RESUMO
SUMMARY In Argentina little is known about the epidemiology of tuberculosis (TB) infection in swine. We characterized the epidemiological dynamics of Mycobacterium avium complex (MAC) infection in a swine population of Argentina using molecular tools and spatial analysis techniques. Isolates (n = 196) obtained from TB-like lesions (n = 200) were characterized by polymerase chain reaction. The isolates were positive to either M. bovis (IS6110) (n = 160) or M. avium (IS1245) (n = 16) while the remaining 20 (10.2%) isolates were positive to both M. bovis and M. avium. The detection of both bacteria together suggests co-infection at the animal level. In addition, MAC-positive isolates (n = 36) were classified as M. avium subsp. avium (MAA) (n = 30) and M. avium subsp. hominissuis (MAH) (n = 6), which resulted in five genotypes when they were typed using mycobacterial interspersed repetitive unit, variable number of tandem repeats (MIRU-VNTR). One significant (P = 0.017) spatial clustering of genotypes was detected, in which the proportion of MAH isolates was larger than expected under the null hypothesis of even distribution of genotypes. These results show that in Argentina the proportion of TB cases in pigs caused by M. avium is larger than that reported in earlier studies. The proportion of M. bovis-MAC co-infections was also higher than in previous reports. These results provide valuable information on the epidemiology of MAC infection in swine in Argentina.
Assuntos
Coinfecção/veterinária , DNA Bacteriano/análise , Complexo Mycobacterium avium/genética , Infecção por Mycobacterium avium-intracellulare/veterinária , Mycobacterium bovis/genética , Doenças dos Suínos/epidemiologia , Tuberculose/veterinária , Animais , Argentina/epidemiologia , Coinfecção/epidemiologia , Coinfecção/microbiologia , Repetições Minissatélites , Complexo Mycobacterium avium/isolamento & purificação , Infecção por Mycobacterium avium-intracellulare/epidemiologia , Mycobacterium bovis/isolamento & purificação , Suínos , Doenças dos Suínos/microbiologia , Tuberculose/epidemiologiaRESUMO
With 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)-gamma and inducible nitric oxide synthase (iNOS). In contrast, the more virulent strains induced lower expression of IFN-gamma, tumour necrosis factor-alpha 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.
Assuntos
Mycobacterium bovis/genética , Mycobacterium bovis/imunologia , Tuberculose Pulmonar/imunologia , Animais , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Ensaio de Unidades Formadoras de Colônias , Progressão da Doença , Variação Genética , Granuloma/microbiologia , Interferon gama/genética , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Óxido Nítrico Sintase Tipo II/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia , Fator de Necrose Tumoral alfa/genética , Virulência , beta-Defensinas/genéticaRESUMO
Paratuberculosis or Johne's disease is a chronic infectious disorder caused by Mycobacterium avium subsp. paratuberculosis (Map). The disease produces diarrhea and weight loss in cattle and other animal species, and it is characterized by granulomatous enteritis and lymphadenitis. Histopathology and in situ techniques can be used as a diagnostic test, but the performance of these methods was not previously compared. The aim of this paper was to evaluate the ability of immunohistochemistry and in situ hybridization to detect Map in formalin-fixed tissue samples from infected cattle. Samples (ileum or ileocecal lymph node) from four animals that had positive Map culturing, lesions and detectable acid fast bacilli, as well as from two control animals, were tested by immunohistochemistry and in situ hybridization. Immunostaining and positive hybridization were observed in areas with lesions from infected animal samples, inside the cytoplasm of macrophages, epithelioid and giant cells. Immunostaining was intense in three samples and weak in one, while hybridization was weak in all cases. In situ hybridization was positive in negative areas of tissues analyzed by immunohistochemistry, which could be related to spheroplast detection as it was previously described for this method. Control samples resulted negative by these two methods. Both techniques were able to detect Map in formalin fixed and paraffin embedded tissues, however immunohistochemistry produced higher intensity staining and was easier to perform. Therefore, we believe that immunohistochemistry and in situ hybridization to be useful for the post-mortem diagnosis and research of Paratuberculosis.
