Seroprevalence and seroincidence of Leptospira infection in dogs during a one-year period in an endemic urban area in Southern Brazil

Introduction: Leptospirosis is a zoonosis that affects both humans and animals. Dogs may serve as sentinels and indicators of environmental contamination as well as potential carriers for Leptospira. This study aimed to evaluate the seroprevalence and seroincidence of leptospirosis infection in dogs in an urban low-income community in southern Brazil where human leptospirosis is endemic. Methods: A prospective cohort study was designed that consisted of sampling at recruitment and four consecutive trimestral follow-up sampling trials. All households in the area were visited, and those that owned dogs were invited to participate in the study. The seroprevalence (MAT titers >= 100) of Leptospira infection in dogs was calculated for each visit, the seroincidence (seroconversion or four-fold increase in serogroup-specific MAT titer) density rate was calculated for each follow-up, and a global seroincidence density rate was calculated for the overall period. Results: A total of 378 dogs and 902.7 dog-trimesters were recruited and followed, respectively. The seroprevalence of infection ranged from 9.3% (95% CI; 6.7 - 12.6) to 19% (14.1 - 25.2), the seroincidence density rate of infection ranged from 6% (3.3 - 10.6) to 15.3% (10.8 - 21.2), and the global seroincidence density rate of infection was 11% (9.1 - 13.2) per dog-trimester. Canicola and Icterohaemorraghiae were the most frequent incident serogroups observed in all follow-ups. Conclusions: Follow-ups with mean trimester intervals were incapable of detecting any increase in seroprevalence due to seroincident cases of canine leptospirosis, suggesting that antibody titers may fall within three months. Further studies on incident infections, disease burden or risk factors for incident Leptospira cases should take into account the detectable lifespan of the antibody.

Tissue apoptosis in mice infected with Leptospira interrogans serovar Icterohaemorrhagiae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

TNF-alpha and IL-6 immunohistochemistry in rat renal tissue experimentaly infected with Leptospira interrogans serovar Canicola

Leptospirosis is a public health problem worldwide and its etiology remains unclear. Its pathogenesis involves a complex interaction between host and infecting microorganism. The inflammatory reaction that controls the infection process also underscores many pathophysiological events occurring in leptospirosis. We investigated the presence of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in renal tissues by immunohistochemical and histopathological examination in animals experimentally inoculated with Leptospira serovar Canicola. All the tests were carried out 2, 7, 14, 21 or 28 days after inoculation. Although TNF-alpha and IL-6 had been detected in tissues throughout the observation period, these cytokines appeared more intensely during the initial phase of infection. Therefore, both TNF-alpha and IL-6 were associated with the immunopathogenesis of leptospirosis. This profile suggests a high immunocellular response throughout the early infection stages followed by subsequent humoral response.

Perfil de Aglutininas Anti-leptospira e anti-brucella e condições sanitárias de ovinos da região noroeste do Estado de São Paulo, Brasil

The sheep industry has become increasingly prominent in agribusiness, transforming the stage production of Brazil, and thus contributes more to the socio-economic development of the country. The work aimed to verify the occurrence of brucellosis and leptospirosis in sheep from northwestern São Paulo state. In addition to determining the prevalence of major Leptospira in the region and to trace the diagnosis of sheep breeding in this part of the country. All the 1222 sheep serum samples from 49 properties did not react serologically to evidentiary testing for brucellosis, compared to antigens of B. abortus and B. ovis used in the testing of 2-ME and IDGA, respectively. The Microscopic Agglutination Test (MAT) test revealed that 19.14% (232/1212) of samples were positive for one or more serovars, with titles ranging from 100 to 800. The most frequent serovar was hebdomadis in the region, with Sentot and Sherman (18.10%, 11.64% and 8.62%, respectively). By profiling the system of sheep farming in the region, we found that most herds are composed of more than one race being the main purpose is for the court. According to the scheme adopted immunoprophylactics there is a homogeneous set schedule. It adopts the use of anti-helminth, non-prescription veterinary antibiotic. There is also the presence of diarrhea and abortions and the lack of criteria for los ovinos on management, making it the need for emergency development of programs for disease control, schema immunoprophylactics adequate sanitation and hygienic measures in sheep breeding.

