Molecular detection of Leptospira spp. in small wild rodents from rural areas of São Paulo State, Brazil.

BACKGROUND: Leptospirosis represents a One Health issue, affecting humans and animals. This study investigated pathogenic leptospires in small wild rodents in São Paulo, Brazil. METHODS: Kidney samples from 164 rodents underwent qPCR testing, targeting pathogenic Leptospira spp. RESULTS: Thirty-five animals (21.34%) tested positive, including five rodent species: Akodon montensis (2/21; 9.5%), Necromys lasiurus (1/4; 25%), Oligoryzomys nigripes (24/92; 26.1%), Oligoryzomys flavescens (5/26; 19.2%), and Sooretamys angouya (3/14; 21.4%). Botucatu municipality exhibited the highest prevalence, with 42.5% (20/47) of the animals testing positive. CONCLUSIONS: The presence of Leptospira spp. in wild rodents suggests they may be chronic carriers, contaminating the environment.

Molecular detection of Leptospira spp. in small wild rodents from rural areas of São Paulo State, Brazil

ABSTRACT Background: Leptospirosis represents a One Health issue, affecting humans and animals. This study investigated pathogenic leptospires in small wild rodents in São Paulo, Brazil. Methods: Kidney samples from 164 rodents underwent qPCR testing, targeting pathogenic Leptospira spp. Results: Thirty-five animals (21.34%) tested positive, including five rodent species: Akodon montensis (2/21; 9.5%), Necromys lasiurus (1/4; 25%), Oligoryzomys nigripes (24/92; 26.1%), Oligoryzomys flavescens (5/26; 19.2%), and Sooretamys angouya (3/14; 21.4%). Botucatu municipality exhibited the highest prevalence, with 42.5% (20/47) of the animals testing positive. Conclusions: The presence of Leptospira spp. in wild rodents suggests they may be chronic carriers, contaminating the environment.

Exposure of free-ranging capybaras (Hydrochoerus hydrochaeris) to the vaccinia virus.

The aim of this study was to evaluate the possibility of free-ranging animals/hunting dogs as sources of infection in the vaccinia virus (VACV) transmission chain. Serological, cell culture and molecular assays were conducted in 56 free-ranging animals and 22 hunting dogs. ELISA/neutralizing assays showed that two (2.5%) capybaras (Hydrochoerus hydrochaeris) had anti-OPV positive antibodies, while all samples tested negative through PCR/cell culture. After being hit by cars on roads, capybaras that exhibited neither clinical signs nor any association with bovine outbreaks had neutralizing antibodies against the Orthopoxvirus, as detected through plaque-reduction neutralizing tests and ELISA. Evidence exists regarding peridomestic capybaras acting as a source of the virus and serving as a link between wild and urban environments, thus contributing to viral maintenance.

Real-time quantitative PCR-based detection of Coxiella burnetii in unpasteurized cow's milk sold for human consumption.

Coxiella burnetii is a zoonotic pathogen with a worldwide distribution that is responsible for Q fever in humans. It is a highly infectious bacterium that can be transmitted from cattle to humans through the consumption of unpasteurized milk. We report the molecular identification of C. burnetii in raw cow's milk being sold directly for human consumption in Brazil without official inspection or pasteurization. One hundred and twelve samples of raw milk were analysed by real-time quantitative PCR (qPCR), and C. burnetii was detected in 3.57% (4/112) of the samples at a concentration ranging from 125 to 404 bacteria per millilitre. The identification of this zoonotic pathogen in raw milk sold directly for human consumption is a public health concern since C. burnetii can be transmitted through the oral route. This result indicates that health education and other preventive measures should be officially implemented in Brazil to prevent the spread of infection. To our knowledge, this is the first qPCR-based detection of C. burnetii in raw milk samples from cows sold in Brazil that do not undergo official inspection or pasteurization.

Author Correction: Septicemia due to Streptococcus dysgalactiae subspecies dysgalactiae in vampire bats (Desmodus rotundus).

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Septicemia due to Streptococcus dysgalactiae subspecies dysgalactiae in vampire bats (Desmodus rotundus).