Assuntos
Íleo/microbiologia , Imuno-Histoquímica/veterinária , Hibridização In Situ/veterinária , Linfonodos/microbiologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/diagnóstico , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/microbiologia , Paratuberculose/microbiologia , Álcool Feniletílico/análogos & derivados , Sensibilidade e EspecificidadeRESUMO
A batch of 28 llama (Lama gama) sera from Jujuy province in Argentina was studied in order to identify immune reactive antigens to Leptospira interrogans. Different antigenic preparations from the bacterium were used to study the immune reactivity by the microagglutination (MAT), ELISA and Western immunoblot tests. A control pool of positive bovine sera was used. All the llama sera were negative to MAT as well as to ELISA. Two of the llama sera and the positive bovine sera pool rendered positive results when evaluated by Western immunoblot, allowing the identification of immune reactive proteins. These proteins were identified by MALDI-TOF. A periplasmic flagellin of Leptospira interrogans serovar Lai STR called FlaB1 was identified from the reactive llama sera, and an external membrane lipoprotein of Leptospira interrogans serovar Ballum called LipL21 was identified from the pool of bovine positive sera. These proteins could be used in a new ELISA applied to the early diagnosis of leptospirosis in different kind of cattle or wild reservoirs.
Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Camelídeos Americanos/imunologia , Epitopos/imunologia , Flagelina/imunologia , Leptospira interrogans/imunologia , Leptospirose/veterinária , Lipoproteínas/imunologia , Animais , Antígenos de Bactérias/isolamento & purificação , Argentina/epidemiologia , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Western Blotting , Camelídeos Americanos/sangue , Bovinos , Ensaio de Imunoadsorção Enzimática , Epitopos/isolamento & purificação , Flagelina/isolamento & purificação , Leptospirose/epidemiologia , Leptospirose/imunologia , Lipoproteínas/isolamento & purificação , Testes Sorológicos/veterinária , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The presence of microbiological agents in waters intended for human consumption, irrigation and recreational activity, is a significant risk for the human health. Recent epidemiological data demonstrate an increase of the diseases caused by waterborne pathogens, especially of the gastroenteritis infections, in the industrialized countries. Numerous studies have evidenced the contamination of deep and surface waters; bacteria within contaminated water represent a highly diversified group, that are usually encountered in wastewater and sewage. In particular the pollution of the groundwater is much dangerous, from the viewpoint of drinking water safety. In fact, the groundwaters whenever polluted, have an insufficient self-purification. The aim of this paper was to estimate the presence of waterborne pathogens in groundwater intended for the irrigation. Well water samples were collected at eighteen different agricultural areas located in Calabria and analyzed for the presence of waterborne pathogens. The results shows a diffuse pollution of the wells in all of the monitorated zones. Then out of eighteen examined samples were positive for total coliforms and Enterococcus spp. and the value of total microbial contamination were higher than 100 UFC/ml; eight of these ten wells were contaminated from Salmonella spp. and Shigella spp. (minimal value 2x10(1) UFC/l; maximum value 2x10(3) UFC/l).
Assuntos
Doenças dos Trabalhadores Agrícolas/microbiologia , Bactérias/isolamento & purificação , Microbiologia da Água , Água Doce , Humanos , Fatores de RiscoRESUMO
The operating room is a complex environment, traditionally considered at high infectious risk, for both the patients and the health care workers, they can contract diseases, because of the exposure for relatively long times to various dangerous chemical, physical and biological factors. The biological contamination in the operating rooms is mostly imputable to airborne and bloodborne microorganisms, whose primary source represent the staff: patients and operating team, while either secondary sources are the contaminate air introduced from the VCCC system and the use of the infect instruments. About 10% of the hospital infections are determined by airborne bacteria and a variable fraction of these, not only in immunocompromised patients but also in healthy people, may cause the respirators pathologies. The aim of this paper was to estimate the microbial contamination, in 20 hospitals located in three regions of the South Italy, for a total 81 operating rooms. The results show that 17 of the 20 operating units and 45 out of 81 operating rooms examined are contaminated. Periodic inspections should be carried out in order to control and lower the biological risk for both the patients and the health care workers.
