Marcelo Marques da Silveira

Development and application of polymerase chain reaction test for detection of Conidiobolus lamprauges

ABSTRACT.- Silveira M.M., Paula D.A.J., Silva M.C., Pitchenin L.C., Cruz R.A.S., Colodel E.M., Dutra V. & Nakazato L. 2013. Development and application of polymerase chain reaction test for detection of Conidiobolus lamprauges . Pesquisa Veterinaria Brasileira 33(12):1448-1452. Departamento de Clinica Medica Veterinaria, Faculdade de Agronomia, Medicina Ve-terinaria e Zootecnia, Universidade Federal de Mato Grosso, Av. Fernando Correa da Costa 2673, Bairro Boa Esperanca, Cuiaba, MT 78068-900, Brazil. E-mail: lucnak@ufmt.brConidiobolomycosis is a granulomatous disease caused by the fungus Conidiobolus spp. in humans and animals. Traditional technique for diagnosis of the disease is isolation of the agent associated with the presence of typical clinical signs and pathological conditions. The aim of this study was to describe the development of a specific polymerase chain reaction (PCR) test for Conidiobolus lamprauges to detect the fungus in clinical samples. Samples from suspected animals were collected and submitted to isolation, histopathological analy-sis and amplification by PCR. DNA from tissues was subjected to PCR with fungi universal primers 18S rDNA gene, and specific primers were designed based on the same gene in

A phylogenetic study of canine parvovirus type 2c in midwestern Brazil

Since the late 1970s, canine parvovirus type 2 (CPV-2) has emerged as a causative agent of fatal severe acute hemorrhagic enteritis in dogs. To date, three antigenic types of CPV-2 were described worldwide (CPV-2a/b/c). This study was conducted to determine the variants of CPV-2 circulating in dogs from the Cuiaba Municipality in Midwestern Brazil. Out of 50 fecal samples, collected between 2009 and 2011, 27 tested positive for CPV-2. A 583 bp fragment of the VP2 gene was amplified by PCR, 13 representative samples were analyzed further by DNA sequencing. All strains were characterized as CPV-2c, displayed a low genetic variability although observed several amino acid substitution. These findings indicated that CPV-2c has been circulating in dogs from the Cuiaba Municipality in Midwestern Brazil.

Genes termorregulados diferencialmente expressos em Conidiobolus lamprauges

Conidiobolus lamprauges is a pathogen zygomycetes fungi of humans and animals, responsible for conidiobolomycosis, which is characterized by a severe granulomatous chronic rhinosinusitis. The ability to adapt and grow at high temperatures is suggested as an attribute of virulence in fungi that infect animals and humans, however regarding C. lamprauges little information is available about this aspect. This paper aims to identify differential expression genes in C. lamprauges grown at 30°C and 37°C through the technique of Representational Difference Analysis (RDA). After the analysis and sequencing of a set of 120cDNAs, it was identified enolase, a glycolytic enzyme, differentially expressed at 37°C. This gene performs functions related to pathogenicity during host-pathogen interaction process in several pathogenic microorganisms. showing a potential involvement in host-pathogen relationship, and virulence in C. lamprauges.

Expressão diferencial de proteínas do fungo Conidiobolus lamprauges cultivado em diferentes temperaturas

To survive at the body temperature of their hosts, pathogenic fungi have developed important molecular mechanisms, such as protein expression associated with growth at high temperatures. Thus, the aim of this study was to analyze the in vitro growth of Conidiobolus lamprauges at different temperatures and compare proteins expressed by two-dimensional electrophoresis (2D), for the pathogen cultivated at low (28°C) and high (37°C) temperatures. For the analysis of growth temperatures, five isolates of C. lamprauges from sick sheep were incubated at 20, 25, 30, 35 and 40°C and radial growth was measured every 24 hours. For the analysis of differential expression, protein extraction and two-dimensional polyacrylamide gel electrophoresis were performed with C. lamprauges cultivated at 28°C and 37°C for 48 hours. The average radial growth was different at the temperatures tested, and 35°C was found to be the best growth temperature for all isolates. The optimum adjusted temperature ranged between 33.3°C and 34.8°C. The upper and lower limits of growth inhibition were 18°C and 42°C, respectively. Upon expression analysis, a total of 16 spots were differentially expressed, seven (7/16) proteins were downregulated and nine (9/16) were over-expressed at 37oC compared to 28°C. In addition, eight spots were present only at 28oC and six were present only at 37oC. It is suggested that C. lamprauges produces a profile of proteins that is related to thermoregulation triggered by the high temperature of the host.

Detection of noroviruses in free-ranging jaguars (Panthera onca) in the Pantanal, Mato Grosso, Brazil

Nine free-ranging jaguars (Panthera onca) were captured, and rectal swabs were collected in the Pantanal of Cáceres, Mato Grosso, Brazil. Reverse transcription polymerase chain reaction specific for noroviruses was performed. Six jaguars (66.6%) tested positive for norovirus genotype GII.11.

