Advances in Bovine Tuberculosis Diagnosis and Pathogenesis: What Policy Makers Need to Know

The mainstay of tuberculosis diagnosis in cattle and deer has been the tuberculin skin test. Recent advances have allowed the incorporation of blood based assays to the diagnostic arsenal for both cattle and deer. Use of defined and specific antigens has allowed for improved specificity of cell mediated assays in both cattle and deer and advances in antibody tests for tuberculosis have potential for use in free-ranging and captive cervid populations. Combined use of blood-based assays with skin testing will require further understanding of the effect of skin testing on the accuracy of blood based assays. Models of experimental infection of cattle have allowed for increased understanding of natural disease pathogenesis. Differences likely exist; however, between cattle and deer in both disease distribution and primary route of inoculation in naturally infected animals.

Aerosol Exposure of White-Tailed Deer (Odocoileus virginianus) to Mycobacterium Bovis

Tuberculosis due to Mycobacterium bovis affects both captive and free-ranging Cervidae in the United States. Various animal models have been developed to study tuberculosis of both humans and animals. Generally, tuberculosis is transmitted by aerosol and oral routes. Models of aerosol exposure of large animals to M. bovis are uncommon. In order to develop a reliable method of aerosol exposure of white-tailed deer (Odocoileus virginianus) to M. bovis, 12 healthy white-tailed deer, aged 8–10 mo, were infected by aerosol exposure to 2x105 to 1x106 colony forming units (CFU) (high dose, n=4) of M. bovis or 6x102 to 1.6 x 103 CFU (low dose, n=8) of M. bovis. Tuberculous lesions were more widely disseminated in deer receiving the high dose, while lesions in deer receiving the low dose were more focused on the lungs and associated lymph nodes (tracheobronchial and mediastinal). Aerosol delivery of M. bovis to white-tailed deer results in a reliable manner of experimental infection that may be useful for studies of disease pathogenesis, immune response, mycobacterial shedding, and vaccine efficacy.

INFLUENZA VIRUSES IN ADULT DOGS RAISED IN RURAL AND URBAN AREAS IN THE STATE OF SAO PAULO, BRAZIL

In 1970, searching for the interspecies transmission of influenza viruses led to the first study on influenza viruses in domestic animals. Birds and mammals, including human beings, are their natural hosts; however, other animals may also play a role in the virus epidemiology. The objective was to investigate the incidence of influenza viruses in adult dogs raised in rural (9, 19.56%) and urban (37, 80.43%) areas in the state of Sao Paulo, Brazil. Dog serum samples were examined for antibodies to influenza viruses by the hemagglutination inhibition (HI) test using the corresponding antigens from the circulating viruses in Brazil. Dogs from rural areas presented antibodies to influenza A H3N2, and influenza A H7N7 and H3N8. In rural areas, dog sera displayed mean titers as 94.37, 227.88, 168.14, 189.62 HIU/25 mu L for subtypes H1N1, H3N2, H7N7, H3N8, respectively. About 84% and 92% of dogs from urban areas exhibited antibodies to human influenza A H1N1 and H3N2, respectively, with statistical difference at p < 0.05 between the mean titers of antibodies to H1N1 and H3N2. About 92% and 100% were positive for H7N7 and H3N8, respectively. In dogs from urban areas, the mean titers of antibodies against influenza A H1N1, H3N2, H7N7 and H3N8, were 213.96, 179.42, 231.76, 231.35 HIU/25 mu L respectively. The difference among them was not statistically significant at p > 0.05. In conclusion, these dogs were positive for both human and equine influenza viruses. The present study suggests the first evidence that influenza viruses circulate among dogs in Brazil.