Assuntos
Microbiologia do Ar , Bactérias/isolamento & purificação , Fungos/isolamento & purificação , Pessoal de Saúde , Exposição Ocupacional , Salas Cirúrgicas , Humanos , Fatores de RiscoRESUMO
The aim of this work has been to determine the occupational exposure to the biological agents and airborne dust in a sewage treatment plant in south Italy. The air samplings were performed in a sewage treatment plant in Calabria, in two different seasons (spring and summer) at 5 sites associated with various phases of sewage treatment process. In addition we have estimated the concentration of airborne endotoxins and PNOC (Particles Not Otherwise Classified) by using personal samplers. The results showed a significant variation in exposure to bioaerosols, endotoxins and PNOC depending on the sampling season: the PNOC concentration increase as much as the endotoxins concentration in spring and decrease in summer
Assuntos
Microbiologia do Ar , Poluentes Ocupacionais do Ar/efeitos adversos , Doenças Profissionais/prevenção & controle , Exposição Ocupacional/efeitos adversos , Material Particulado/análise , Esgotos/microbiologia , Poluentes Ocupacionais do Ar/análise , Endotoxinas/isolamento & purificação , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Itália , Exposição Ocupacional/análise , Tamanho da Partícula , Medição de Risco , Fatores de Risco , Estações do Ano , Local de Trabalho/normasRESUMO
P36 is a member of a family of secreted proteins distributed throughout the genus Mycobacterium. The central domain of these proteins contains several amino acid PGLTS repeats, which differ considerably between species. P36, also called exported repetitive protein (Erp) in M. tuberculosis, has been shown to be associated with virulence since the disruption of its gene impaired multiplication of both virulent M. tuberculosis and M. bovis BCG in cultured macrophages and immunocompetent mice. In order to demonstrate that P36 is a putative virulence factor of wild-type Mycobacterium bovis we generated a P36 mutant by gene disruption and we evaluated its replication in spleen and lungs of infected mice. In this study, the mutant strain displays low levels of multiplication in mice, indicating that the P36 gene is important for in vivo growth of M. bovis.
Assuntos
Bacillus/genética , Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Mutação/genética , Mycobacterium bovis/genética , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Different experimental approaches were evaluated for their ability to detect stx genes by PCR and identify Shiga toxin-producing Escherichia coli (STEC) in bovine fecal samples. One hundred and sixty fecal samples from steers in Argentina were processed by protocols that involved: (1) enrichment of fecal samples and DNA extraction using a commercially available kit (Protocol A); (2) plating on selective media after enrichment of the fecal sample followed by heat-lysis DNA extraction from the confluent growth zone (Protocol B); (3) analysis of individual colonies isolated from direct fecal culture on MacConkey agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (Protocol C), used as Gold Standard. PCR performed on bacteria from the confluent growth zone (Protocol B) proved to be the most sensitive methodology. In addition, enrichment for greater than 6h, enhanced sensitivity. Among eight STEC isolates, four were O8:H19 and four were stx2/eae-negative. An STEC isolate was characterized as O26:H11 with a stx1/eae/EHEC-hlyA genotype, often associated with human disease. Finally, no STEC O157 strains were isolated using these methods.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/metabolismo , Reação em Cadeia da Polimerase/veterinária , Toxina Shiga I/biossíntese , Toxina Shiga II/biossíntese , Animais , Argentina , Bovinos , DNA Bacteriano/química , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Masculino , Antígenos O/sangue , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Toxina Shiga I/genética , Toxina Shiga II/genética , VirulênciaRESUMO
A commercial kit intended for Taq polymerase inhibitor removal was tested to detect Shiga-toxigenic Escherichia coli (STEC) by polymersase chain reaction (PCR) directly from cattle fecal samples. Forty-five samples were analysed for the presence of stx genes. Results were compared to those obtained by two other methods: amplification of DNA purified by a non-commercial procedure (heat lysis protocol), and amplification of DNA from samples cultured in solid media, commonly used in our lab. Identical numbers of positive samples (33/45, 73 %) were obtained with the QIAamp DNA stool purification kit and the culturing procedure, suggesting an adequate removal of inhibitors that interfere in PCR amplification from the feces. Besides, the number of positive samples detected using DNA purified by the non-commercial protocol was lower, 25/39 (64%) than that achieved by using the kit. In conclusion, the use of the QIAamp DNA stool purification kit provided a rapid stx gene detection by PCR in bovine fecal samples.