Detecção de anticorpos contra Leptospira spp. em animais de vida livre do Pantanal do Mato Grosso do Sul

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Soroprevalência, isolamento e diagnóstico molecular para leptospirose em bovinos de fazendas de cria e de centrais de receptoras de embrião da região de Bauru- SP: Diagnóstico de leptospirose bovina em Bauru - SP

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Soroprevalência de anticorpos anti- Leptospira spp. e anti- Toxoplasma gondii em quatis (Nasua nasua) provenientes do Parque Ecológico do Tietê, São Paulo, Brasil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

HEALTH RISKS TO HUMANS AND DOMESTIC LIVESTOCK POSED BY FERAL PIGS (Sus scrofa) IN NORTH QUEENSLAND

A sample of 608 adult pigs from Cape York and adjacent islands was examined for parasites and their serum tested for livestock diseases associated with the Queensland tropics. Feral pigs from North Queensland pose a significant health threat to humans with the incidence of Spargana (the plerocercoid of Spirometra erinacei) through the consumption of undercooked pork. Meliodosis (Pseudomonas pseudomalleO. Leptospirosis (L. yar. pomona). and Brucellosis (Brucella suis) are capable of infecting humans directly during unhygienic butchering of infected carcasses. In North Queensland, the widespread intermingled distribution of feral pigs and cattle increases the potential for the transmission of Actinobacillus, Leptospirosis, and Brucellosis from feral pigs to cattle. Both Europeans and Aborigines on Cape York also raise wild-caught feral pigs for meat. It is important to realize that parasites and diseases are present in young pigs and that poor husbandry practices increase the risk of infection from several parasites, i.e., Lungworm (Metastrongylus sp.) Stomach worm (Physocephalus sexalatus. Hvostrongvlus rubidus). Thorny headed worm (Macracanthorrhynchus hirudinaceus) and Kidney worm (Stephanurus dentatus). Heavy infection of these parasites reduce growth rates and cause unthriftiness in infected ani¬mals.

Prevalência e fatores de risco de leptospirose bovina no Estado do Maranhão

Silva F.J., Conceicao W. L. F., Fagliari J.J., Girio R.J.S., Dias R. A., Borba M. R. & Mathias L. A. 2012. [Prevalence and risk factors of bovine leptospirosis in the State of Maranhao, Brazil.] Prevalencia e fatores de risco de leptospirose bovina no Estado do Maranhao. Pesquisa Veterineria Brasileira 32(4): 303-312. Departamento de Medicina Veterinaria Preventiva e Reproducao Animal, Faculdade de Ciencias Agrarias e Veterinarias, Universidade Estadual Paulista, Via de Acesso Professor Paulo Donato Castellane s/n, Zona Rural, Jaboticabal, SP 14884-900, Brazil. E-mail: fjsepi@gmail.com Prevalence and risk factors of bovine leptospirosis in the State of Maranhao were investigated. Based on production parameters that vary across different production systems, management practices, the purpose of exploitation, the average size of herds and market systems, the state was divided in four sampling circuits. The study aimed to investigate the epidemiological features of bovine leptospirosis in the State of Maranhao, in order to determine the prevalence of the infection in cattle and herds, to determine the occurrence of serovars of Leptospira spp., to identify risk factors associated with leptospirosis in cattle and to differentiate the livestock circuits itself regarding the prevalence of leptospirosis. The survey was conducted in 136 herds in the circuit I, in which 841 >= 24 months old females were analyzed; 238 in the circuit II and 2,582 females were analyzed; 122 in the circuit III and 869 females were analyzed; 77 in the circuit IV and 540 females were analyzed; a total of 573 herds and 4,832 females were analyzed. The presence of antibodies against Leptospira spp. was verified by microscopic agglutination test (MAT). Of the 4,832 cows examined, 1,904 (35.94%, CI 95% = 33.01% -38.98%) were positive. Of the 573 herds, 380 (64.81%, CI 95% = 61.10% -68.35%) were positive. Serovars Hardjo and Wolffi were the most frequent in the state. The circuit III showed the lowest prevalence of leptospirosis in all comparisons. The variables presence of horses (p = 0.000), presence of capybaras (p = 0.034) and herds with up to 32 adult females (p = 0.002) were identified as risk factors for leptospirosis.

OmpL1 Is an Extracellular Matrix- and Plasminogen-Interacting Protein of Leptospira spp.

Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical beta-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with KD (equilibrium dissociation constant) values of 2,099.93 +/- 871.03 nM and 1,239.23 +/- 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a KD of 368.63 +/- 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection.

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coil BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K-D) of 292 +/- 24 nM and 157 +/- 35 nM, respectively. Moreover, the Lsa30 is a plasminogen (PLC) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system. (C) 2012 Elsevier Ltd. All rights reserved.