Epidemiology of Brazilian spotted fever in the Atlantic Forest, state of Sao Paulo, Brazil

The tick-borne bacterium Rickettsia rickettsii is the aetiological agent of Brazilian spotted fever (BSF). The present study evaluated tick infestations on wild and domestic animals, and the rickettsial infection in these animals and their ticks in 7 forest areas adjacent to human communities in the Sao Paulo Metropolitan Area (SPMA). The results were compared to ecological traits of each sampled area. Two main tick species, Amblyomma aureolatum and Rhipicephalus sanguineus, were collected from dogs. The major ticks found on small mammals and birds were Ixodes loricatus and Amblyomma longirostre, respectively. Both anti-R. rickettsii antibodies and R. rickettsii-infected ticks were detected on dogs from only 2 areas in the southern part of the SPMA, which were considered to be endemic for BSF; the remaining 5 areas were considered to be non-endemic. Ecologically, the BSF-endemic areas clearly differed from the non-endemic areas by the presence of significantly more degraded forest patches in the former. The present results corroborate historical observations that have indicated that all human cases of BSF in the SPMA were contracted in the southern part of this metropolitan area. However, not all forest patches in the southern part of the SPMA were shown to be associated with BSF endemism.

Exposure of small mammals to ticks and rickettsiae in Atlantic Forest patches in the metropolitan area of Recife, North-eastern Brazil

Between December 2007 and March 2009, small mammals were captured in 6 Atlantic Forest patches in Brazil. We assessed tick-host associations and whether they differ among forest strata, sites, seasons, and host age classes or between sexes. Moreover, we assessed the exposure of animals to Rickettsia spp. In total, 432 animals were captured and 808 ticks were found on 32-9% of them. Significant differences were found among host species, collection sites, and forest strata; microhabitat preference was a strong risk factor for tick infestation. The highest tick density rates were recorded in forest fragments settled in rural areas; 91.3% of the ticks were collected from animals trapped in these forest fragments. A high prevalence (68.8%) of antibodies to Rickettsia spp. was detected among animals. This study suggests that disturbed Atlantic Forest fragments provide an environment for ticks and small mammals, which are highly exposed to rickettsiae. It also indicates that forest patches settled in rural areas are usually associated with higher small mammal diversity as well as with higher tick density rates.

Pathogenesis of reproductive failure induced by Trypanosoma vivax in experimentally infected pregnant ewes

The present study was aimed at investigating the effect of experimental infection by Trypanosoma vivax in different stages of pregnancy, determining the pathogenesis of reproductive failure, and confirming transplacental transmission. We used 12 pregnant ewes distributed into four experimental groups: G1, was formed by three ewes infected with T. vivax in the first third of pregnancy (30 days); G2 comprised three infected ewes in the final third of pregnancy (100 days); G3 and G4 were composed of three non-infected ewes with the same gestational period, respectively. Each ewe of G1 and G2 was inoculated with 1.25 × 105 tripomastigotes. Clinical examination, determination of parasitemia, serum biochemistry (albumin, total protein, glucose, cholesterol, and urea), packed cell volume (PCV), serum progesterone, and pathological examination were performed. Placenta, amniotic fluid, blood and tissues from the fetuses and stillbirths were submitted to PCR. Two ewes of G1 (Ewe 1 and 3) presented severe infection and died in the 34th and 35th days post-infection (dpi), respectively; but both fetuses were recovered during necropsy. In G2, Ewe 5 aborted two fetuses on the 130th day (30 dpi) of pregnancy; and Ewe 6 aborted one fetus in the 140th day (40 dpi) of gestation. Ewes 2 and 4 delivered two weak lambs that died five days after birth. Factors possibly involved with the reproductive failure included high parasitemia, fever, low PCV, body score, serum glucose, total protein, cholesterol, and progesterone. Hepatitis, pericarditis, and encephalitis were observed in the aborted fetuses. The presence of T. vivax DNA in the placenta, amniotic fluid, blood, and tissues from the fetuses confirms the transplacental transmission of the parasite. Histological lesion in the fetuses and placenta also suggest the involvement of the parasite in the etiopathogenesis of reproductive failure in ewes.

