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.

Associação de variáveis ambientais à ocorrência de leptospirose canina e humana na cidade de São Paulo

Foi realizada uma análise espacial da ocorrência de leptospirose humana e canina na Supervisão de Vigilância em Saúde do Butantã, situada no município de São Paulo, no ano de 2007, associada a variáveis ambientais de risco, tais como: focos de enchente e áreas de desratização. Foram encontrados aglomerados espaciais de pontos de alagamentos em 12 setores censitários e de casos de leptospirose humana em quatro setores censitários, sem correlação entre ambos. Não foram encontrados agrupamentos de casos em cães, possivelmente devido à subnotificação. As proporções casos humanos de leptospirose : população humana dentro e fora da área de desratização foram 7:199.600 e 9:257.980, respectivamente. Conclui-se que medidas de controle de roedores como a desratização foram responsáveis pela minimização dos efeitos dos fatores de risco para a transmissão de leptospirose para humanos.

Detection of porcine circovirus genotypes 2a and 2b in aborted foetuses from infected swine herds in the State of São Paulo, Brazil

Background Porcine circovirus type 2 (PCV2) has been associated with several disease complexes, including reproductive failure. The aim of this study was to identify the subtypes of PCV2 that are associated with reproductive failure in pigs from the State of São Paulo, Brazil and to investigate co-infections with other infectious organisms. Findings Samples of 168 aborted foetuses or mummified foetuses from five farrow-to-finish swine farms known to be infected with PCV2 and located in the State of São Paulo were tested for PCV2 by polymerase chain reaction (PCR). Positive samples were additionally tested for porcine parvovirus (PPV), Leptospira spp. and Brucella spp. by PCR. PCV2 was detected in 18 of the samples (10.7%). PPV, Brucella spp. and Leptospira spp were found in 2, 10 and 0 cases, respectively. Eleven PCV2 strains were sequenced and determined to be either genotype 2a (n = 1) or 2b (n = 10). Conclusions The findings indicate that the frequency of PCV2 infections in aborted porcine foetuses from the State of São Paulo is rather low (10.7%) and that co-infection with other pathogens is common and may be involved in PCV2 associated reproductive failure. No repeatable, characteristic amino acid motifs for regions of the PCV2 capsid protein seemed to be associated with abortion in sows.

Prevalência de leptospirose em caprinos leiteiros do semiárido paraibano

Este trabalho teve como objetivos determinar a prevalência de propriedades de caprinos leiteiros positivas e de animais soropositivos para leptospirose no semiárido paraibano. A amostragem foi delineada para a determinação da prevalência de propriedades positivas (focos) e de animais soropositivos para a infecção por Leptospira spp. No total, foram colhidas amostras de sangue de 975 animais procedentes de 110 propriedades leiteiras localizadas no Munícipio de Monteiro, microrregião do Cariri Ocidental, Estado da Paraíba. Para o diagnóstico da infecção por Leptospira spp., foi utilizado o teste de soroaglutinação microscópica (SAM), utilizando como antígenos 24 sorovares. Uma propriedade foi considerada foco quando apresentou pelo menos um animal soropositivo. As prevalências de propriedades positivas e de animais soropositivos foram de 43,6% (IC 95% = 34,2-53,4%) e de 8,7% (IC 95% = 5,7-12,9%), respectivamente. Nos animais, os sorovares mais frequentes foram Autumnalis (1,74%; IC 95% = 0,97-3,09%), Sentot (1,71%; IC 95% = 0,82-3,52%) e Whitcomb (1,39%; IC 95% = 0,65-2,93%), e nas propriedades, os sorovares Autumnalis (10,9%; IC 95% = 5,8-18,3%), Whitcomb (8,2%; IC 95% = 3,8-15,0%) e Sentot e Patoc (7,3%; IC 95% = 3,2-13,8%) foram os mais frequentes. Sugere-se que a infecção está distribuída em caprinos da região, e que há necessidade de implantação de medidas de controle e prevenção, com o intuito de reduzir a ocorrência da infecção e, consequentemente, diminuir perdas econômicas ocasionadas e bloquear a possível transmissão do agente aos seres humanos.