Beta-hemolytic Streptococcus dysgalactiae is a well-known pathogen for a wide range of animals and humans. Two subspecies are recognized: (i) equisimilis, associated to disease in horses and humans, and (ii) dysgalactiae mainly isolated from animal illness with only a few humans' cases. This study describes the isolation and characterization of Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) from vampire bats, maintained in captivity for research proposes. Animals presented neurologic, respiratory and gastroenteric symptoms and sudden death. Beta-hemolytic Gram-positive cocci were isolated in blood agar plates and further characterized as Lancefield group C. All isolates were identified as S. dysgalactiae by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and subspecies dysgalactiae was confirmed by 16S rRNA sequencing and phylogenetic analysis. Genotyping through SE-ALFP resulted in three profiles (A1-A3) with one bat being infected by profiles A1 and A3. This is the first report of SDSD causing illness in bats and especially in Desmodus rotundus species.

Brucellosis Prevalence in Brazilian Slaughterhouses with Different Meat Inspection Systems.

In Brazil, meat inspection occurs in a decentralized manner and consists of three types: (i) federal inspection (SIF), (ii) state inspection (SISP), and (iii) municipal inspection (SIM). The objective of this work was to discuss the three current inspection systems through the apparent prevalence of bovine brucellosis, a zoonosis that has an eradication program implemented by the Brazilian government. Nine abattoirs from federal, state, and municipal inspection systems were assessed and 1,490 animals were sampled. Serology for brucellosis was determined by the rose bengal test and the complement fixation test. The overall apparent prevalence (and 95% confidence interval) of brucellosis was 2.2% (1.5 to 2.9%). Apparent prevalence stratified by inspection system for SIF, SISP, and SIM was 0.4% (0.0 to 0.9%), 2.0% (0.8 to 3.2%), and 4.3% (2.5 to 6.1%), respectively. Multivariable logistic regression analysis revealed the odds ratio for finding an animal positive for brucellosis among inspection systems. A statistical difference ( P < 0.0015) was observed among surveillance systems, with SISP × SIF, SIM × SISP, and SIM × SIF having an odds ratio of 4,996, 2,304, and 11,494, respectively. Hence, the need for increasing official surveillance in state and municipal inspection systems seems to be necessary and could assist in the surveillance of bovine brucellosis and other diseases of interest to the federation. In addition, an increase in official presence would help to improve the selection of slaughtered cattle during ante- and postmortem inspection, with consequent impact on food safety and public health.

Ticks infesting wild small rodents in three areas of the state of São Paulo, Brazil / Carrapatos infestando pequenos roedores silvestres em três municípios do estado de São Paulo, Brasil

ABSTRACT: From May to September 2011, a total of 138 wild rodents of the Cricetidae family were collected in the cities of Anhembi, Bofete and Torre de Pedra, in São Paulo State. All animals were examined for the presence of ticks, which were collected and identified at species level in the laboratory by morphological examination (for adults, nymphs and larvae) and molecular biology, by sequencing of a fragment of the 16S mitochondrial rDNA (for larvae). A total of 47 ticks (21 larvae and 26 nymphs) were collected from rodents, identified as 21 larvae and 23 nymphs of Amblyomma ovale (collected on Oligoryzomys nigripes, Oligoryzomys flavescens, Sooretamys agouya and Nectomys squamipes), two nymphs of Ixodes schulzei (O. nigripes and O. flavescens) and an Amblyomma sculptum nymph in S. agouya. The overall prevalence of parasitism by A. ovale was 4.3% for larvae, and 10.1% for nymphs. The mean intensity of parasitism was 3.5 larvae/parasitized animal, and 1.6 nymphs/parasitized animal. One O. nigripes was found parasitized by both larva and nymph of A. ovale, and another O. nigripes was parasitized simultaneously by an I. schulzei nymph and five A. ovale nymphs. This research reports the following unpublished records: A. ovale on O. flavescens, O. nigripes and S. agouya; A. sculptum on S. agouya; and I. schulzei on O. flavescens and O. nigripes.

RESUMO: De maio a setembro de 2011, um total de 138 roedores silvestres da família Cricetidae foram coletados nos municípios de Anhembi, Bofete e Torre de Pedra, no estado de São Paulo. Todos os animais foram examinados quanto à presença de carrapatos, os quais foram coletados e identificados ao nível de espécie em laboratório, através de análises morfológicas (para adultos, ninfas e larvas) e por biologia molecular para confirmar estas análises, através do sequenciamento de um fragmento do gene 16S rDNA mitocondrial (para uma parte das larvas). Um total de 47 carrapatos (21 larvas e 26 ninfas) foi coletado dos roedores, sendo identificados como 21 larvas e 23 ninfas de Amblyomma ovale (coletados sobre Oligoryzomys nigripes, Oligoryzomys flavescens, Sooretamys agouya e Nectomys squamipes), duas ninfas de Ixodes schulzei (em O. nigripes e O. flavescens) e uma ninfa de Amblyomma sculptum em S. agouya. A prevalência geral de parasitismo por A. ovale foi de 4,3% e de 10,1% para larvas e ninfas, respectivamente. As intensidades médias de parasitismo foram de 3,5 larvas/animal parasitado e de 1,6 ninfas/animal parasitado. Um O. nigripes foi encontrado parasitado simultaneamente por larva e ninfa de A. ovale, e outro O. nigripes estava parasitado simultaneamente por uma ninfa de I. schulzei e cinco ninfas de A. ovale. O presente trabalho reporta os seguintes registros inéditos: A. ovale em O. flavescens, O. nigripes e S. agouya; A. sculptum em S. agouya; e I. schulzei em O. flavescens e O. nigripes.

