Guido Fontgalland Coelho Linhares

Assessment of primers designed for the subspecies-specific discrimination among Babesia canis canis, Babesia canis vogeli and Babesia canis rossi by PCR assay

Canine babesiosis is an infectious disease caused by either Babesia gibsoni or Babesia canis protozoans. The latter is also classified under three different phylogenetic groups, referred to as subspecies B. canis canis, B. canis vogeli and B. canis rossi. The objective of the present study was to validate and standardize a PCR assay to discriminate the organisms at the subspecies level. First, the reference sequences of the 18S rRNA, 5.8S rRNA and 28S rRNA genes, including the internal transcribed spacer 1 (ITS1) and 2 (ITS2) of the most common species and subspecies of the genus Babesia were retrieved from the GenBank database. Subspecies-specific primers (BAB3, BAB4 and BAB5) and one genus-specific primer were designed from the alignment of the sequences. The PCR assays were evaluated in three different combinations of primer pairs in order to assure complete specificity for each reaction. The results of the tests had demonstrated effectiveness of the novel primer pairs BAB1/BAB3, BAB1/BAB4 and BAB1/BAB5 for the amplification of the subspecies-specific target fragments of 746 bp (B. c. canis), 546 bp (B. c. vogeli) and 342 bp (B. c. rossi) by PCR. The original enzymatic amplification assays with novel primers reported in this paper were confirmed to be a reliable tool for the specific discrimination among B. canis subspecies by single-step PCR assays.

Seasonal dynamics of Rhipicephalus sanguineus (Acari: Ixodidae) in dogs from a police unit in Goiânia, Goiás, Brazil

The seasonal dynamics of Rhipicephalus sanguineus ticks was developed in dogs from a Police Unit in Goiânia, Goias, Brazil, from July 2001 to July 2002. The study was carried out on seven naturally infested dogs (two English Cocker Spaniels and five mongrel dogs), with ages between six months and 10 years. Every two weeks, the numbers of feeding larvae, nymphs, and adults were determined. Dogs showing infestation levels above 500 adult ticks received three acaricide treatments. Considering that the treatments had affected the development of some peaking populations of ticks, it was inferred the occurrence of the following peaks: - larvae (four peaks): from August to November, from November to February, from March to May, and from May to July; - nymphs (five peaks): from July to September, from October to December, from December to February, from March to May, and from June to July; - adults (four peaks): from July to October, from October to January, from January to March, and from April to July. The occurrence of these consecutive peaks of activity of each stage of R. sanguineus may indicate that this tick can develop up to four generations per year in Goiânia. On the other hand, if the acaricide treatment did not interfere with the development of R. sanguineus peaks, more than four peaks of each stage have occurred on the dogs. In this case, it is acceptable to infer that more than one population of R. sanguineus was developing within the kennel concomitantly. The mean numbers of each tick stage was similar in the different seasons. The main attachment sites were located on the neck, chest, forelegs, armpits, ears, between toes and on the head. The number of adult ticks feeding on English Cocker Spaniel dogs was 1.4 to 11.5 times higher than that feeding on mongrel dogs.

Diferenciação específica entre Taenia saginata e Taenia solium por ensaio de PCR e duplex-PCR

ABSTRACT This study was conducted to evaluate a protocoland to select novel primers for the species-specific identificationof Taenia saginata and Taenia solium by PCR and duplex-PCR assays. Sequences of the LSU rRNA gene of taenids wereobtained from the GenBank ( T. saginata access n ° AB020399and T. solium access n ° AB020395). The sequences were alignedand then used for primer design. The generic primer TBR3 (5’-ggcttgtttgaatggtttgacg- 3’) was selected from a conservedregion. The T. saginata specific primer TBR-4 (5’-cgactcatgaagataaacaaggt-3’) as well as T. solium specificprimers TBR-5 (5’-cggtcgaacagaccataaatct-3’) and TBR-6 (5’-gctactacacctaaattctaacc- 3’) were selected from different semi-conserved regions. The selected sequences were examined infor similarities with other organisms through the GenBank Blastprocedure and experimentally by PCR using total DNA (tDNA) 1 Programa de Pos-graduacao da Escola de Veterinaria da Universidade Federal de Goias Universidade Federal de Goias (UFG),Goiania, GO, Brasil.