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

Foram investigados a prevalência e os fatores de risco da leptospirose bovina no Estado do Maranhão. O Estado foi dividido em quatro circuitos amostrais com base em parâmetros de produção distintos que variam conforme os diferentes sistemas de produção, as práticas de manejo, a finalidade de exploração, o tamanho médio dos rebanhos e os sistemas de comercialização. Objetivou-se estudar as características epidemiológicas da leptospirose bovina no Estado do Maranhão, de modo a determinar a prevalência em bovinos e em rebanhos, detectar as sorovariedades de Leptospira spp. presentes, identificar os fatores de risco eventualmente associados à leptospirose em bovinos e diferenciar os circuitos pecuários entre si no que se refere à prevalência de leptospirose. A pesquisa foi realizada em 136 propriedades rurais pertencentes ao circuito I, no qual 841 fêmeas bovinas com idade igual ou superior a 24 meses foram analisadas; 238 do circuito II, com 2.582 fêmeas analisadas; 122 do circuito III, com 869 fêmeas analisadas; e 77 do circuito IV, com 540 fêmeas analisadas; no total, 573 propriedades e 4.832 fêmeas foram estudadas. A presença de anticorpos contra Leptospira spp. foi verificada pela técnica de soroaglutinação microscópica (SAM). Das 4.832 fêmeas bovinas analisadas, 1.904 (35,94%; IC 95% = 33,01% - 38,98%) foram reagentes. Das 573 propriedades analisadas, 380 (64,81%; IC 95% = 61,10% - 68,35%) foram consideradas positivas. As sorovariedades Hardjo e Wolffi foram as mais frequentes em todo o Estado. O circuito III foi o que apresentou menor prevalência de leptospirose em todas as comparações. As variáveis identificadas como fatores de risco de leptospirose foram: presença de equinos (p = 0,000), presença de capivaras (p = 0,034) e rebanhos bovinos com 32 ou mais fêmeas adultas (p = 0,002).

Epizootiologic Investigations of Selected Abortive Agents in Free-Ranging Alpine Ibex (Capra Ibex Ibex) in Switzerland

In the early 2000s, several colonies of Alpine ibex (Capra ibex ibex) in Switzerland ceased growing or began to decrease. Reproductive problems clue to infections with abortive agents might have negatively affected recruitment. We assessed the presence of selected agents of abortion in Alpine ibex by serologic, molecular, and culture techniques and evaluated whether infection with these agents might have affected population densities. Blood and fecal samples were collected from 651 ibex in 14 colonies throughout the Swiss Alps between 2006 and 2008. All samples were negative for Salmonella. spp., Neospora caninum, and Bovine Herpesvirus-1. Antibodies to Coxiella burnetii, Leptospira spp., Chlamydophila abortus, Toxoplasma gondii, and Bovine Viral Diarrhea virus were detected in at least one ibex. Positive serologic results for Brucella spp. likely were false. Overall, 73 samples (11.2%) were antibody-positive for at least one abortive agent. Prevalence was highest for Leptospira spp. (7.9%, 95% CI=5.0-11.7). The low prevalences and the absence of significant differences between colonies with opposite population trends suggest these pathogens do not play a significant role in the population dynamics of Swiss ibex. Alpine ibex do not seem to be a reservoir for these abortive agents or an important source of infection for domestic livestock in Switzerland. Finally, although interactions on summer pastures occur frequently, spillover from infected livestock to free-ranging ibex apparently is uncommon.

Prebreeding Immunization of Beef Cows

A number of infectious agents are potential threats to the fetus of a pregnant cow and may result in abortion. These agents include Leptospira sp., Campylobacter fetus and viruses such as infectious bovine rhinotracheitis (IBR) and bovine virus diarrhea (BVD). Maintenance in the cow of a high level of immunity to these agents during pregnancy can insure protection of the fetus. In particular, vaccines against BVD and IBR viruses can establish protective immunity throughout gestation. An appropriate vaccination regimen prior to breeding is required to establish this protective immunity. This can be achieved with a single dose of certain modified live virus vaccines, but those vaccines must be administered at least 30 days prior to breeding to avoid interference with conception. We have evaluated an oil-adjuvanted inactivated virus vaccine in cattle with differing immunological histories. Two doses of the vaccine administered 30 days apart to serologically negative animals induced appreciable levels of BVD and IBR anti-viral antibodies with persisting titers throughout gestation. In other experiments a single dose of the vaccine was administered to: (1) animals given two doses of the vaccine several months earlier, (2) animals previously vaccinated with other inactivated virus vaccines, or (3) animals previously vaccinated with modified live virus vaccine. The vaccine consistently induced marked anamnestic responses in these animals. Not only did mean titers rise, but a vast majority of individual animals responded. This contrasts with efforts to boost titers with modified live virus vaccines where the effect may be erratic among animals. The safety and efficacy of selected inactivated viral vaccines argues for their use in prebreeding immunization of beef cows.