Pathogenesis of reproductive failure induced by Trypanosoma vivax in experimentally infected pregnant ewes

The present study was aimed at investigating the effect of experimental infection by Trypanosoma vivax in different stages of pregnancy, determining the pathogenesis of reproductive failure, and confirming transplacental transmission. We used 12 pregnant ewes distributed into four experimental groups: G1, was formed by three ewes infected with T. vivax in the first third of pregnancy (30 days); G2 comprised three infected ewes in the final third of pregnancy (100 days); G3 and G4 were composed of three non-infected ewes with the same gestational period, respectively. Each ewe of G1 and G2 was inoculated with 1.25 × 105 tripomastigotes. Clinical examination, determination of parasitemia, serum biochemistry (albumin, total protein, glucose, cholesterol, and urea), packed cell volume (PCV), serum progesterone, and pathological examination were performed. Placenta, amniotic fluid, blood and tissues from the fetuses and stillbirths were submitted to PCR. Two ewes of G1 (Ewe 1 and 3) presented severe infection and died in the 34th and 35th days post-infection (dpi), respectively; but both fetuses were recovered during necropsy. In G2, Ewe 5 aborted two fetuses on the 130th day (30 dpi) of pregnancy; and Ewe 6 aborted one fetus in the 140th day (40 dpi) of gestation. Ewes 2 and 4 delivered two weak lambs that died five days after birth. Factors possibly involved with the reproductive failure included high parasitemia, fever, low PCV, body score, serum glucose, total protein, cholesterol, and progesterone. Hepatitis, pericarditis, and encephalitis were observed in the aborted fetuses. The presence of T. vivax DNA in the placenta, amniotic fluid, blood, and tissues from the fetuses confirms the transplacental transmission of the parasite. Histological lesion in the fetuses and placenta also suggest the involvement of the parasite in the etiopathogenesis of reproductive failure in ewes.

Pathogenicity of Nc-Bahia and Nc-1 strains of Neospora caninum in experimentally infected cows and buffaloes in early pregnancy

Neospora caninum is a protozoan parasite known as an important cause of bovine abortion worldwide. Little is currently known about how different strains of N. caninum vary in their pathogenicity. In this study, we compared a Brazilian strain, Nc-Bahia, with the first isolate of this coccidian, Nc-1. Eight cows and seven buffaloes were submitted to fixed-time artificial insemination protocols for a better control of pregnancy. Group 1 was inoculated with Nc-Bahia (n=8; five cows and three buffaloes), and Group 2 was inoculated with Nc-1 (n=5; two cows and three buffaloes). One nonpregnant female of each species was left uninfected as sentinel controls for potential environmental infection. All inoculated animals received 5×108 tachyzoites of N. caninum, by intravenous route, on the 70th day of gestation. Uninfected animals remained seronegative throughout the experiment, indicating no exogenous infection, whereas all inoculated animals became seropositive to N. caninum. In Group 1, abortion was found in only one cow on 42 days postinfection (dpi; frequency of abortion=12.5 %), whilst all animals from Group 2 aborted on 35 dpi (frequency of abortion= 100 %). Parasite DNA was detected by seminested PCR in maternal, foetal and placental tissues, confirming vertical transmission in Groups 1 and 2, although histological lesions had different frequencies and degrees of severity between the groups. There was evidence of lower pathogenicity of Nc- Bahia compared to Nc-1 when used in experimental infection, as it caused fewer abortions, as well as less frequent and milder histological lesions. This was the first time Nc-Bahia has been used for experimental infection.