Induction of TNF-alfa and CXCL-2 mRNAs in different organs of mice infected with pathogenic Leptospira

The role of innate immune response in protection against leptospirosis is poorly understood. We examined the expression of the chemokine CXCL2/MIP-2 and the cytokine TNF-alpha. in experimental resistant and susceptible mice models, C3H/HeJ, C3H/HePas and BALB/c strains, using a virulent strain of Leptospira interrogans serovar Copenhageni. Animals were infected intraperitoneally with 107 cells and the development of the disease was followed. Mortality of C3H/HeJ mice was observed whereas C3H/HePas presented jaundice and BALB/c mice remained asymptomatic. The infection was confirmed by the presence of leptospiral DNA in the organs of the animals, demonstrated by PCR. Sections of the organs were analyzed, after H&E stain. The relative expression of mRNA of chemokine CXCL2/MIP-2 and cytokine TNF-alpha was measured in lung, kidney and liver of the mice by qPCR. The concentrations of these proteins were measured in extracts of tissues and in serum of the animals, by ELISA. Increasing levels of transcripts and protein CXCL2/MIP-2 were detected since the first day of infection. The highest expression was observed at third day of infection in kidney, liver and lung of BALB/c mice. In C3H/HeJ the expression of CXCL2/MIP-2 was delayed, showing highest protein concentration in lung and kidney at the 5th day. Increasing in TNF-alpha transcripts were detected after infection, in kidney and liver of animals from the three mice strains. The expression of TNF-alpha protein in C3H/HeJ was also delayed, being detected in kidney and lung. Our data demonstrated that Leptospira infection stimulates early expression of CXCL2/MIP-2 and TNF-alpha in the resistant strain of mice. Histological analysis suggests that the expression of those molecules may be related to the influx of distinct immune cells and plays a role in the naturally acquired protective immunity. (C) 2012 Elsevier Ltd. All rights reserved.

Brazilian spotted fever: Real-time PCR for diagnosis of fatal cases

Suspicion of Brazilian spotted fever (BSF) should occur in endemic regions upon surveillance of the acute febrile icteric hemorrhagic syndrome (AFIHS). However, limitations associated with currently available laboratory tests pose a challenge to early diagnosis, especially in fatal cases. Two real-time PCR (qPCR) protocols were evaluated to diagnose BSF in 110 fatal AFIHS cases, collected in BSF-endemic regions in 2009-2010. Of these, 24 were positive and 86 negative by indirect immunofluorescence (IFA) assay (cutoff IgG and/or IgM >= 128). DNA from these samples was used in the qPCR protocols: one to detect Rickettsia spp. (Citrate synthase gene) and another to determine spotted fever group (SFG) Rickettsia species (OmpA gene). Of the 24 IFA-positive samples, 5 (21%) were positive for OmpA and 9 (38%) for citrate synthase. In the IFA-negative group (n = 86), OmpA and citrate synthase were positive in 23 (27%) and 27 (31%), respectively. These results showed that the 2 qPCR protocols were about twice as sensitive as the IFA test alone (93% concordance). In conclusion, qPCR is a sensitive method for the diagnosis of fatal BSF cases and should be considered for routine surveillance of AFIHS in places like Brazil, where spotted fever-related lethality is high and other endemic diseases like dengue and leptospirosis can mislead diagnosis. (C) 2012 Elsevier GmbH. All rights reserved.

Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions

Background: Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results: We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (K-D) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a K-D of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions: We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

OCCURRENCE OF ANTIBODIES TO TOXOPLASMA GONDII AND LEPSTOSPIRA SPP. IN MANATEES (TRICHECHUS INUNGUIS) OF THE BRAZILIAN AMAZON

The presence of Toxoplasma gondii and Leptospira spp. antibodies was investigated in 74 manatees (Trichechus inunguis [Mammalia: Sirenia]) kept in captivity in two rescue units in the northern region of Brazil. Antibodies to T gondii were detected in 29 (39.2%) of 74 animals by using the modified agglutination test (titer, 1:25). For antibodies against Leptospira spp., sera were diluted 1:50 and tested against 24 strains of leptospires by microscopic agglutination microtechnique, and positive samples were end titrated. Twenty-three (31.1%) of 74 animals were reactive to four serovars (Patoc 21/23, Castellonis 2/23, Icterohaemorrhagiae 1/23, and Butembo 1/23), with titers ranging from 100 to 1,600. This is the first report of antibodies against T gondii and Leptospira spp. in T. inunguis from the Brazilian Amazon.