Serial CT features of pulmonary leptospirosis in 10 dogs.

Leptospirosis pulmonary haemorrhage syndrome (LPHS) is a frequent manifestation of Leptospira infection in dogs and is associated with a high morbidity and mortality. Three helical 16-slice thoracic CT scans were performed in 10 dogs naturally infected with Leptospira, within 24 hours of admission, and three and seven days later. Patients were sedated and scanned without breathhold, with a protocol adapted for rapid scanning. One dog died of respiratory failure on the morning following the first scan. On the initial scan, imaging features of LPHS included ground-glass nodules (10/10), peribronchovascular interstitial thickening (10/10), diffuse or patchy ground-glass opacity (9/10), solid nodules (8/10) and consolidation (7/10). Temporary bronchiolar dilation was observed in all dogs in association with peribronchovascular interstitial thickening, which had completely resolved at day 7. Nodules were with few exceptions assigned to the centrilobular region. Regression of lesion severity was observed after each subsequent scan. Consolidation and solid nodules changed over time into lesions of ground-glass attenuation. Pleural effusion (3/10) and mediastinal effusion (2/10) were mild and transient. Lesion severity appeared unassociated with survival to discharge.

Evaluation of the diagnostic value of serologic microagglutination testing and a polymerase chain reaction assay for diagnosis of acute leptospirosis in dogs in a referral center.

OBJECTIVE To determine the diagnostic value of a serologic microagglutination test (MAT) and a PCR assay on urine and blood for the diagnosis of leptospirosis in dogs with acute kidney injury (AKI). DESIGN Cross-sectional study. Animals-76 dogs with AKI in a referral hospital (2008 to 2009). PROCEDURES Dogs' leptospirosis status was defined with a paired serologic MAT against a panel of 11 Leptospira serovars as leptospirosis-associated (n = 30) or nonleptospirosis-associated AKI (12). In 34 dogs, convalescent serologic testing was not possible, and leptospirosis status was classified as undetermined. The diagnostic value of the MAT single acute or convalescent blood sample was determined in dogs in which leptospirosis status could be classified. The diagnostic value of a commercially available genus-specific PCR assay was evaluated by use of 36 blood samples and 20 urine samples. RESULTS Serologic acute testing of an acute blood sample had a specificity of 100% (95% CI, 76% to 100%), a sensitivity of 50% (33% to 67%), and an accuracy of 64% (49% to 77%). Serologic testing of a convalescent blood sample had a specificity of 92% (65% to 99%), a sensitivity of 100% (87% to 100%), and an accuracy of 98% (88% to 100%). Results of the Leptospira PCR assay were negative for all samples from dogs for which leptospirosis status could be classified. CONCLUSIONS AND CLINICAL RELEVANCE Serologic MAT results were highly accurate for diagnosis of leptospirosis in dogs, despite a low sensitivity for early diagnosis. In this referral setting of dogs pretreated with antimicrobials, testing of blood and urine samples with a commercially available genus-specific PCR assay did not improve early diagnosis.

Detection and quantification of leptospires in urine of dogs: a maintenance host for the zoonotic disease leptospirosis.

Leptospirosis is a global zoonotic disease. Pathogenic Leptospira species, the causative agent of leptospirosis, colonize the renal tubules of chronically infected maintenance hosts such as dogs, rats and cattle. Maintenance hosts typically remain clinically asymptomatic and shed leptospires into the environment via urine. In contrast, accidental hosts such as humans can suffer severe acute forms of the disease. Infection results from direct contact with infected urine or indirectly, through contaminated water sources. In this study, a quantitative real-time PCR specific for lipL32 was designed to detect the urinary shedding of leptospires from dogs. The sensitivity and specificity of the assay was evaluated using both a panel of pathogenic Leptospira species and clinical microbial isolates, and samples of urine collected from experimentally infected rats and non-infected controls. The lower limit of detection was approximately 3 genome equivalents per reaction. The assay was applied to canine urine samples collected from local dog sanctuaries and the University Veterinary Hospital (UVH) at University College Dublin. Of 525 canine urine samples assayed, 37 were positive, indicating a prevalence of urinary shedding of leptospires of 7.05%. These results highlight the need to provide effective canine vaccination strategies and raise public health awareness.