Assuntos
Bovinos/microbiologia , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Reação em Cadeia da Polimerase/métodos , Kit de Reagentes para Diagnóstico , Toxinas Shiga/análise , Animais , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Contaminação de Alimentos/prevenção & controle , Reto/microbiologia , Sensibilidade e Especificidade , Taq Polimerase/antagonistas & inibidoresRESUMO
Bovine tuberculosis is a highly prevalent animal disease in Argentina. In this work evidence was obtained showing that a major Mycobacterium bovis group in Argentina had been introduced with the bovine bulls imported from the United Kingdom at the end of the XIX century. This evidence came from two sources: historical, obtained by bibliographical references, and from laboratory results, using a molecular typing method called spoligotyping. These strains are also present in other countries that introduced cattle from the same origin.
Assuntos
Mycobacterium bovis/classificação , Tuberculose Bovina/microbiologia , Criação de Animais Domésticos/história , Animais , Argentina/epidemiologia , Técnicas de Tipagem Bacteriana , Bovinos , Comércio , DNA Bacteriano/análise , Europa (Continente)/epidemiologia , Genótipo , História do Século XIX , Masculino , Mycobacterium bovis/genética , Mycobacterium bovis/isolamento & purificação , Nova Zelândia/epidemiologia , Prevalência , Estudos Retrospectivos , África do Sul/epidemiologia , América do Sul/epidemiologia , Tuberculose Bovina/epidemiologia , Tuberculose Bovina/história , Tuberculose Bovina/transmissão , Reino Unido/epidemiologia , Estados Unidos/epidemiologiaRESUMO
Culture filtrate and cell extracts from Mycobacterium bovis cultures contain molecules which could promote protective immunity to tuberculosis in animals. Different protein fractions of M. bovis cultures were obtained by elution electrophoresis and were tested in experimentally infected cattle. The fractions that elicited gamma interferon (IFN-gamma) responses were resolved by two-dimensional gel electrophoresis, and individual proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The open reading frames were cloned, expressed as their recombinant forms, and retested with naturally and experimentally infected animals. Eleven protein fractions were highly reactive, from which the Rv1636, HspX, Rv0138, Rv2524, EsxI, and Rv3740 recombinant proteins were obtained. EsxI and HspX were the antigens most recognized by the IFN-gamma release assay. In summary, a proteomic approach allowed the identification of novel antigens useful for the diagnosis of bovine tuberculosis.
