Identification of a novel protein in the genome sequences of Leptospira interrogans with the ability to interact with host's components.

BACKGROUND: Leptospirosis is an infectious disease that affects humans and animals worldwide. The etiological agents of this disease are the pathogenic species of the genus Leptospira. The mechanisms involved in the leptospiral pathogenesis are not full understood. The elucidation of novel mediators of host-pathogen interaction is important in the detection of virulence factors involved in the pathogenesis of leptospirosis. OBJECTIVE: This work focused on identification and characterization of a hypothetical protein of Leptospira encoded by the gene LIC10920. METHODS: The protein of unknown function was predicted to be surface exposed. Therefore, the LIC10920 gene was cloned and the protein expressed in Escherichia coli BL21 (DE3) Star pLysS strain. The recombinant protein was purified by metal affinity chromatography and evaluated with leptospirosis human serum samples. The interaction with host components was also performed. RESULTS: The recombinant protein was recognized by antibodies present in leptopsirosis human serum, suggesting its expression during infection. Immunofluorescence and intact bacteria assays indicated that the bacterial protein is surface-exposed. The recombinant protein interacted with human laminin, in a dose-dependent and saturable manner and was named Lsa24.9, for Leptospiral surface adhesin, followed by its molecular mass. Lsa24.9 also binds plasminogen (PLG) in a dose-dependent and saturable fashion, fulfilling receptor ligand interaction. Moreover, Lsa24.9 has the ability to acquire PLG from normal human serum, exhibiting similar profile as observed with the human purified component. PLG bound Lsa24.9 was able of generating plasmin, which could increase the proteolytic power of the bacteria. CONCLUSIONS: This novel leptospiral protein may function as an adhesin at the colonization steps and may help the invasion process by plasmin generation at the bacterial cell surface.

Identification of a novel protein in the genome sequences of Leptospira interrogans with the ability to interact with host's components

Background Leptospirosis is an infectious disease that affects humans and animals worldwide. The etiological agents of this disease are the pathogenic species of the genus Leptospira. The mechanisms involved in the leptospiral pathogenesis are not full understood. The elucidation of novel mediators of host-pathogen interaction is important in the detection of virulence factors involved in the pathogenesis of leptospirosis. Objective This work focused on identification and characterization of a hypothetical protein of Leptospira encoded by the gene LIC10920. Methods The protein of unknown function was predicted to be surface exposed. Therefore, the LIC10920 gene was cloned and the protein expressed in Escherichia coli BL21 (DE3) Star pLysS strain. The recombinant protein was purified by metal affinity chromatography and evaluated with leptospirosis human serum samples. The interaction with host components was also performed. Results The recombinant protein was recognized by antibodies present in leptopsirosis human serum, suggesting its expression during infection. Immunofluorescence and intact bacteria assays indicated that the bacterial protein is surface-exposed. The recombinant protein interacted with human laminin, in a dose-dependent and saturable manner and was named Lsa24.9, for Leptospiral surface adhesin, followed by its molecular mass. Lsa24.9 also binds plasminogen (PLG) in a dose-dependent and saturable fashion, fulfilling receptor ligand interaction. Moreover, Lsa24.9 has the ability to acquire PLG from normal human serum, exhibiting similar profile as observed with the human purified component. PLG bound Lsa24.9 was able of generating plasmin, which could increase the proteolytic power of the bacteria. Conclusions This novel leptospiral protein may function as an adhesin at the colonization steps and may help the invasion process by plasmin generation at the bacterial cell surface.

Identification of a novel protein in the genome sequences of Leptospira interrogans with the ability to interact with host's components