Immunohistochemical detection of IgM and IgG in lung tissue of dogs with leptospiral pulmonary haemorrhage syndrome (LPHS).

Leptospiral pulmonary haemorrhage syndrome (LPHS) is a severe form of leptospirosis. Pathogenic mechanisms are poorly understood. Lung tissues from 26 dogs with LPHS, 5 dogs with pulmonary haemorrhage due to other causes and 6 healthy lungs were labelled for IgG (n=26), IgM (n=25) and leptospiral antigens (n=26). Three general staining patterns for IgG/IgM were observed in lungs of dogs with LPHS with most tissues showing more than one staining pattern: (1) alveolar septal wall staining, (2) staining favouring alveolar surfaces and (3) staining of intra-alveolar fluid. Healthy control lung showed no staining, whereas haemorrhagic lung from dogs not infected with Leptospira showed staining of intra-alveolar fluid and occasionally alveolar septa. Leptospiral antigens were not detected. We conclude that deposition of IgG/IgM is demonstrable in the majority of canine lungs with naturally occurring LPHS, similar to what has been described in other species. Our findings suggest involvement of the host humoral immunity in the pathogenesis of LPHS and provide further evidence to support the dog as a natural disease model for human LPHS.

Comparative proteomic analysis of lung tissue from guinea pigs with leptospiral pulmonary haemorrhage syndrome (LPHS) reveals a decrease in abundance of host proteins involved in cytoskeletal and cellular organization

Leptospiral pulmonary haemorrhage syndrome (LPHS) is a particularly severe form of leptospirosis. LPHS is increasingly recognized in both humans and animals and is characterized by rapidly progressive intra-alveolar haemorrhage leading to high mortality. The pathogenic mechanisms of LPHS are poorly understood which hampers the application of effective treatment regimes. In this study a 2-D guinea pig proteome lung map was created and used to investigate the pathogenic mechanisms of LPHS. Comparison of lung proteomes from infected and non-infected guinea pigs via differential in-gel electrophoresis revealed highly significant differences in abundance of proteins contained in 130 spots. Acute phase proteins were the largest functional group amongst proteins with increased abundance in LPHS lung tissue, and likely reflect a local and/or systemic host response to infection. The observed decrease in abundance of proteins involved in cytoskeletal and cellular organization in LPHS lung tissue further suggests that infection with pathogenic Leptospira induces changes in the abundance of host proteins involved in cellular architecture and adhesion contributing to the dramatically increased alveolar septal wall permeability seen in LPHS. BIOLOGICAL SIGNIFICANCE The recent completion of the complete genome sequence of the guinea pig (Cavia porcellus) provides innovative opportunities to apply proteomic technologies to an important animal model of disease. In this study, the comparative proteomic analysis of lung tissue from experimentally infected guinea pigs with leptospiral pulmonary haemorrhage syndrome (LPHS) revealed a decrease in abundance of proteins involved in cellular architecture and adhesion, suggesting that loss or down-regulation of cytoskeletal and adhesion molecules plays an important role in the pathogenesis of LPHS. A publically available guinea pig lung proteome map was constructed to facilitate future pulmonary proteomics in this species.

Markers of endothelial cell activation and immune activation are increased in patients with severe leptospirosis and associated with disease severity