Assuntos
Antígenos de Bactérias/análise , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/imunologia , Mycobacterium bovis/química , Mycobacterium bovis/imunologia , Animais , Bovinos , Eletroforese em Gel Bidimensional , Imunoensaio , Interferon gama/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tuberculose Bovina/diagnósticoRESUMO
Johne's disease or paratuberculosis is widespread in almost all countries and remains difficult to eradicate. Nowadays, diagnosis of Mycobacterium avium subsp. paratuberculosis (MPTB) infection is one of the main concerns. In this work, we evaluated the expression, biochemical properties and antigenicity of the Apa antigen, encoded by the gene annotated as MAP1569, in the MPTB genome. We confirmed its expression in MPTB and its glycosylation by the ConA binding assay. Although the MPTB-Apa is not an immunodominant antigen, MPTB-infected cattle showed a strong humoral response to recombinant Apa by Western blot and ELISA. Milk was also a suitable sample to be tested by ELISA. We comparatively analysed the humoral cross-reactivity to the Apa from MPTB (MPTB-Apa) and the orthologue from Mycobacterium tuberculosis (MT-Apa, identical to that from Mycobacterium bovis) in both infected and control cows. Response of M. bovis- and MPTB-infected animals against MT-Apa was similar (P=0.6985) but the response of the M. bovis-infected ones to MPTB-Apa was differential, being significantly diminished (P<0.0001). Although 6 out 45 animals from MPTB-infected herds responded to MPTB-Apa stimulation in the IFNgamma release assay, we found no significant differences when compared infected herds with non-infected ones (P=0.34). This antigen, in contrast to bovine Purified Protein Derivative (PPDb), was strongly represented in avian PPD (PPDa), as shown by the recognition of BALB/c mice hyperimmune sera against MPTB-Apa by Dot-blot immunoassay. We therefore demonstrated the antigenicity of Apa in MPTB-infected animals and a differential response to the recombinant antigen when compared to M. bovis-infected animals. These traits herein described, added to the usefulness of milk samples to detect IgG anti-Apa, could be important for routine screening in dairy cattle, considering a multiantigenic approach to overcome the lack of immunodominance.
Assuntos
Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias , Bovinos/imunologia , Bovinos/microbiologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/microbiologia , Reações Cruzadas , Feminino , Genes Bacterianos , Glicosilação , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Leite/imunologia , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/patogenicidade , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/imunologia , Paratuberculose/imunologia , Paratuberculose/microbiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Especificidade da Espécie , Tuberculina/imunologiaRESUMO
A Mycobacterium avium subsp. paratuberculosis expression library in lambda ZAP was screened with immunized mice sera. One clone was selected, sequenced and further characterized. The sequence analysis of the hypothetical open-reading frame (ORF) predicts a protein of 20.8 kDa with a probable signal sequence compatible with Cys-acylation at Cys24, characteristic of lipoproteins. In consequence, the protein was termed Lpp34. Recombinant expression of Lpp34 was achieved by cloning the lpp34 gene into the histidine-tag expression vector pRSET-A. Western blot analysis showed a protein band with a molecular weight of 34 kDa. The native protein was localized in the membrane fraction of M. avium subsp. paratuberculosis and extracted in the detergent phase of Triton X-114. Southern blot and polymerase chain reaction showed that the gene is absent from all the non-M. avium complex mycobacterial genomes tested. Humoral reactivity using bovine sera demonstrated that this protein is widely recognized by both the infected and non-infected animals. This could partly be due to the conserved sequence in close-related environmental bacteria such as M. avium subsp. avium and to the presence of a conserved epitope in other bacteria such as Escherichia coli. In conclusion, these findings show that Lpp34 is a membrane protein and a putative lipoprotein present in M. avium complex mycobacteria and absent in the M. tuberculosis complex.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Lipoproteínas/isolamento & purificação , Mycobacterium avium subsp. paratuberculosis/imunologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/imunologia , Sequência de Bases , Western Blotting/veterinária , Bovinos , Clonagem Molecular , DNA Bacteriano/análise , Lipoproteínas/classificação , Lipoproteínas/imunologia , Dados de Sequência Molecular , Peso Molecular , Mycobacterium avium subsp. paratuberculosis/classificação , Fases de Leitura Aberta , Reação em Cadeia da Polimerase/veterinária , Proteínas Recombinantes/classificação , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNA , Análise de Sequência de ProteínaRESUMO
Se estudió un lote de 28 sueros de llama (Lama gama) de la provincia de Jujuy, Argentina, a fin de identificar antígenos inmunorreactivos contra Leptospira interrogans. Se utilizaron distintas preparaciones antigénicas de la bacteria para estudiar la inmunorreactividad mediante microaglutinación (MAT), ELISA y Western inmunoblot. Un pool de sueros bovinos positivos a la MAT fue empleado como control. Todos los sueros de llama fueron negativos mediante MAT e igual resultado se observó mediante ELISA. Dos de los 28 sueros de llama y el pool de sueros bovinos positivos, al ser evaluados por Western inmunoblot, arrojaron resultados positivos y permitieron identificar proteínas inmunorreactivas. Por MALDI-TOF se logró establecer que la proteína asociada a los dos sueros de llama inmunorreactivos era una flagelina periplásmica de Leptospira interrogans serovar Lai STR, mientras que la asociada al pool de sueros bovinos positivos a Leptospira sp. se trataba de una lipoproteína de la membrana externa de Leptospira interrogans serovar Ballum, LipL21. Estas proteínas podrían ser utilizadas en el diseño de un nuevo ELISA aplicado al diagnóstico temprano de leptospirosis, ya sea en distintos tipos de ganado como así también en reservorios silvestres.