Profile of Cytokines and Chemokines Triggered by Wild-Type Strains of Rabies Virus in Mice.

Rabies is a lethal infectious disease that causes 55,000 human deaths per year and is transmitted by various mammalian species, such as dogs and bats. The host immune response is essential for avoiding viral progression and promoting viral clearance. Cytokines and chemokines are crucial in the development of an immediate antiviral response; the rabies virus (RABV) attempts to evade this immune response. The virus's capacity for evasion is correlated with its pathogenicity and the host's inflammatory response, with highly pathogenic strains being the most efficient at hijacking the host's defense mechanisms and thereby decreasing inflammation. The purpose of this study was to evaluate the expression of a set of cytokine and chemokine genes that are related to the immune response in the brains of mice inoculated intramuscularly or intracerebrally with two wild-type strains of RABV, one from dog and the other from vampire bat. The results demonstrated that the gene expression profile is intrinsic to the specific rabies variant. The prompt production of cytokines and chemokines seems to be more important than their levels of expression for surviving a rabies infection.

Evaluation of short-interfering RNAs treatment in experimental rabies due to wild-type virus

ABSTRACTWe have evaluated the efficacy of short-interfering RNAs targeting the nucleoprotein gene and also the brain immune response in treated and non-treated infected mice. Mice were inoculated with wild-type virus, classified as dog (hv2) or vampire bat (hv3) variants and both groups were treated or leaved as controls. No difference was observed in the lethality rate between treated and non-treated groups, although clinical evaluation of hv2 infected mice showed differences in the severity of clinical disease (p = 0.0006). Evaluation of brain immune response 5 days post-inoculation in treated hv2 group showed no difference among the analyzed genes, whereas after 10 days post-inoculation there was increased expression of 2',5'-oligoadenylate synthetase 1, tumor necrosis factor alpha, interleukin 12, interferon gamma, and C-X-C motif chemokine 10 associated with higher expression of Ngene in the same period (p < 0.0001). In hv2 non-treated group only higher interferon beta expression was found at day 5. The observed differences in results of the immune response genes between treated and non-treated groups is not promising as they had neither impact on mortality nor even a reduction in the expression of N gene in siRNA treated animals. This finding suggests that the use of pre-designed siRNA alone may not be useful in rabies treatment.

Fluorescent antibody test, quantitative polymerase chain reaction pattern and clinical aspects of rabies virus strains isolated from main reservoirs in Brazil

ABSTRACTRabies virus (RABV) isolated from different mammals seems to have unique characteristics that influence the outcome of infection. RABV circulates in nature and is maintained by reservoirs that are responsible for the persistence of the disease for almost 4000 years. Considering the different pattern of pathogenicity of RABV strains in naturally and experimentally infected animals, the aim of this study was to analyze the characteristics of RABV variants isolated from the main Brazilian reservoirs, being related to a dog (variant 2),Desmodus rotundus (variant 3), crab eating fox, marmoset, and Myotis spp. Viral replication in brain tissue of experimentally infected mouse was evaluated by two laboratory techniques and the results were compared to clinical evolution from five RABV variants. The presence of the RABV was investigated in brain samples by fluorescent antibody test (FAT) and real time polymerase chain reaction (qRT-PCR) for quantification of rabies virus nucleoprotein gene (N gene). Virus replication is not correlated with clinical signs and evolution. The pattern of FAT is associated with RABV replication levels. Virus isolates from crab eating fox and marmoset had a longer evolution period and higher survival rate suggesting that the evolution period may contribute to the outcome. RABV virus variants had independent characteristics that determine the clinical evolution and survival of the infected mice.

Evaluation of short-interfering RNAs treatment in experimental rabies due to wild-type virus.

