosing the high standard for meat quality. Given that, the present research attempted for the first time the isolation of pathogenic strains from local small mammals, in view of a more specific contribution to the pioneer research about the role of these renal Leptospira chronic carriers, in Maronesa breeding smallholdings. A total of 308 small mammals, mainly rodents, were trapped on cattle smallholdings of Trás-os-Montes region, with the following distribution: Mus musculus (n=255), M. spretus (n=24), Rattus norvegicus (n=19), R. rattus (n=3), Apodemus sylvaticus (n=4) and Crocidura russula (n=3) (Paiva-Cardoso et al., 2005a). Seventeen rodent-isolates were obtained by culture in selective media as follows: 13 strains from Mus musculus, three from Mus spretus and one from Rattus norvegicus. Of these, only 14 strains were forwarded for identification by microscopic agglutination test (MAT), monoclonal antibodies and restriction endonuclease analyses, being further used in the reference MAT under the small mammals and cattle serosurveys. Eleven strains have been identified as belonging to Ballum serogroup (serovar Ballum), one to Sejroe group (serovar Saxkoebing) and the remaining two strains to Pomona group (serovar Kunming?). This latest isolated-serovar is still unknown in Portugal (Paiva-Cardoso et al., 2005a) and showed reactivity profiles very different from those obtained with Mozdok and Tsaratsovo strains previously isolated in Portugal (Collares-Pereira et al., 1996, 1997a, 1997b, 2000a, 2000b; Rocha, 1990; Rocha, 2004). Once the most closely related serovar to these Pomona isolated-strains has only been found in China (Zhang et al., 1987; Brenner et al., 1999; Salaün et al., 2006), there is a distinct possibility that these isolates represent a new serovar, whose clinical meaning to cattle health needs further evaluation. Regarding Sejroe serogroup, the isolation of a serovar (Saxkoebing) from the house-mouse Mus musculus is a very important epidemiological finding, not only because it‟s the first report in Portugal, but also in the Iberian Peninsula (Paiva-Cardoso et al., 2005a). To overcome the reduced information gathered by culturing, for the renal carrier state evaluation of the small mammals analysed, a polymerase chain reaction (PCR) was performed on 278 (90.3%) kidney samples, using three pairs of primers: G1/G2 derived from the sequence of the recombinant plasmid pLIPs60; a primer set designed from LipL21 gene sequence and a Southern Blotting using a 257 bp probe; and a primer set designed from LipL32 gene sequence with a 423 bp probe in the Southern Blotting. Leptospiral DNA was detected in 54% (150+/278) of the small mammals examined, that is, near 10 times greater than the percentage of 5.5 % (17+/308) obtained by culture. These bacteriological data confirm the generalized occurrence of Leptospira infection in the rodents‟ population studied, and anticipate the increased importance of these vertebrates in the transmission of leptospires to cattle, especially concerning accidental infections (Paiva-Cardoso et al., 2005b). Two hundred and eighty two sera from small mammals (91.6%) were tested by the microscopic agglutination test. The seroreactivity rate (55%) was similar to that found in the molecular approach for the search of leptospiral DNA, meaning that a spirochete infection took place in a recent past. The use of local isolated field-strains, as MAT live-antigens, contributed to increase the diagnostic value of this laboratory approach in animals with a potentially reduced immune response due to their chronic renal carrier condition of these leptospires. In parallel, the study of anti-Leptospira seroreactivity in cattle, aiming at the indirect evaluation of the occurrence of accidental infections by murine transmitted leptospires, evidenced a high (61.0%) percentage of reactive animals at low titres, equal or higher than 1: 10 (geometric mean titre 1:17). As expected, the anti-Sejroe titres were the most common, representing 26.7% of the agglutinations detected. This finding may not only be due to the high number of bovines with an anti-Hardjo response, as an endemic cattle infection, but also to the contact with serovar Saxkoebing isolated from the house-mice Mus musculus present in the same ecosystem. The seroreactivity of 12.1% with Ballum serogroup was also important, suggesting that exposure of cattle to rodent maintained-serovars of this serogroup is high in this region. Serogroup Pomona registered a serological prevalence of 11.8%, which was compatible with the existence of incidental infections with its serovars, namely serovar Kunming (?) firstly isolated in Portugal. Similarly to rodents, the greatest immunogenicity of the antigens (isolates) used in MAT, certainly contributed to the important detection of specific agglutinins in the cattle surveyed. The intensive management systems for bovines have shown to be an increased risk for the greater contact with infected rodents, especially Mus musculus, the most trapped animal species and the one with a higher number of isolates obtained. However, Leptospira transmission risk by other species was also high, due to the strong seropositivity detected in Rattus rattus (100%), Apodemus sylvaticus (75%), Mus spretus (56.5%) and Rattus norvegicus (55.7%). These findings assess the need for an effective control of rodents in the smallholdings studied, due to the wide inexistence of cattle protective measures, in particular, in Bragança district, where the presence of anti-Leptospira agglutinins was significantly higher (p<0.0001) than the one for Vila Real district. Amongst the pointed reasons to justify this observation, genetic factors of the breed (Mirandesa) with a higher density in that geographical area are admitted, but other eco-epidemiological studies are needed to explain the differences observed. On the contrary, Maronesa showed to be less sensitive to leptospirosis than the remaining non-Maronesa breeds, being genetic factors together with the type of cattle management also suggested as potential reasons to explain this feature. In conclusion, Leptospira infection is widely spread in rodents of Trás-os-Montes region and the percentage of renal chronic carriers is sufficiently important to increase awareness regarding cattle production of indigenous breeds, including Maronesa, though its (apparent) higher resistance to disease. Thus, there is an urgent need towards the implementation of surveillance and control measures of leptospirosis, in order to reduce both the consequences for human and animal health, besides the inherent social and economic effects in this Northern Portuguese region. |
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