A batch of 28 llama (Lama gama) sera from Jujuy province in Argentina was studied in order to identify immune reactive antigens to Leptospira interrogans. Different antigenic preparations from the bacterium were used to study the immune reactivity by the microagglutinattion (MAT), ELISA and Western immunoblot tests. A control pool of positive bovine sera was used. All the llama sera were negative to MAT as well as to ELISA. Two of the llama sera and the positive bovine sera pool rendered positive results when evaluated by Western immunoblot, allowing the identification of immune reactive proteins. These proteins were identified by MALDI-TOF. A periplasmic flagellin of Leptospira interrogans serovar Lai STR called FlaB1 was identified from the reactive llama sera, and an external membrane lipoprotein of Leptospira interrogans serovar Ballum called LipL21 was identified from the pool of bovine positive sera. These proteins could be used in a new ELISA applied to the early diagnosis of leptospirosis in different kind of cattle or wild reservoirs.
Assuntos
Animais , Bovinos , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Camelídeos Americanos/imunologia , Epitopos/imunologia , Flagelina/imunologia , Leptospira interrogans/imunologia , Leptospirose/veterinária , Lipoproteínas/imunologia , Antígenos de Bactérias/isolamento & purificação , Argentina/epidemiologia , Western Blotting , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Camelídeos Americanos/sangue , Ensaio de Imunoadsorção Enzimática , Epitopos/isolamento & purificação , Flagelina/isolamento & purificação , Leptospirose/epidemiologia , Leptospirose/imunologia , Lipoproteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Testes Sorológicos/veterináriaRESUMO
Paratuberculosis (Ptbc) has a high prevalence in Argentina, that affects dairy and beef cattle. The culture is the gold standard to the diagnosis of the disease. Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis), the aetiological agent, is difficult to isolate and grow in culture. In this study, 24 randomly selected cows of the Fresian breed from a dairy herd with a history of Ptbc were used to evaluate the performance of different diagnostic techniques. These animals did not show clinical signs of the disease. However, another animal from this herd presented evidence of clinical disease at the moment of the present study. This animal was necropsied and one strain of M. paratuberculosis was isolated from faeces, lymph nodes and intestine. Serum for indirect absorbed enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) tests and whole blood samples to perform gamma interferon (gammaIFN) release assays were obtained from each animal. Faeces and milk samples to carry out bacteriological cultures, PCR identification of M. paratuberculosis, and direct examinations of smears with Ziehl-Neelsen's (ZN) stain were also collected. Tuberculin test with bovine purified protein derivative (PPD) in the caudal fold was performed. The results showed that 10 out of 24 animals (41.6%) were positive to ELISA. Eight strains of M. paratuberculosis were isolated, six from faeces, two from milk. Five of the animals that excreted the bacteria through faeces were ELISA-positive, whereas the excreters through milk were negative to ELISA. No positive samples by AGID were obtained in clinical asymptomatic animals. Seven samples gave positive gammaIFN results with avian PPD, but only two of these animals were confirmed with culture. Direct PCR, to detect IS900 (M. paratuberculosis) in faeces and milk samples, was negative, but PCR using material taken from faecal and milk cultures gave positive results before visualizing the colonies. No sample was positive by PCR directed to IS6110 (M. tuberculosis complex). There was not always agreement between isolations and ZN in the studied samples. In conclusion, the absorbed ELISA was useful to detect positive animals and excreters through faeces but not through milk. PCR applied to cultures with incipient development before the visualization of colonies was effective to specifically determine the presence of M. paratuberculosis. The gammaIFN test was not able to detect the most positive animals confirmed by culture. The importance of using ELISA and cultures is emphasized by this study but it is necessary to continue with the gammaIFN test development for early detection of the disease.