Parapoxvirus: basi genomiche della patogenesi

Parapoxvirus (PPV) are member of a genus in the family poxviridae which currently encompasses four species: the prototype orf virus (OV), bovine papular stomatitis virus (BPSV), pseudocowpox virus (PCPV) and parapoxvirus of New Zealand red deer (PVNZ). PPVs cause widespread, but localized diseases of small and large ruminants and they can also be transmitted to man. Knowledge of the molecular biology of PPV is still limited as compared to orthopoxviruses, especially vaccinia virus (VACV). The PPV genome displays a high G+C content and relatively small size for poxvirus. Coventional electron microscopy displays PPV virions with ovoid shape and slightly smaller in size than the brickshaped orthopoxviruses. The most striking feature, which readily enables identification of PPV, is a tubule-like structure that surrounds the particle in a spiral fashion. PPV genome organization and content is very similar to that of other poxviruses, the central region contain 88 genes which are present in all poxviruse, in contrast the terminal regions are variable and contain a set of genes unique to the genus PPV. Genes in the near-terminal regions of the genome are frequently not essential for growth in cultured cells encoding factors with important roles in virushost interactions including modulating host immune responses and determining host range. Recently it was suggested that the open reading frames (ORFs) 109 and 110 of the OV genome have a major role in determining species specificity during natural infection in sheep and goats. This hypothesis is based on the analysis of a few number of sequences of different sheep and goats viral isolates. PPV replicate into the cytoplasm of infected cells and produce three structurally different infectious particles: the intracellular mature virions (IMV), intracellular enveloped virions (IEV) and the extracellular enveloped virions (EEV). The vaccinia A33R and A34R hotologue proteins encoded by the ORFS 109 and 110 are expressed in the envelope of the IEV and EEV. The F1L immunodominant protein of orf virus is the major component of the surface tubule structure of the IMV and can post-translationaly insert into membranes via Cterminal, hydrofobic anchor sequence like its orthologue VACV H3L protein. Moreover the F1L protein binds to glycosaminoglycans on the cell surface and has an important role in IMV adsorption to mammalian cells. In this study we investigated the morphogenesis of the PPV through the construction of a mutant virus deleted of the F1L protein. A study of the deleted virus life cycle was conducted in different type of cells and its morphology was observed with electron microscopy. It was demonstared that F1L protein have important role in morphogenesis and infectivity. Moreover it is essential to determine the spiral fashion of the tubule like structure of the virion surface. Some pathogenetic aspects of the PPV infection were studied, in particular the protein implicated in the host range were analysed in detail. An experimental infection with OV and PCPV was conducted in goats and sheep. After infection, the severity of the lesions were comparable in both the animal species. The OV did not result in severe disease neither in sheep nor in goats, suggesting that host factors, rather than virus strain characteristics, may play an important role in the pathogenesis of the Parapoxvirus infections. The PCPV failed to produce any lesion in both sheep and goats, ruling out the possibility of any recombination between PCPV and OV during natural infection in these animal species. The phylogenetic analysis of the ORFs 109 and 110 from several goats and sheep viral isolates showed a clustering based on the antigenic content of the protein that was independent from species and geographic origin.

Molecular survival strategies of Echinococcus multilocularis in the murine host

Larval infection with Echinococcus multilocularis starts with the intrahepatic postoncospheral development of a metacestode that-at its mature stage-consists of an inner germinal and an outer laminated layer (GL ; LL). In certain cases, an appropriate host immune response may inhibit parasite proliferation. Several lines of evidence obtained in vivo and in vitro indicate the important bio-protective role of the LL. For instance, the LL has been proposed to protect the GL from nitric oxide produced by periparasitic macrophages and dendritic cells, and also to prevent immune recognition by surrounding T cells. On the other hand, the high periparasitic NO production by peritoneal exsudate cells contributes to periparasitic immunosuppression, explaining why iNOS deficienct mice exhibit a significantly lower susceptibility towards experimental infection. The intense periparasitic granulomatous infiltration indicates a strong host-parasite interaction, and the involvement of cellular immunity in control of the metacestode growth kinetics is strongly suggested by experiments carried out in T cell deficient mouse strains. Carbohydrate components of the LL, such as Em2(G11) and Em492, as well as other parasite metabolites yield immunomodulatory effects that allow the parasite to survive in the host. I.e., the IgG response to the Em2(G11)-antigen takes place independently of alpha-beta+CD4+T cells, and in the absence of interactions between CD40 and CD40 ligand. Such parasite molecules also interfere with antigen presentation and cell activation, leading to a mixed Th1/Th2-type response at the later stage of infection. Furthermore, Em492 and other (not yet published) purified parasite metabolites suppress ConA and antigen-stimulated splenocyte proliferation. Infected mouse macrophages (AE-MØ) as antigen presenting cells (APC) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells when compared to normal MØ. As AE-MØ fully maintain their capacity to appropriately process antigens, a failure in T cell receptor occupancy by antigen-Ia complex or/and altered co-stimulatory signals can be excluded. Studying the status of accessory molecules implicated in T cell stimulation by MØ, it could be shown that B7-1 (CD80) and B7-2 (CD86) remained unchanged, whereas CD40 was down-regulated and CD54 (=ICAM-1) slightly up-regulated. FACS analysis of peritoneal cells revealed a decrease in the percentage of CD4+ and CD8+T cells in AE-infected mice. Taken together the obstructed presenting-activity of AE-MØ appeared to trigger an unresponsiveness of T cells leading to the suppression of their clonal expansion during the chronic phase of AE infection. Interesting information on the parasite survival strategy and potential can be obtained upon in vitro and in vivo treatment. Hence, we provided very innovative results by showing that nitazoxanide, and now also, respectively, new modified compounds may represent a useful alternative to albendazole. In the context of chemotherapeutical repression of parasite growth, we searched also for parasite molecules, whose expression levels correlate with the viability and growth activity of E. multilocularis metacestode. Expression levels of 14-3-3 and II/3-10, relatively quantified by realtime reverse transcription-PCR using a housekeeping gene beta-actin, were studied in permissive nu/nu and in low-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro-treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide for a period of 8 days resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels,