Background Leptospirosis is an infectious disease that affects humans and animals worldwide. The etiological agents of this disease are the pathogenic species of the genus Leptospira. The mechanisms involved in the leptospiral pathogenesis are not full understood. The elucidation of novel mediators of host-pathogen interaction is important in the detection of virulence factors involved in the pathogenesis of leptospirosis. Objective This work focused on identification and characterization of a hypothetical protein of Leptospira encoded by the gene LIC10920. Methods The protein of unknown function was predicted to be surface exposed. Therefore, the LIC10920 gene was cloned and the protein expressed in Escherichia coli BL21 (DE3) Star pLysS strain. The recombinant protein was purified by metal affinity chromatography and evaluated with leptospirosis human serum samples. The interaction with host components was also performed. Results The recombinant protein was recognized by antibodies present in leptopsirosis human serum, suggesting its expression during infection. Immunofluorescence and intact bacteria assays indicated that the bacterial protein is surface-exposed. The recombinant protein interacted with human laminin, in a dose-dependent and saturable manner and was named Lsa24.9, for Leptospiral surface adhesin, followed by its molecular mass. Lsa24.9 also binds plasminogen (PLG) in a dose-dependent and saturable fashion, fulfilling receptor ligand interaction. Moreover, Lsa24.9 has the ability to acquire PLG from normal human serum, exhibiting similar profile as observed with the human purified component. PLG bound Lsa24.9 was able of generating plasmin, which could increase the proteolytic power of the bacteria. Conclusions This novel leptospiral protein may function as an adhesin at the colonization steps and may help the invasion process by plasmin generation at the bacterial cell surface.

Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira.

Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the ß-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.

Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira

Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the ß-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.

Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira

Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the ß-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.

Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant.

Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigA(C)) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigA(C), either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigA(C) or LigA(C) coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen.

Evaluation of the elastinolytic activity and protective effect of Leptallo I, a protein composed by metalloprotease and FA5/8C domains, from Leptospira interrogans Copenhageni.

Leptospirosis is a re-emergent zoonosis, caused by pathogenic spirochetes from the genus Lepstospira. To date, there is no protein described to be involved in leptospiral hemorrhagic manifestations, although several proteases have been reported for other bacterial infections. In this study we identified 12 putative metalloproteases from the genome of Leptospira interrogans, and characterized for the first time a putative metalloprotease, here named Leptallo I, as a potential Zn(2+) dependent glycylglycine protease belonging to the M23 metalloendopeptidase family. The native protein was detected in extracts from several pathogenic Leptospira species and further shown to be secreted to the culture medium. We expressed the recombinant protein and its C-terminal fragment containing the metalloprotease domain, and both presented regular secondary structures. The sera of humans with leptospirosis were able to recognize rLeptallo I, indicating that the native protein is expressed and presented to the immune system during infection. The recombinant proteins displayed a significant, though relatively low, elastinolytic activity, and the challenge of hamsters immunized with rLeptallo I conferred 33% protection, suggesting a significant importance of this protein in the pathogenesis. The elastinolytic activity may be important for leptospires-host interaction, because elastin constitutes a significant proportion of total lung and blood vessel proteins.

Anticorpos anti-Leptospira spp. em ovinos do município de Monte Negro, estado de Rondônia / Anti-Leptospira spp. antibodies in ovines from Monte Negro municipality, Rondonia state, Brazil

O presente estudo determinou a prevalência de anticorpos anti-Leptospira spp. em ovinos do Município de Monte Negro, RO. Foram examinados soros de 141 ovinos de raça, idade e sexo variados provenientes de 15 fazendas, pela técnica de Soroaglutinação Microscópica. Doze (80,0%) propriedades apresentaram pelo menos um animal reagente. Títulos de anticorpos iguais ou superiores a 100 foram detectados em 47 (33,3%) animais, e os sorovares mais frequentes foram Patoc (29,7%), Autumnalis (14,8%), Pyrogenes (10,6%), Australis (4,2%), Bratislava (4,2%), Hardjo (4,2%), Icterohaemorrhagiae (4,2%), Castellonis (2,1%) e Hebdomadis (2,1%). Em 11 (23,4%) soros não foi possível a determinação do provável sorovar envolvido na reação. Alerta-se também para a possibilidade de infecção no homem, tendo em vista as características regionais de fronteira agrícola amazônica.

The present study determined the prevalence of anti-Leptospira spp.antibodies in 141 ovines from 15 farms of the Monte Negro Municipality, Rondonia State, Brazil, by the microscopic agglutination test. Twelve (80.0%) farms presented at least 1 reactive animal. Antibodies titers of ? 100 were detected in 47 (33.3%) animals, the most frequent serovars being Patoc (29.7%), Autumnalis (14.8%), Pyrogenes (10.6%), Australis (4.2%), Bratislava (4.2%), Hardjo (4.2%), Icterohaemorrhagiae (4.2%), Castellonis (2.1%) and Hebdomadis (2.1%). In 11 (23.4%) sera it was not possible to determine the most frequent serovar involved. The results raise a warning as to the possibility of infection in the human being by Leptospira in light of the regional characteristics of the Amazon agricultural frontier.

Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis.

The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.

Tuberculose em caprinos do semiárido brasileiro / Tuberculosis in goats from a semiarid region in Brazil

No período de janeiro a outubro de 2007, foram tuberculinizadas 1.866 fêmeas caprinas de diferentes raças e idades distribuídas em 84 propriedades da microrregião de Monteiro, semiárido da Paraíba. Nove cabras (0,48%) foram positivas, duas delas foram sacrificadas e, à necropsia foram observadas lesões sugestivas de tuberculose nos pulmões e no fígado. No exame histopatológico, foram evidenciados granulomas típicos. Foi procedido o cultivo e isolamento de micobactérias, sendo observado o crescimento do agente no meio de cultura de Stonebrink a partir de 36 dias de incubação, com um tempo médio de crescimento de 42 dias. Também foram encontrados resultados positivos na pesquisa de bacilos álcool-ácido resistentes (BAAR) pela coloração de Ziehl-Neelsen. O cultivo de Corynebacterium pseudotuberculosis foi negativo. O presente relato, com isolamento e visualização do agente, bem como verificação de lesões macro e microscópicas, é o primeiro caso de tuberculose confirmado em caprinos do semiárido brasileiro.

From January to October 2007, 1,866 female goats of different breeds and ages from 84 herds of the Monteiro microregion, in a semiarid area of Paraíba State, Brazil, were tuberculinized. Nine goats (0.48%) were positive and two of them were slaughtered, and at necropsy tuberculosis-like lesions were found in lungs and liver. At the histopathologic examination, typical tuberculosis granulomas were observed. Micobacteria culture and isolation were carried out, and there was growth of the agent on the Stonebrink medium culture from 36 days of inoculation with an average growth time of 42 days. Positive results were also found in the research of acid-fast bacilli by Ziehl-Neelsen staining. The culture of Corynebacterium pseudotuberculosis was negative. This report, with isolation and visualization of the agent, as well as verification of gross and microscopic lesions, is the first confirmed case of tuberculosis in goats of a semiarid region of Brazil.

Evaluation of the expression and protective potential of leptospiral sphingomyelinases

Leptospirosis is a zoonotic disease of global distribution, which affects both animals and humans. Pathogenic leptospires, the bacteria that cause this disease, require iron for their growth, and these spirochetes probably use their hemolysins, such as the sphingomyelinases, as a way to obtain this important nutrient from host red blood cells during infection. We expressed and purified the leptospiral sphingomyelinases Sph1, Sph2, Sph4, and SphH in a heterologous system. However, the recombinant proteins were not able to lyse sheep erythrocytes, despite having regular secondary structures. Transcripts for all sphingomyelinases tested were detected by RT-PCR analyses, but only Sph2 and SphH native proteins could be detected in Western blot assays using Leptospira whole extracts as well as in renal tubules of infected hamsters. Moreover, antibodies present in the serum of a human patient with laboratory-confirmed leptospirosis recognized Sph2, indicating that this sphingomyelinase is expressed and exposed to the immune system during infection in humans. However, in an animal challenge model, none of the sphingomyelinases tested conferred protection against leptospirosis.

Fatores de risco para a leptospirose em fêmeas bovinas em idade reprodutiva no Estado de São Paulo / Risk factors to leptospirosis in cows in São Paulo state, Brazil

Risk factors associated to leptospirosis were identified in cows from São Paulo State. The State was divided into seven productive circuits from which 8,216 cows older than 24 months of age from 1,021 herds were sampled. For serological diagnosis of Leptospira spp. infection, the microscopic agglutination test (MAT) was carried out. Of the investigated herds, 718 (71.3%; 95% CI = 68.5% - 74.0%) presented at least one reactant animal at MAT to any serovar. Serovar Hardjo was the most prevalent, with 55.2% (95% CI = 51.4% - 58.9%) of the positive herds. Herd size, animal purchase, share pasture, presence of ovine and swine, and utilization of artificial insemination were identified as risk factors. Utilization of maternity pens was a protective factor against leptospirosis.