Assuntos
Doenças dos Bovinos/diagnóstico , Ensaio de Imunoadsorção Enzimática/veterinária , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Paratuberculose/diagnóstico , Animais , Argentina/epidemiologia , Cruzamento , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/microbiologia , Indústria de Laticínios , Eletroforese em Gel de Ágar/veterinária , Ensaio de Imunoadsorção Enzimática/normas , Fezes/microbiologia , Feminino , Interferon gama , Masculino , Leite/microbiologia , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/epidemiologia , Paratuberculose/microbiologia , Reação em Cadeia da Polimerase/veterinária , Valor Preditivo dos TestesRESUMO
Faecal samples from 76 diarrhoeic calves belonging to 36 farms located in the Pampas plain, Argentina, were examined for Shiga toxin-producing Escherichia coli (STEC). A total of 15 STEC strains were isolated from 12 (15.8%) calves which came from six different farms. All stx positive strains assayed by PCR were also positives in the Vero cell cytotoxicity test. The majority (60.0%) of the STEC strains carried the stx(1) gene. Twelve (80.0%) of the STEC isolates which belonged to serotypes O5:H- (n = 4), O26:H11 (n = 4), O26:H- (n = 1), O111:H- (n = 2), and O123:H38 (n = 1) were also enterohaemolysin (EHly) positive and carried the gene encoding for intimin (eae). All the stx positive strains were negative for the bfpA gene. Localized adherence to HEp-2 cells were observed in 83.3% of the eae+ STEC strains. STEC belonging to serotype O5:H- showed atypical biochemical properties, including urease production. Urease was also produced by two strains belonging to serotypes O153:H? and non-typeable, respectively. Resistance to three or more antibiotics was observed in 12 (80.0%) of the STEC isolates. Most of the serotypes of STEC recovered in this survey carried virulence traits that are associated with increased human and bovine pathogenicity. The present study shows that highly virulent STEC strains are being shed by diarrhoeic calves from farms located in a high incidence area of human STEC infections.
Assuntos
Anti-Infecciosos/farmacologia , Doenças dos Bovinos/epidemiologia , Diarreia/veterinária , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Animais , Animais Recém-Nascidos , Argentina/epidemiologia , Aderência Bacteriana , Bovinos , Doenças dos Bovinos/etiologia , Doenças dos Bovinos/microbiologia , Primers do DNA , DNA Bacteriano/análise , Diarreia/epidemiologia , Farmacorresistência Bacteriana , Escherichia coli/genética , Escherichia coli/fisiologia , Testes de Sensibilidade Microbiana , Fenótipo , Reação em Cadeia da Polimerase/veterinária , Toxinas ShigaRESUMO
Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) which relies on the amplification of a 439-bp portion of the hsp65 gene present in all mycobacteria, followed by two distinct digestions (with BstEII and HaeIII) of the PCR product, offers a rapid and easy alternative that allows identification of the species without the need for specialized equipment. Wild leprosy in the nine-banded armadillo (Dasypus novemcinctus) is characterized by the presence of multiple bacilli in internal organs such as lymph nodes, spleen and liver, as well as in nerves and skin. We could observe this in 9 out of 132 animals captured in Corrientes, Argentina, an area endemic for leprosy in humans. Mycobacterium leprae were recognized in those naturally infected animals through different techniques. Three samples of extracted DNA of the mycobacteria present in the spleen, liver and popliteal lymph node of a naturally infected animal during the Experimental Program in Armadillo (PEA) and three samples of human lepromas were processed by PRA. The patterns of the six samples analyzed were identical and were characteristic of M. leprae. These studies, made for the first time in Argentina, corroborate the initial discoveries in South America made by our investigative group on the detection of armadillos naturally infected with the Hansen bacillus.