We have evaluated the efficacy of short-interfering RNAs targeting the nucleoprotein gene and also the brain immune response in treated and non-treated infected mice. Mice were inoculated with wild-type virus, classified as dog (hv2) or vampire bat (hv3) variants and both groups were treated or left as controls. No difference was observed in the lethality rate between treated and non-treated groups, although clinical evaluation of hv2 infected mice showed differences in the severity of clinical disease (p=0.0006). Evaluation of brain immune response 5 days post-inoculation in treated hv2 group showed no difference among the analyzed genes, whereas after 10 days post-inoculation there was increased expression of 2',5'-oligoadenylate synthetase 1, tumor necrosis factor alpha, interleukin 12, interferon gamma, and C-X-C motif chemokine 10 associated with higher expression of N gene in the same period (p<0.0001). In hv2 non-treated group only higher interferon beta expression was found at day 5. The observed differences in results of the immune response genes between treated and non-treated groups is not promising as they had neither impact on mortality nor even a reduction in the expression of N gene in siRNA treated animals. This finding suggests that the use of pre-designed siRNA alone may not be useful in rabies treatment.

Fluorescent antibody test, quantitative polymerase chain reaction pattern and clinical aspects of rabies virus strains isolated from main reservoirs in Brazil.

Rabies virus (RABV) isolated from different mammals seems to have unique characteristics that influence the outcome of infection. RABV circulates in nature and is maintained by reservoirs that are responsible for the persistence of the disease for almost 4000 years. Considering the different pattern of pathogenicity of RABV strains in naturally and experimentally infected animals, the aim of this study was to analyze the characteristics of RABV variants isolated from the main Brazilian reservoirs, being related to a dog (variant 2), Desmodus rotundus (variant 3), crab eating fox, marmoset, and Myotis spp. Viral replication in brain tissue of experimentally infected mouse was evaluated by two laboratory techniques and the results were compared to clinical evolution from five RABV variants. The presence of the RABV was investigated in brain samples by fluorescent antibody test (FAT) and real time polymerase chain reaction (qRT-PCR) for quantification of rabies virus nucleoprotein gene (N gene). Virus replication is not correlated with clinical signs and evolution. The pattern of FAT is associated with RABV replication levels. Virus isolates from crab eating fox and marmoset had a longer evolution period and higher survival rate suggesting that the evolution period may contribute to the outcome. RABV virus variants had independent characteristics that determine the clinical evolution and survival of the infected mice.

Serological study of vaccinia virus reservoirs in areas with and without official reports of outbreaks in cattle and humans in São Paulo, Brazil.

Vaccinia virus (VACV), the etiological agent of an exanthematic disease, has been associated with several bovine outbreaks in Brazil since the end of the global vaccination campaign against smallpox. It was previously believed that the vaccine virus used for the WHO global campaign had adapted to an unknown wild reservoir and was sporadically re-emerging in outbreaks in cattle and milkers. At present, it is known that Brazilian VACV is phylogenetically different from the vaccinia virus vaccinal strain, but its origin remains unknown. This study assessed the seroprevalence of orthopoxviruses in domestic and wild animals and farmers from 47 farms in three cities in the southwest region of the state of São Paulo with or without official reports of outbreaks in cattle or humans. Our data indicate a low seroprevalence of antibodies in wild animals and raise interesting questions about the real potential of wild rodents and marsupials as VACV reservoirs, suggesting other routes through which VACV can be spread.

Rabies virus distribution in tissues and molecular characterization of strains from naturally infected non-hematophagous bats.

Bats are main reservoirs for Lyssavirus worldwide, which is an important public health issue because it constitutes one of the big challenges in rabies control. Yet, little is known about how the virus is maintained among bats, and the epidemiological relationships remain poorly understood. The aim of the present study was to investigate the distribution of the rabies virus (RABV) in bat tissues and organs and to genetically characterize virus isolates from naturally infected non-hematophagous bats. The heminested reverse transcriptase polymerase chain reaction (hnRT-PCR) and sequencing using primers to the nucleoprotein coding gene were performed. The results showed a dissemination of the RABV in different tissues and organs, particularly in the salivary glands, tongue, lungs, kidneys, bladder, intestine and feces, suggesting other possible forms of RABV elimination and the possibility of transmission among these animals. The phylogenetic analysis confirmed that different variants of RABV are maintained by non-hematophagous bats in nature and have similar tissue distribution irrespective of bat species and phylogenetic characterization.