Letícia Camara Pitchenin

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

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.

Presence of Ureaplasma diversum in the genital tracts of female dairy cattle in Mato Grosso State, Brazil

Ureaplasma diversum infection in bovine females may result in various reproductive problems, including granular vulvovaginitis, abortion, weak calves, salpingitis, and spontaneous abortion. The presence of U. diversum in a dairy bovine population from midwestern Brazil has not been established. The aim of this study was to determine whether U. diversum was present in dairy cattle from midwestern Brazil using polymerase chain reaction (PCR). Vulvovaginal mucus was analyzed from 203 cows located in six municipalities in the north region of Mato Grosso State, Brazil. A total of 25% of dairy cows with vulvovaginitis were positive for U. diversum. The factors evaluated were included in a multivariable logistic regression model with the presence of at least one positive cow in the herd serving as the dependent variable. Three variables were significantly associated with a U. diversum-positive PCR and were included in the final multivariable model: number of parities, vulvar lesions, and reproductive problems. For each new parity, the chance of U. diversum infection decreased 0.03-fold, indicating that cows with the highest number of parities were more protected. The presence of vulvar lesions was increased 17.6-fold in females positive for U. diversum, suggesting that this bacterium could be related to the red granular lesions in the vulvar mucosa, whereas reproductive problems were increased 7.6-fold. However, further investigations should be conducted to ascertain the effects of U. diversum in association with other mycoplasma species in the herds studied.

Molecular assessment of the transplacental transmission of Toxoplasma gondii, Neospora caninum, Brucella canis and Ehrlichia canis in dogs

Given the fact that numerous microbial species can be detected in pregnant female dogs, the objective of this study was to assess the transplacental transmission of Brucella canis, Ehrlichia canis, Neospora caninum and Toxoplasma gondii in stillborn puppies. This study involved 41 stillborn puppies, 78.6% of which were positive for T. gondii, 52.4% for N. caninum and 59.5% for B. canis. E. canis was not detected in any of the analyzed puppies. Pregnancy is an important physiological condition for the transmission of infectious agents to puppies and transplacental transmission may be epidemiologically relevant in the spread of these opportunistic agents.

Padronização da técnica de nanopartícula de ouro não modificada (AuNPs) para detecção de Actinobacillus pleuropneumoniae em pulmões de suínos

Based on diagnostic tests for the detection of nucleic acids without amplification through the use of gold nanoparticles (AuNPs) have been described for various diseases. This study aimed to develop a technique of unmodified AuNPs to detect Actinobacillus pleuropneumoniae (App). We used 70 lung samples from pigs, 17 with and 53 without characteristic lesions of pneumonia, to detect App. The primer used was based on ApxIV gene. The AuNPs test had a sensitivity of 93.8% and specificity of 84.6% when compared with PCR detection. The results showed good agreement between AuNPs and PCR testing, and the technique can be used as an alternative to conventional tests, since it is quick and easy, and does not require implementation infrastructure and skilled labor.

Porphyromonas gingivalis in the Oral Cavity of English Bulldog Newborn Puppies

Background: Periodontal disease (PD) is the most common disease of the oral cavity in cats and dogs, and it affects up to 80% of these animals. PD begins with the accumulation of bacteria on the surface of the teeth, and it poses a risk for the health of pets. Research on PD in dogs has focused on the identification and characterization of bacterial communities present in the oral cavity. Porphyromonas gingivalis is highly prevalent in the oral cavity. Therefore, the aim of this study was to detect P. gingivalis before and after dental eruption in 15 English bulldog newborn puppies, hoping to contribute to early guidance of oral hygiene management and prevent future PD. Materials, Methods & Results: Fifteen English bulldog newborn puppies were used in this study. Two groups (G1 and G2) were formed with eight and seven puppies, respectively. Oral swab samples were taken from the maxillary incisor region of animals from G1 and G2 10 days after birth (T10). At this moment, the clinical evaluation of the oral cavity showed healthy gums with a thin, shiny, pinkish, and firm margin, without any odor or granular appearance, and with no tooth eruption. On postnatal day 25 (T25), a subgingival sample was collected with a Gracey curette from the maxillary incisors; the oral cavity examination revealed healthy gums and presence of gingival sulcus. Bilateral subgingival samples were also collected from the maxillary canines and fourth premolars of the dams at T10 and T25. All newborn puppies were fed maternal breast milk and supplementation exclusively with commercial milk for dogs in individual bottles. The dams were fed commercial dry food. The average weight of G1 and G2 at T10 was 625.87 ± 85.26 g and 543.50 ± 92.88 g, respectively, and 100% (15/15) of the animals were negative for PG as assessed by polymerase chain reaction (PCR) on oral swab samples. At T25, puppies from groups G1 and G2 weighed 1.465 ± 194 g and 1.206 ± 201 g, respectively, and 100% (15/15) of the puppies were positive for P. gingivalis as assessed by PCR on subgingival samples collected with a Gracey curette. The dams of the puppies in G1 and G2 were positive for PG at T10 and T25 as determined by PCR on subgingival samples. Discussion:An important finding of this study was that the dams of the puppies in G1 and G2 were positive for P. gingivalis at T10. Several species of bacteria that cause periodontal disease can be transmitted from humans to pets; therefore, transmission from dam to puppy would be possible, but was not observed in this study at T10, when 100% (15/15) of the animals were negative for P. gingivalis. Subgingival microbiota associated with periodontitis consists essentially of Porphyromonas spp., and the presence of gingival sulcus and dental eruption are determinant factors for the presence of P. gingivalis in the oral cavity. Nevertheless, the hygiene habits of dogs, with the dam licking the puppies after dental eruption, could have been a relevant factor for transmission and appearance of P. gingivalis in the subgingival sample in 100% (15/15) of the puppies at T25. The oral microbiota is closely related to many diseases, and resident pathogenic oral bacteria can be transferred by close contact. Certain species of bacteria present in the subgingival biofilm exhibit higher etiologic relevance during the onset and progression of periodontitis, and Porphyromonas spp. is among the most important of these species. It is important to keep in mind that age is a relevant factor to prevent periodontitis. Therefore, providing owners with instructions for thorough dental brushing of animals when they still have deciduous teeth can prevent the appearance of future PD.

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.