Canine Visceral Leishmaniasis: Dermatological Aspects and Associated Dermatoses

Background: Visceral leishmaniasis (VL) is a zoonotic disease caused by Leishmania chagasi (syn. L. infantum). The dog is the main reservoir for this infectious agent in the urban environment. Among the various systemic signs of viscerotropic infection by L. chagasi, cutaneous lesions, including exfoliative dermatitis, cutaneous ulcers and nodules, alopecia, papular or pustular dermatitis, and onychogryphosis, are the most common in dogs. This study aimed to describe the major cutaneous lesions, evaluate the skin parasite L. chagasi by PCR, and investigate the main dermatoses associated with this zoonosis. Materials, Methods & Results: This study evaluated 50 seropositive dogs of various breeds and sizes for VL by ELISA and IFA and for the dermatological signs associated with VL. Moreover, molecular analysis of skin fragments was performed with primers 150 and 152 for the genus Leishmania, and the species was verified as L. chagasi with RV1 and RV2 primers. Deep skin scraping for mites and fungal culture analysis were performed in all dogs. Of the 50 dogs, 15 (30%) were free of systemic or cutaneous signs; however, changes in skin and annexes were observed in 35 (70%) dogs. Thirty-one dogs (62%) presented infection with dermatophytes, 26 (83.9%) with Microsporum sp., and 5 (16.1%) with Trichophyton sp.; only one dog showed parasitism by Sarcoptes scabiei. A statistically significant association was observed between skin alterations and dermatological infection by dermatophytes (P = 0.61). Of the 29 dogs from which skin fragments were used to perform PCR with specific primers, 19 (65.5%) showed L. chagasi DNA amplification. Discussion: Symptomatic dogs are more common than asymptomatic ones; therefore, sampling was set up in the hospital for reagents dogs with clinical suspicion of this zoonosis. Dermatological signs accounted for 70% of the clinical symptoms presented by the dogs, which were evaluated as described by other authors. Exfoliative dermatitis was the most common skin lesion followed by onychogryphosis and alopecia. This is because of granulomatous or pyogranulomatous inflammation, inflammation in different structures of the skin, or deposition of immune complexes. Only few studies have described the co-existence of VL and dermatophytosis in dogs. We found dermatophyte fungal infection in more than half of the dogs (70%), most frequently Microsporum sp. followed by infection with Trichophyton sp. Regarding the clinical signs, no statistical difference was observed between the dogs with and without dermatophyte infection, which reinforces the lack of specificity in clinical signs that may hinder the diagnosis of both diseases when present as co-morbidities or in isolation. The high frequency of dermatophytosis in dogs with VL may result from a compromised immune system. L. chagasi DNA detected in 65.5% of samples tested by conventional PCR can be related to the host’s immune response, as well as to the uneven distribution of the parasite in different tissues. These results support the high frequency of skin changes and concomitant skin diseases like ringworm observed in dogs with LV, highlighting the importance of researching other differential diagnoses in endemic areas.

Proteínas imunorreativas de Conidiobolus lamprauges isoladas de ovinos infectados naturalmente

The study of sheep conidiobolomycosis has been carried out in its clinical, epidemiological, pathological and molecular aspects. Information, however, about the host immune response in infection Conidiobolus lamprauges is absent. This study aimed to identify immunoreactive proteins that may play an important role in the immune response of sheep naturally infected by C.lamprauges. For protein and immunological characterization, C. lamprauges (strain FIOCRUZ-INCQS 40316) isolated from a sheep with clinical signs of conidiobolomycosis in the MT state and five sera samples of naturally infected sheep were used. The presence of IgG antibody was observed in all patients with reagent titers in dilutions up to 1:1600. In immunoblot technique, the antigenic profile against infected sheep sera showed twelve reactive bands with molecular weights ranging from 35 to 198 kDa. Among them, the 198 kDa protein was reactive against sera from three sheep and the 53 kDa showed increased intensity compared to other bands probably being immunodominant. Healthy animal serum samples showed no reactivity demonstrating the specificity of the technique. The presence of antigenic proteins of C. lamprauges and specific IgG in sheep sera observed in this study may assist in the development of early diagnostic methods and the use of protein as candidate vaccines for the control and prevention of infection in animals and human.

Pneumonia bacteriana em jabuti-piranga (Chelonoidis carbonaria): aspectos clínicos, microbiológicos, radiológicos e terapêutica

Pneumonia is a common respiratory disease in clinical of reptiles. Infectious agents are capable of causing primary pneumonia in reptiles maintained in captivity, but in most cases are secondary to problems of management, hygiene and nutrition. The aim of this study was to report the occurrence of bacterial pneumonia in red-footed tortoise (Chelonoidis carbonaria), and describe the clinical, microbiologic, radiographic and therapeutic management. The animal showed signs of respiratory disorders and has been described in the clinical history before diagnosis of pneumonia. The radiographic findings were suggestive of pneumonia/pulmonary edema. Based on the displayed radiographic examination and clinical signs began treatment with administration of chloramphenicol (40mg/kg/SID/IM) for ten days. Were isolated Klebsiella spp. and Citrobacter spp. bacterial culture done collecting endotracheal lavage. Both with multiple antibiotic resistance profile tested. Treatment protocol was instituted using gentamicin (5mg/kg/IM) applications into seven intervals of 72h. There was improvement in clinical signs of the animal, but the presence of nasal secretion was still observed. New radiographic examination, demonstrating slight decrease in the opacity of the right lung field and no significant change in the left lung field in craniocaudal projection was performed. Because of the persistence of clinical signs presented new collection endotracheal material was performed, and there was isolation of Citrobacter spp. and Enterobacter spp. From the results obtained in the antibiogram, was instituted new protocol with the use of amikacin (2.5mg/kg/IM) applications into seven intervals of 72h. After antibiotic therapy, other radiological examination was performed, and showed satisfactory reduction in pulmonary function and clinical signs.