Road-killed wild animals: a preservation problem useful for eco-epidemiological studies of pathogens

Road-killed wild animals have been for years used for surveillance of vectors of zoonotic pathogens and may offer new opportunities for eco-epidemiological studies. In the current study, fungal infection was evaluated by PCR and nested-PCR in tissue samples collected from 19 road-killed wild animals. The necropsies were carried out and samples were collected for DNA extraction. Results, using PCR with a panfungal primer and nested PCR with specific primers, indicated that some animals are naturally infected with Amauroascus aureus, Metarhizium anisopliae, Aspergillus flavus, Aspergillus oryzae, Emmonsia parva, Paracoccidioides brasiliensis or Pichia stipitis. The approach employed herein proved useful for detecting the environmental occurrence of several fungi, as well as determining natural reservoirs in wild animals and facilitating the understanding of host-pathogen relationships.(FAPESP) São Paulo Research Foundatio

PHYTOHAEMAGGLUTININ’S EFFECT ON NEOSPORA CANINUM ANTIGEN PRODUCTION

The parasite Neospora caninum affects mainly cattle and dogs. This study aimed to evaluate the effect of phytohaemagglutinin (PHE) in antigen production of N. caninum NC-1 strain in gerbils (Meriones unguiculatus) and in vitro. 20 gerbils were used, 10 inoculated intraperitoneally with 1 x 106 tachyzoites and 10 with 1 x 106 tachyzoites plus 300 µL/mL of PHE. 16 bottles of Vero cell culture were inoculated, 8 with 1.5 x 105 tachyzoites and 8 with 1.5 x 105 tachyzoites plus 30 µL/mL of PHE. Serology of gerbils was performed on day 0 and before euthanasia. Tachyzoites present in peritoneal fluid and cell culture bottles were quantified by Neubauer chamber and by real-time PCR (qPCR). PHE has not interfered in the production of tachyzoites of N. caninum in intraperitoneal inoculated gerbils and the effect of PHE in cell culture had a negative impact, considering the qPCR technique as the gold standard

Importance of xenarthrans in the eco-epidemiology of Paracoccidioides brasiliensis

Abstract\ud Background\ud Several pathogens that cause important zoonotic diseases have been frequently associated with armadillos and other xenarthrans. This mammal group typically has evolved on the South American continent and many of its extant species are seriously threatened with extinction. Natural infection of armadillos with Paracoccidioides brasiliensis in hyperendemic areas has provided a valuable opportunity for understanding the role of this mammal in the eco-epidemiology of Paracoccidioidomycosis (PCM), one of the most important systemic mycoses in Latin America.\ud \ud Findings\ud This study aimed to detect P. brasiliensis in different xenarthran species (Dasypus novemcinctus, Cabassous spp., Euphractus sexcinctus, Tamandua tetradactyla and Myrmecophaga tridactyla), by molecular and mycological approaches, in samples obtained by one of the following strategies: i) from road-killed animals (n = 6); ii) from naturally dead animals (n = 8); iii) from animals that died in captivity (n = 9); and iv) from living animals captured from the wild (n = 2). Specific P. brasiliensis DNA was detected in several organs among 7/20 nine-banded armadillos (D. novemcinctus) and in 2/2 anteaters (M. tridactyla). The fungus was also cultured in tissue samples from one of two armadillos captured from the wild.\ud \ud Conclusion\ud Members of the Xenarthra Order, especially armadillos, have some characteristics, including a weak cellular immune response and low body temperature, which make them suitable models for studying host-pathogen interaction. P. brasiliensis infection in wild animals, from PCM endemic areas, may be more common than initially postulated and reinforces the use of these animals as sentinels for the pathogen in the environment.This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP-n° 05/56771-9, 06/03597-4). We also thank the Departamento de Estradas de Rodagem do Estado de São Paulo (DER), as well as Prof. Dr. Reinaldo José da Silva and Juliana Griese for information about the road-killed animals.This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESPn° 05/567719, 06/035974). We also thank the Departamento de Estradas de Rodagem do Estado de São Paulo (DER), as well as Prof. Dr. Reinaldo José da Silva and Juliana Griese for information about the roadkilled animals

FIRST ISOLATION OF Leptospira kirschneri SEROVAR CANICOLA AND Leptospira interrogans SEROVAR PYROGENES IN URINE SAMPLES FROM SLAUGHTERED CATTLE IN MIDWEST REGION OF SÃO PAULO STATE, BRAZIL

