No abstract

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Bovine coronavirus (BCoV) causes enteric and respiratory dis- orders in calves and dysentery in cows. In this study, 51 stool samples of calves from 10 Brazilian dairy farms were analysed by an RT-PCR that amplifies a 488-bp fragment of the hypervariable region of the spike glycoprotein gene. Maximum parsimony genealogy with a heuristic algorithm using sequences from 15 field strains studied here and 10 sequences from GenBank and bredavirus as an outgroup virus showed the existence of two major clusters (1 and 2) in this viral species, the Brazilian strains segregating in both of them. The mean nucleotide identity between the 15 Brazilian strains was 98.34%, with a mean amino acid similarity of 98%. Strains from cluster 2 showed a deletion of 6 amino acids inside domain II of the spike protein that was also found in human coronavirus strain OC43, supporting the recent proposal of a zoonotic spill- over of BCoV. These results contribute to the molecular characterization of BCoV, to the prediction of the efficiency of immunogens, and to the definition of molecular markers useful for epidemiologic surveys on coronavirus-caused diseases.

BackgroundDiarrhea in piglets directly affects commercial swine production. The disease results from the interaction of pathogens with the host immune system and is also affected by management procedures. Several pathogenic agents such as Campylobacter spp., Clostridium perfringens, Escherichia coli, Salmonella spp., group A rotavirus (RV-A), coronaviruses (transmissible gastroenteritis virus; porcine epidemic diarrhea virus), as well as nematode and protozoan parasites, can be associated with disease cases.ResultsAll bacterial, viral, protozoan, and parasitic agents here investigated, with the exception of Salmonella spp. as well as both coronaviruses, were detected in varying proportions in piglet fecal samples, and positive animals were equally distributed between case and control groups. A statistically significant difference between case and control groups was found only for Cystoisospora suis (p = 0.034) and Eimeria spp. (p = 0.047). When co-infections were evaluated, a statistically significant difference was found only for C. perfringens β2 and C. suis (p = 0.014).ConclusionsThe presence of pathogens in piglets alone does not determine the occurrence of diarrhea episodes. Thus, the indiscriminate use of antibiotic and anthelminthic medication should be re-evaluated. This study also reinforces the importance of laboratory diagnosis and correct interpretation of results as well as the relevance of control and prophylactic measures.

Canine rangeliosis, caused by the piroplasmid protozoon Rangelia vitalii, is currently recognized as a reemerging disease that affects domestic dogs in Brazil. In the present study, piroplasmid infection was searched in wild canids (20 Cerdocyon thous and 4 Lycalopex gymnocercus) in Brazil. Molecular analysis, based on PCR and DNA sequencing of a portion of the 18S rRNA gene, revealed that 30% (6/20) C. thous were infected by R. vitalii. Blood and bone marrow samples from one of the R. vitalii-infected C. thous were inoculated into a domestic dog, which developed clinical rangeliosis that was confirmed by molecular tests. However, the C. thous donor showed no clinical, hematological or biochemical alterations, even though its R. vitalii infection status was confirmed for at least 80 days. These observations suggest that R. vitalii is not as highly pathogenic for C. thous as it is for domestic dogs. Phylogenetic analysis inferred by the 18S rRNA gene placed R. vitalii embedded in the clade 'Babesia sensu stricto', consisting of a number of species that represent truly the genus Babesia. It is proposed that the species R. vitalii should be transferred to the genus Babesia. The present study expands our knowledge on the natural history of R. vitalii, suggesting that it might have a natural cycle involving the wild canid C. thous. Further studies are needed to confirm that C. thous is a natural reservoir of R. vitalii in Brazil.

Polymerase chain reaction can be a method of great potential for the detection of leptospires at artificial insemination centres.

No abstract

No abstract