Situação epidemiológica da brucelose bovina no Estado do Espírito Santo / Epidemiological situation of bovine brucellosis in the State of Espírito Santo, Brazil

Realizou-se um estudo para caracterizar a situação epidemiológica da brucelose bovina no Estado do Espírito Santo. O Estado foi dividido em dois circuitos produtores. Em cada circuito foram amostradas aleatoriamente cerca de 300 propriedades e, dentro dessas, foi escolhido de forma aleatória um número pré-estabelecido de animais, dos quais foi obtida uma amostra de sangue. No total, foram amostrados 5.351 animais, provenientes de 622 propriedades. Em cada propriedade amostrada foi aplicado um questionário epidemiológico para verificar o tipo de exploração e as práticas de criação e sanitárias que poderiam estar associadas ao risco de infecção pela doença. O protocolo de testes utilizado foi o da triagem com o teste do antígeno acidificado tamponado e o reteste dos positivos com o teste do 2-mercaptoetanol. O rebanho foi considerado positivo quando pelo menos um animal foi reagente às duas provas sorológicas. Para o Estado, as prevalências de focos e de animais infectados foram, respectivamente, de 9,0 por cento [7,0-11,6 por cento] e 3,5 por cento [1,9-6,4 por cento]. Para os circuitos, as prevalências de focos e de animais infectados foram, respectivamente, de: circuito 1, 6,8 por cento [4,5-10,2 por cento] e 3,4 por cento [1,3-8,6 por cento]; circuito 2, 10,9 por cento [7,9 por cento-14,8 por cento] e 3,7 por cento [2,1-6,3 por cento]. Os fatores de risco (odds ratio, OR) associados à condição de foco foram: utilização de inseminação artificial (OR = 7,05 [2,51-19,82]) e confinamento/semiconfinamento dos animais (OR = 2,98 [1,22-7,26]). A vacinação de fêmeas entre três e oito meses de idade foi um fator protetor (OR = 0,03 [0,01-0,1]).

A study to characterize the epidemiological status of brucellosis was carried out in the State of Espírito Santo. The State was divided in two regions. Three hundred herds were randomly sampled in each region and a pre-established number of animals were sampled in each of these herds. A total of 5,351 serum samples from 622 herds were collected. In each herd, it was applied an epidemiological questionnaire focused on herd traits as well as husbandry and sanitary practices that could be associated with the risk of infection. The serum samples were screened for antibodies against Brucella spp. by the Rose-Bengal Test (RBT), and all positive sera were re-tested by the 2-mercaptoethanol test (2-ME). The herd was considered positive if at least one animal was positive on both RBT and 2-ME tests. The prevalence of infected herds and animals in the State were, respectively, 9.0 percent [7.0-11.6 percent] and 3.5 percent [1.9-6.4 percent]. The prevalence of infected herds and animals in the regions were, respectively: region 1, 6.8 percent [4.5-10.2 percent] and 3.4 percent [1.3-8.6 percent]; and region 2, 10.9 percent [7.9-14.8 percent] and 3.7 percent [2.1-6.3 percent]. The risk factors (odds ratio, OR) associated with the presence of the infection were: use of artificial insemination (OR = 7.05 [2.51-19.82]) and intensive/semi-intensive management systems (OR = 2.98 [1.22-7.26]). Vaccination of heifers from three to eight months of age was a protective factor (OR = 0.03 [0.01-0.1]).

Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface-exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection.

Identification of Leptospira spp. carriers among seroreactive goats and sheep by polymerase chain reaction.

Few studies were conducted on the diagnosis and control of small ruminants' leptospirosis. Thirteen goat herds and seven sheep flocks located in the state of Rio de Janeiro, Brazil, were screened for leptospirosis. From the three herds and three flocks with greatest seroreactivity by MAT (Microscopic Agglutination Test), 19 and 40 seropositive goats and sheep, respectively, were selected, and urine samples were collected for bacteriology and PCR. For both species of animals, the most prevalent reactions were due to serogroups Sejroe and Shermani. Although leptospires were observed by darkfield microscopy in eight samples, pure isolates were obtained by bacteriological culture from only two samples. However, twelve urine samples (six goats and six sheep) were positive by PCR. Based on these findings, we consider that the combined use of MAT as a screening test followed by urine PCR for the direct detection of Leptospira spp. DNA was adequate for the identification of carrier animals among goats and sheep. These are valuable tools for the control of leptospirosis in small ruminants.