Immunohistochemical demonstration of the putative canine distemper virus receptor CD150 in dogs with and without distemper

Signaling lymphocyte activation molecule (SLAM) or CD150 can function as a receptor for the canine distemper virus (CDV) in vitro. The expression of SLAM was studied using immunohistochemistry in order to evaluate the presence and distribution of the receptor in dogs in vivo. Additionally, receptor expression was assessed after experimental infection of dogs with CDV. In 7 control dogs without distemper virus, the receptor was found in various tissues, mostly on cells morphologically identified as lymphocytes and macrophages. In 7 dogs with early distemper lesions characterized by presence of the virus, higher numbers of SLAM-expressing cells were found in multiple tissues recognized as targets of CDV compared with those in control dogs. These findings suggest that SLAM, a putative distemper receptor, is expressed in dogs in vivo. Additionally, virus infection is associated with up-regulation of SLAM, potentially causing an amplification of virus in the host.

Proteome-wide screening of the European porcine reproductive and respiratory syndrome virus reveals a broad range of T cell antigen reactivity.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly evolving and diversifying pathogen necessitating the development of improved vaccines. Immunity to PRRSV is not well understood although there are data suggesting that virus-specific T cell IFN-γ responses play an important role. We therefore aimed to better characterise the T cell response to genotype 1 (European) PRRSV by utilising a synthetic peptide library spanning the entire proteome and a small cohort of pigs rendered immune to PRRSV-1 Olot/91 by repeated experimental infection. Using an IFN-γ ELISpot assay as a read-out, we were able to identify 9 antigenic regions on 5 of the viral proteins and determine the corresponding responder T cell phenotype. The diversity of the IFN-γ response to PRRSV proteins suggests that antigenic regions are scattered throughout the proteome and no one single antigen dominates the T cell response. To address the identification of well-conserved T cell antigens, we subsequently screened groups of pigs infected with a closely related avirulent PRRSV-1 strain (Lelystad) and a divergent virulent subtype 3 strain (SU1-Bel). Whilst T cell responses from both groups were observed against many of the antigens identified in the first study, animals infected with the SU1-Bel strain showed the greatest response against peptides representing the non-structural protein 5. The proteome-wide peptide library screening method used here, as well as the antigens identified, warrant further evaluation in the context of next generation vaccine development.

Manipulation and potentiation of antimycobacterial immunity using recombinant bacille Calmette-Guérin strains that secrete cytokines.