OBJECTIVES Previous studies concluded that haemorrhage is one of the most accurate prognostic factors of mortality in leptospirosis. Therefore, endothelial cell activation was investigated in relation to disease severity in severe leptospirosis. METHODS Prospective cohort study of severe leptospirosis patients. Plasma levels of sE-selectin and Von Willebrand factor (VWF) were determined. Consequently, an in vitro endothelial cell model was used to assess endothelial activation after exposure to virulent Leptospira. Finally, immune activation, as a potential contributing factor to endothelial cell activation, was determined by soluble IL2-receptor (sIL-2r) and soluble Fas-ligand (sFasL) levels. RESULTS Plasma levels of sE-selectin and VWF strongly increased in patients compared to healthy controls. Furthermore, sE-selectin was significantly elevated (203 ng/ml vs. 157 ng/ml, p < 0.05) in survivors compared to non-survivors. Endothelial cells exposed to virulent Leptospira showed increased VWF expression. E-selectin and ICAM-1 expression did not change. Immunohistochemistry revealed the presence of intracellular Leptospira and qPCR suggested replication. In vivo analysis showed that increased levels of sFasL and sIL-2r were both strongly associated with mortality. Furthermore sIL-2r levels were increased in patients that developed bleeding and significantly correlated to duration of hospital stay. DISCUSSION Markers of endothelial activation and immune activation were associated with disease severity in leptospirosis patients.

Multiple infections of rodents with zoonotic pathogens in Austria

Rodents are important reservoirs for a large number of zoonotic pathogens. We examined the occurrence of 11 viral, bacterial, and parasitic agents in rodent populations in Austria, including three different hantaviruses, lymphocytic choriomeningitis virus, orthopox virus, Leptospira spp., Borrelia spp., Rickettsia spp., Bartonella spp., Coxiella burnetii, and Toxoplasma gondii. In 2008, 110 rodents of four species (40 Clethrionomys glareolus, 29 Apodemus flavicollis, 26 Apodemus sylvaticus, and 15 Microtus arvalis) were trapped at two rural sites in Lower Austria. Chest cavity fluid and samples of lung, spleen, kidney, liver, brain, and ear pinna skin were collected. We screened selected tissue samples for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, Leptospira, Borrelia, Rickettsia, Bartonella spp., C. burnetii, and T. gondii by RT-PCR/PCR and detected nucleic acids of Tula hantavirus, Leptospira spp., Borrelia afzelii, Rickettsia spp., and different Bartonella species. Serological investigations were performed for hantaviruses, lymphocytic choriomeningitis virus, orthopox viruses, and Rickettsia spp. Here, Dobrava-Belgrade hantavirus-, Tula hantavirus-, lymphocytic choriomeningitis virus-, orthopox virus-, and rickettsia-specific antibodies were demonstrated. Puumala hantavirus, C. burnetii, and T. gondii were neither detected by RT-PCR/PCR nor by serological methods. In addition, multiple infections with up to three pathogens were shown in nine animals of three rodent species from different trapping sites. In conclusion, these results show that rodents in Austria may host multiple zoonotic pathogens. Our observation raises important questions regarding the interactions of different pathogens in the host, the countermeasures of the host's immune system, the impact of the host–pathogen interaction on the fitness of the host, and the spread of infectious agents among wild rodents and from those to other animals or humans.

Cluster of Leptospirosis Acquired Through River Surfing in Switzerland

Background.  In Switzerland, leptospirosis is still considered as a travel-associated disease. After the surprising diagnosis of leptospirosis in a patient who was initially suspected as having primary human immunodeficiency virus infection, we recognized that acquisition of leptospirosis occurred through recreational activities and we identified additional affected individuals. Methods.  Detailed anamnesis, excluding occupational exposure, acquisition abroad, and pet contacts, enabled us to detect the source of infection and identify a cluster of leptospirosis. Convalescent sera testing was performed to confirm Leptospira infection. Microscopic agglutination tests were used to determine the infecting serovar. Results.  We identified a cluster of leptospirosis in young, previously healthy persons. Acquisition of leptospirosis was traced back to a surfing spot on a river in Switzerland (Reuss, Aargau). Clinical presentation was indistinct. Two of the 3 reported cases required hospitalization, and 1 case even suffered from meningitis. Serologic tests indicated infection with the serovar Grippotyphosa in all cases. With the exception of the case with meningitis, no antibiotics were administered, because leptospirosis was diagnosed after spontaneous resolution of most symptoms. Despite a prolonged period of convalescence in 2 cases, full recovery was achieved. Recent reports on beavers suffering from leptospirosis in this region underline the possible water-borne infection of the 3 cases and raise the question of potential wildlife reservoirs. Conclusions.  Insufficient awareness of caregivers, which may be promoted by the missing obligation to report human leptospirosis, combined with the multifaceted presentation of the disease result in significant underdiagnosis. More frequent consideration of leptospirosis as differential diagnosis is inevitable, particularly as veterinary data suggest re-emergence of the disease.