In LipL32, the major leptospiral lipoprotein, the C terminus is the primary immunogenic domain and mediates interaction with collagen IV and plasma fibronectin

LipL32 is the major leptospiral outer membrane lipoprotein expressed during infection and is the immunodominant antigen recognized during the humoral immune response to leptospirosis in humans. In this study, we investigated novel aspects of LipL32. In order to define the immunodominant domains(s) of the molecule, subfragments corresponding to the N-terminal, intermediate, and C-terminal portions of the LipL32 gene were cloned and the proteins were expressed and purified by metal affinity chromatography. Our immunoblot results indicate that the C-terminal and intermediate domains of LipL32 are recognized by sera of patients with laboratory-confirmed leptospirosis. An immunoglobulin M response was detected exclusively against the LipL32 C-terminal fragment in both the acute and convalescent phases of illness. We also evaluated the capacity of LipL32 to interact with extracellular matrix (ECM) components. Dose-dependent, specific binding of LipL32 to collagen type IV and plasma fibronectin was observed, and the binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin-binding and 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. Taken together, our results provide evidence that the LipL32 C terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins.

Detection of Leptospira spp. in semen and vaginal fluids of goats and sheep by polymerase chain reaction.

Thirteen goat herds and seven sheep flocks in the state of Rio de Janeiro, Brazil were screened for leptospirosis. From the three herds and three flocks with greatest seroreactivity, 19 goats (16 females and three bucks) and 40 sheep (26 ewes and 14 rams) that were seropositive (specific anti-Leptospira titres > or =400, based on a microscopic agglutination test), were selected for more detailed studies. From those animals, samples of vaginal fluids or semen were collected for bacteriological and molecular assays. For both species of animals, the most prevalent reactions were to serovars Hardjo, Shermani, and Grippotyphosa. Although leptospires were detected by darkfield microscopy in three vaginal fluid samples (from two goats and one ewe), pure isolates were not obtained by bacteriological culture of vaginal fluids or semen. However, seven vaginal fluid samples (from four goats and three ewes) and six semen samples (all from rams) were positive on polymerase chain reaction (PCR). Based on these findings, in addition to analogous findings in cattle, we inferred that there is potential for venereal transmission of leptospirosis in small ruminants.

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches.

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.

Infecção por leptospira em bovinos e sua associação com transtornos reprodutivos e condições climáticas / Leptospiral infection in bovine and its association with reproductive failure and climatic conditions

Realizou-se estudo sorológico, por meio do teste de soroaglutinação microscópica, utilizando antígenos vivos, para pesquisa de aglutininas anti-leptospiras em bovinos de leite na Microrregião de Parnaíba, PI, em dois períodos do ano: estações seca e chuvosa. Das 1975 amostras analisadas, pertinentes a 16 rebanhos, 1044 (52,9 por cento) mostraram-se positivas a pelo menos um sorovar e todos os rebanhos apresentaram animais reagentes. A prevalência variou entre 25 por cento e 81 por cento. O sorovar de maior prevalência foi Hardjo reagindo em 39,5 por cento, seguido de Wolffi com 26,7 por cento e Hebdomadis com 12,2 por cento. Os sorovares mais freqüentes foram Hardjo e Wolffi presentes em 100 por cento dos rebanhos. Foram ainda encontrados animais reagentes aos sorovares Australis, Bratislava, Autumnalis, Butembo, Icterohaemorrhagiae, Shermani e Patoc. Houve correlação positiva entre a proporção de animais sororreagentes para leptospirose e pluviometria, mas não para temperatura. Foi constatada associação entre infecção e ocorrência de transtornos reprodutivos, e o sorovar que apresentou maior associação foi o Hardjo

This study was carried out by serologic sampling (microscopic agglutination test, living antigens) for anti-leptospiral antibodies in dairy farms of Parnaíba microrregion, state of Piauí, Brazil, during dry and rainny seasons. All 16 flocks were infected and 1044 out of 1975 sera samples (52.9 percent) were positive for at least one serovar. Higher prevalence was serovar Hardjo (39.5 percent) folowed by Wolffi (26.7 percent) and Hebdomadis (12.2 percent). Hardjo and Wolffi were found in all flocks. Serovars Australis, Bratislava, Autumnalis, Butembo, Hebdomadis, Icterohaemorrhagiae, Hardjo, Wolffi, Shermani and Patoc, were also present. A positive correlation between prevalence of infection and pluviometric was detected, but temperature ratios. Association between infection and reproductive failures was evidenced mainly regarding Hardjo serovar