Bacille Calmette-Guérin (BCG) is a live, attenuated strain of Mycobacterium bovis used widely for tuberculosis prophylaxis and bladder cancer immunotherapy, although it has limitations in both contexts. To investigate whether BCG's immunostimulatory properties could be modified, and to gain insight into the interaction between mycobacteria and their hosts, we constructed recombinant BCG strains that secrete functional murine cytokines and studied their properties in mouse models of experimental infection. Cell-mediated immune responses to mycobacterial antigen (purified protein derivative) were assayed using splenocytes from mice inoculated with various BCG recombinants. Antigen-specific proliferation and cytokine release were found to be substantially greater with splenocytes derived from mice injected with cytokine-secreting BCG than with splenocytes from mice injected with BCG lacking cytokines. The most profound effects were induced by BCG secreting interleukin 2, interferon gamma, or granulocyte-macrophage colony-stimulating factor. Thus, cytokine-secreting BCG can enhance immune responses to mycobacterial antigens and may be improved reagents for tuberculosis prophylaxis and cancer immunotherapy.

Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas.

Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy.

Leishmaniose visceral americana: avaliação dos parâmetros da capacidade vetorial de Lutzomyia longipalpis em área urbana do muncípio de Panorama, São Paulo, Brasil

Introdução: A leishmaniose visceral (LV) é um importante problema de saúde pública no Brasil, com cerca 3000 mil casos notificados anualmente. Nos últimos anos, a LV tem ampliado sua distribuição em vários estados do país, associada principalmente aos processos socioambientais, antrópicos e migratórios. A LV é causada pela infecção com Leishmania infantum chagasi, transmitida, principalmente, por Lutzomyia longipalpis (Diptera: Psychodidae). Este flebotomíneo apresenta ampla distribuição nas Américas, todavia, evidências sugerem que se constitui em um complexo de espécies crípticas. A dinâmica de transmissão da LV é modulada por fatores ecológicos locais que influenciam a interação entre populações do patógeno, do vetor e dos hospedeiros vertebrados. Portanto, o estudo das variáveis associadas a esta interação pode contribuir para elucidar aspectos dos elos epidemiológicos e contribuir para a tomada de decisões em saúde pública. Objetivo: Avaliar parâmetros relacionados à capacidade vetorial da população de Lu. longipalpis presente em área urbana do município de Panorama, estado de São Paulo. Métodos: Foram realizadas capturas mensais durante 48 meses para avaliar a distribuição espaço-temporal de Lu. longipalpis e investigar a circulação de Le. i. chagasi. Também foram realizados os seguintes experimentos com o vetor: captura-marcação-soltura-recaptura para estimar a sobrevida da população e a duração do seu ciclo gonotrófico, a atratividade dos hospedeiros mais frequentes em áreas urbanas, a proporção de repasto em cão, infecção experimental e competência vetorial. Resultados: Observou-se que no município de Panorama, Lu. longipalpis apresentou as frequências mais elevadas na estação chuvosa (entre outubro e março), maior densidade em áreas com presença de vegetação e criação de animais domésticos, locais aonde também foi demonstrada a circulação natural de espécimes de Lu. longipalpis infectados com Le. i. chagasi. Além disto, foi corroborado que a população de Lu. longipalpis apresentou hábito hematofágico eclético, altas taxas de sobrevivência e que foi competente para transmitir o agente da LV. Nos experimentos de laboratório foi evidenciada a heterogeneidade na infecção de fêmeas de Lu. longipalpis desafiadas a se alimentarem em cães comprovadamente infectados por L. i. chagasi e o rápido desenvolvimento do parasita neste vetor natural. Conclusões. As observações do presente estudo corroboram a capacidade vetora de Lu. longipalpis para transmitir a Le. i. chagasi e ressaltam a importância da espécie na transmissão do agente etiológico da LV. Ações de manejo ambiental, educação e promoção à saúde são recomendadas às autoridades municipais para diminuir o risco potencial de infecção na população humana e canina, considerando-se o elevado potencial vetor de Lu. longipalpis e a presença de condições que favorecem a interação dos componentes da tríade epidemiológica da LV.