Phylogenetic characterization of Babesia canis vogeli in dogs in the state of Goiás, Brazil.

The genus Babesia comprises protozoa that cause diseases known as babesiosis. Dogs are commonly affected by Babesia canis or Babesia gibsoni. Babesia canis is divided into the subspecies Babesia canis canis, Babesia canis vogeli and Babesia canis rossi. Among these, Babesia canis vogeli predominates in Brazil. The objective of this study was to conduct a phylogenetic analysis on Babesia isolates from dogs in Goiânia, Goiás. Blood samples were obtained from 890 dogs presenting clinical signs suggestive of canine babesiosis that were attended at a veterinary hospital of Goiás. Only samples presenting typical intraerythrocytic parasites were used in the study. These were subjected to DNA extraction and amplification of a fragment of the 18S rRNA, by means of PCR. The PCR products were purified and sequenced. Sequences were obtained from 35 samples but only 17 of these were kept after quality assessment. Similarity analysis using BLASTn demonstrated that all 17 sequences corresponded to B. canis vogeli. Analysis using the Mega4 software showed that the isolates of B. canis vogeli from dogs in Goiânia present a high degree of molecular similarity (99.2 to 100%) in comparison with other reference isolates from other regions of Brazil and worldwide, deposited in GenBank.

Construção de iniciadores e otimização de ensaios de PCR e de nested-PCR para a detecção específica de Tritrichomonas foetus

Tritrichomonas foetus is a pathogenic protozoan that causes a venereal disease in cattle known as bovine genital tricomonosis. In spite of the efficacy to recognize the target genomic DNA, the protocols so far developed for the diagnosis of this organism by PCR promote some inespecific amplifications or they are unable to discriminate T. foetus against other species within the genus. The objective of this study was to assess and optimize PCR and nested-PCR assays for the specific diagnosis of T. foetus, using novel primers selected from the alignment of sequences of the genes 18S rRNA, 5.8S rRNA, 28S rRNA and of the internal transcribed spacers of the rDNA (ITS1 and ITS2). A pair of primers was constructed for the genus-specific amplification of a 648 bp fragment and two others to amplify T. foetus species-specific fragments of 343 and 429 bp. No cross amplification was observed against Bos taurus genomic DNA neither against the DNA of usual bovine genital pathogens. Both, single and nested-PCR assays, presented analytical sensitivity to detect at least two T. foetus organisms.

Design of primer pairs for species-specific diagnosis of Leishmania (Leishmania) infantum chagasi using PCR

The objective of this study was to design and evaluate new primers for species-specific detection of L. infantum chagasi using PCR. Two combinations of primer pairs were established with the aim of obtaining specific amplification products from the L. infantum chagasi 18S rRNA gene. The combinations of the primer pairs and the respective sizes of the PCR products, based on the U422465 GenBank reference sequence of L. infantum chagasi, were: LCS1/LCS3 (259 bp) and LCS2/LCS3 (820 bp). It was concluded that the new PCR assays optimized using the primer pairs LCS1/LCS3 and LCS2/LCS3 were effective for specific detection of L. infantum chagasi, with analytical sensitivity to detect 1 pg/µL of DNA.

Molecular diagnosis of Hepatozoon canis in symptomatic dogs in the city of Goiania, Goiás, Brazil

More than 300 species have been described in the genus Hepatozoon, occurring in different vertebrates. Among these, only Hepatozoon canis and Hepatozoon americanum are seen in dogs. Different methods may be used for laboratory diagnosis. The most common of these is direct parasitological examination of parasite stages in blood smears. The aim of this investigation was to conduct a phylogenetic study on Hepatozoon isolates from symptomatic dogs in the city of Goiânia, Goias, Brazil. Blood samples were obtained from 40 symptomatic dogs that had been referred to the Veterinary Hospital of the Federal University of Goias. Among these, only two samples were positive for Hepatozoon spp. using the direct parasitological method. These samples were then subjected to a DNA extraction process and amplification of a fragment of the 18S rRNA by means of PCR. Subsequently, the PCR products from each sample were purified and sequenced. The sequences obtained were then analyzed using the BLASTn algorithm, which identified both sequences of this study as Hepatozoon canis. By applying the Mega4 software, it was confirmed that these isolates of H. canis from dogs in Goiânia are similar to other reference isolates of the same species from other regions of Brazil and worldwide.