Assuntos
Tatus/microbiologia , Hanseníase/microbiologia , Mycobacterium leprae/classificação , Animais , Argentina , Proteínas de Bactérias/genética , Chaperonina 60 , Chaperoninas/genética , DNA Bacteriano/análise , Proteínas de Choque Térmico/genética , Humanos , Hanseníase/veterinária , Mycobacterium leprae/genética , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Especificidade da EspécieRESUMO
A commercial kit intended for Taq polymerase inhibitor removal was tested to detect Shiga-toxigenic Escherichia coli (STEC) by polymersase chain reaction (PCR) directly from cattle fecal samples. Forty-five samples were analysed for the presence of stx genes. Results were compared to those obtained by two other methods: amplification of DNA purified by a non-commercial procedure (heat lysis protocol), and amplification of DNA from samples cultured in solid media, commonly used in our lab. Identical numbers of positive samples (33/45, 73 %) were obtained with the QIAamp DNA stool purification kit and the culturing procedure, suggesting an adequate removal of inhibitors that interfere in PCR amplification from the feces. Besides, the number of positive samples detected using DNA purified by the non-commercial protocol was lower, 25/39 (64%) than that achieved by using the kit. In conclusion, the use of the QIAamp DNA stool purification kit provided a rapid stx gene detection by PCR in bovine fecal samples.
Un kit comercial diseñado para la eliminación de inhibidores de la polimerasa Taq fue ensayado para la detección de STEC por PCR en muestras fecales de bovinos. Cuarenta y cinco muestras fueron evaluadas por la presencia de genes stx. Los resultados fueron comparados con aquéllos obtenidos por otros dos métodos: amplificación de ADN purificado por un procedimiento no comercial (protocolo de lisis por calor), y amplificación de ADN de muestras cultivadas en medio sólido, comúnmente usado en nuestro laboratorio. El mismo número de muestras positivas (33/45, 73 %), fueron obtenidas con el QIAamp DNA stool purification kit y el procedimiento de cultivo, sugiriendo una eliminación adecuada de inhibidores que interfieren con la amplificación en materia fecal. Por otro lado, el número de muestras positivas detectadas usando ADN purificado por el protocolo no comercial fue menor, 25/39 (64%). En conclusión, el uso del kit QIAamp DNA stool purification permitió una detección rápida de genes stx por PCR en muestras fecales bovinas.
Assuntos
Animais , Bovinos/microbiologia , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Reação em Cadeia da Polimerase/métodos , Kit de Reagentes para Diagnóstico , Toxinas Shiga/análise , Inibidores Enzimáticos/farmacologia , Escherichia coli/metabolismo , Contaminação de Alimentos/prevenção & controle , Reto/microbiologia , Sensibilidade e Especificidade , Taq Polimerase/antagonistas & inibidoresRESUMO
Bovine tuberculosis is a highly prevalent animal disease in Argentina. In this work evidence was obtained showing that a major Mycobacterium bovis group in Argentina had been introduced with the bovine bulls imported from the United Kingdom at the end of the XIX century. This evidence came from two sources: historical, obtained by bibliographical references, and from laboratory results, using a molecular typing method called spoligotyping. These strains are also present in other countries that introduced cattle from the same origin.