A leptospirose é uma zoonose de grande importância em saúde pública. Cento e quatro (104) amostras de urina de bovinos abatidos na região centro-oeste do Estado de São Paulo, Brasil, foram avaliadas para detectar Leptospira spp. As amostras foram mantidas em PBS e em culturas de EMJH e Fletcher. Observamos crescimento de microrganismos em 13 amostras do meio Fletcher, embora não tenha sido observado no meio EMJH. Por PCR, 25 animais (amostras) foram positivos com os primers LEP1 / LEP2, sendo que 16 amostras foram mantidas em PBS, seis em meio Fletcher e três em meio EMJH. As 25 amostras foram submetidas a PCR com o gene LipL32 para verificação da patogenicidade. Uma amostra amplificada e por sequenciamento resultou em 99,77% de similaridade com Leptospira kirschneri sorovar Canicola e Leptospira interrogans sorovar Pyrogenes. Este é o primeiro relato de isolamento de Leptospira kirschneri sorovar Canicola e Leptospira interrogans sorovar Pyrogenes em amostra de urina bovina na Região Centro-Oeste do estado de São Paulo, Brasil. Concluímos a importância da leptospirose no rebanho avaliado e a atenção para o risco ocupacional, devido à possibilidade de urina contaminada nas pastagens e disseminação da infecção para outros animais e pessoas contactantes.Leptospirosis is a zoonosis of great importance in public health. One hundred and four urine samples from slaughtered cattle in Midwest region of São Paulo State, Brazil were evaluated to detect Leptospira spp. The samples were kept in PBS and in cultures of EMJH and Fletcher. We observed growth of microorganisms in 13 samples of Fletcher medium, although it was not observed in EMJH medium. By PCR, 25 animals (samples) were positive with LEP1/LEP2 primers, which of them 16 samples were kept in PBS, six samples in Fletcher medium and three in EMJH medium. The 25 samples were submitted to PCR with the LipL32 gene to verify the pathogenicity. One sample amplified and by sequencing resulted in 99.77% of similarity to Leptospira kirschneri serovar Canicola and Leptospira interrogans serovar Pyrogenes. This is the first report of isolation of Leptospira kirschneri serovar Canicola and Leptospira interrogans serovar Pyrogenes from bovine urine sample in the Midwest Region of São Paulo state, Brazil. We concluded the importance of leptospirosis in the herd evaluated and the attention for the ocupacional risk, because of the possibility of contaminated urine in the pastures and the spread of infection to other animals and contactants persons

Ecological study of Paracoccidioides brasiliensis in soil: growth ability, conidia production and molecular detection

<p>Abstract</p> <p>Background</p> <p><it>Paracoccidioides brasiliensis </it>ecology is not completely understood, although several pieces of evidence point to the soil as its most probable habitat. The present study aimed to investigate the fungal growth, conidia production and molecular pathogen detection in different soil conditions.</p> <p>Methods</p> <p>Soils samples of clayey, sandy and medium textures were collected from ground surface and the interior of armadillo burrows in a hyperendemic area of Paracoccidioidomycosis. <it>P</it>. <it>brasiliensis </it>was inoculated in soil with controlled humidity and in culture medium containing soil extracts. The molecular detection was carried out by Nested PCR, using panfungal and species specific primers from the ITS-5.8S rDNA region.</p> <p>Results</p> <p>The soil texture does not affect fungus development and the growth is more abundant on/in soil saturated with water. Some soil samples inhibited the development of <it>P. brasiliensis</it>, especially those that contain high values of Exchangeable Aluminum (H+Al) in their composition. Some isolates produced a large number of conidia, mainly in soil-extract agar medium. The molecular detection was positive only in samples collected from armadillo burrows, both in sandy and clayey soil.</p> <p>Conclusion</p> <p><it>P. brasiliensis </it>may grow and produce the infectious conidia in sandy and clayey soil, containing high water content, mainly in wild animal burrows, but without high values of H+Al.</p

Extracellular vesicles in infectious diseases caused by protozoan parasites in buffaloes

Background: Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. Methods: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. Results: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. Conclusions: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes

A study of tuberculosis in road traffic-killed badgers on the edge of the British bovine TB epidemic area

The role of badgers in the geographic expansion of the bovine tuberculosis (bTB) epidemic in England is unknown: indeed there have been few published studies of bTB in badgers outside of the Southwest of England where the infection is now endemic in cattle. Cheshire is now on the edge of the expanding area of England in which bTB is considered endemic in cattle. Previous studies, over a decade ago when bovine infection was rare in Cheshire, found no or only few infected badgers in the south eastern area of the county. In this study, carried out in 2014, road-killed badgers were collected through a network of local stakeholders (farmers, veterinarians, wildlife groups, government agencies), and Mycobacterium bovis was isolated from 21% (20/94) badger carcasses. Furthermore, there was strong evidence for co-localisation of M. bovis SB0129 (genotype 25) infection in both badgers and cattle herds at a county scale. While these findings suggest that both badgers and cattle are part of the same geographically expanding epidemic, the direction of any cross-species transmission and the drivers of this expansion cannot be determined. The study also demonstrated the utility of using road-killed badgers collected by stakeholders as a means of wildlife TB surveillance