Detection of Brucella canis and Leptospira interrogans in Canine Semen by Multiplex Nested PCR

Abstract

ABSTRACT. Brucella canis and Leptospira interrogans are pathogenic bacteria that cause brucellosis and leptospirosis in dogs around the world. Both diseases can be diagnosed serologically, but the direct detection of these organisms in canine semen is needed when it is used for artificial reproduction. We have been attempting the artificial reproduction of guide dogs for greater breeding efficiency and for this purpose have developed a multiplex nested PCR technique for the detection of B. canis and L. interrogans in the semen and cryoprotective agent (CPA). Our results demonstrated the high sensitivity and simplicity of this technique in the detection of these organisms in canine semen and that will be useful in routine diagnosis. Since they have been found to stay alive in canine semen and CPA up to 48 hr, canine semen for breeding purposes should be checked for contamination by the PCR assay. KEY WORDS: Brucella canis, detection, Leptospira interrogans, PCR.J. Vet. Med. Sci. 68(6): 615-618, 2006 Canine brucellosis and leptospirosis are widely distributed around the world and they are important diseases due to the economic losses in animal production, and their risks to human health [2]. Reproductive disorders such as abortions and premature births will be the clinical signals of these bacterial diseases in pregnant animals. Though both diseases can be diagnosed from the detection of serum specific antibodies, the direct detection of these organisms in canine semen is needed when the semen is used for artificial reproduction. Bacteriological isolation is usually employed in diagnosis, but this is difficult, time consuming and dangerous [5,8].In Japan, there are 895 guide dogs which provide physical and mental support to the sight impaired. Since the raising of guide dogs is not easy, and the labor and other costs are huge [6], artificial reproduction of guide dogs will be needed to solve these problems. In the present study, we describe a method of detecting Burucella canis and Leptospira interrogans in canine semen for artificial reproduction using multiplex PCR, which is easier to perform and more rapid than other methods.B. canis QE13, which had been maintained as frozen glycerol stocks, were cultured in Brucella broth (Becton Dickinson) or Brucella broth containing 1.5% agar [7]. L. interrogans serotype Pomona (ATCC23478) were grown in liquid EMJH medium [1] at 30°C under aerobic conditions to a density of about 10 8 bacteria per ml when counted by the method of Faine [3]. Semen samples were collected from three brucellosis-free dogs, and then used for bacteriological and PCR amplification assays to detect B. canis or L. interrogans. Specific primer sequences were designed to detect the virB2 gene for Brucella spp. and 16S rRNA gene for Leptospira spp [4,9]. The sequences of oligonucleotide primers for PCR and nested PCR and expected sizes of amplicons are shown in Table 1. The accession number of the chromosome on the NCBI Enterez Genome website was AF226278 for B. abortus and M71241 for L. interrogans...

The first case of Brucella canis in Sweden: background, case report and recommendations from a northern European perspective

“…PCR-analysis of whole blood has a high sensitivity, whereas serum is not suitable for PCR analysis [52]. Semen can be analysed with PCR [53,54]. PCR can be a more sensitive method than bacterial culture because it detects not only viable but also dead bacteria, and because the method is not affected by contamination with other bacteria [55].…”

Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis

“…In dogs, Brucella species DNA has been detected in blood (Keid and others 2007c), semen (Kim and others 2006, Keid and others 2007a) and vaginal swab samples (Keid and others 2007b). Despite the high sensitivity of blood PCR (Keid and others 2007c), the procedure used to extract DNA from whole blood can be time consuming.…”

Nucleic Acid-based Diagnosis and Epidemiology of Infectious Diseases

“…By this method, typically, DNA duplex templates are melted at high temperatures, and two oligonucleotides complementary to the flanking gene target sequence are specifically annealed in a strict temperature dependent on the primer sequence and length. Variations of the technique includes the utilization of a set of oligonucleotides in order to identify different organisms or variants in a single reaction on a biological sample [4][5][6][7]. Also, to increase sensitivity and specificity, a nested and hemi/semi-nested PCR can be performed using an initial PCR product as template [8,9,6,10].…”