A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.     

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 × 10⁰ cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.     

Detection of Brucella canis from inguinal lymph nodes of naturally infected dogs by PCR

The aim of this study was to standardize and evaluate a PCR assay for the detection of Brucella canis (B. canis) in lymph node samples of naturally infected dogs. The performance of the PCR was compared with the results of bacteriological culture as reference method. Forty-eight inguinal lymph node samples were collected from 48 dogs (18 males and 30 females) that died in the city's pound in the years 2007-2008 and were examined by microbiological culture and the PCR assay. B. canis was isolated from 4 (8.3%) of 48 lymph node samples. Forty-four (91.7%) of the samples were bacteriological culture negative. B. canis DNA was directly detected from all culture positive lymph node samples (n = 4) by PCR. All of the culture negative samples were confirmed as negative by PCR. When the culture method was used as a gold standard, sensitivity and specificity of the PCR assay were found to be 100%. The limit of PCR detection of B. canis DNA was 1.4 × 10¹ CFU/g at least. In conclusion, the PCR assay has been shown to have a diagnostic performance equal to bacteriological culture for detection of B. canis. By a non-hazardous protocol for laboratory workers, the assay can be performed in one day.     

Use of enrofloxacin in the treatment of canine brucellosis in a dog kennel (clinical trial)

To date, no totally effective antibiotic for the eradication of canine brucellosis has been found. The purpose of this study was to evaluate the efficacy of enrofloxacin in a kennel infected with Brucella canis. Twelve dogs, 2 males and 10 females (including 1 in estrus, 3 pregnant, and 6 in anestrus) infected with B. canis were given 5 mg/kg of enrofloxacin orally every 12 h for 30 days. Females received additional courses of enrofloxacin during the estral and luteal phases of the subsequent cycles (0-2 cycles). They were repeatedly mated by infected males. A serological follow-up was carried out for 38 months. The clinical, serological and bacteriological findings were recorded. In a trial carried out 14 months after the beginning of this study, all dogs were negative on the Rapid Slide Agglutination Test (RSAT). No abortions were observed. All mated female dogs conceived and gave birth to healthy puppies. Cultures of postpartum vaginal discharges (lochia) were negative for B. canis. Similar to other treatments, although enrofloxacin was not completely efficacious in treating canine brucellosis, it maintained fertility and avoided the recurrence of abortions, transmission of the disease to the puppies and dissemination of microorganisms during parturition. We inferred that enrofloxacin could be used as an alternative drug for the treatment of canine brucellosis.     

Babesia gibsoni: Serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP

The thrombospondin-related adhesive protein of Babesia gibsoni (BgTRAP) is known as an immunodominant antigen and is, therefore, considered as a candidate for the development of a diagnostic reagent for canine babesiosis. The recombinant BgTRAP (rBgTRAP) expressed in Escherichia coli was tested in an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to B. gibsoni in dogs. The ELISA with rBgTRAP clearly differentiated between B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. The sera collected from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, B. canis rossi, and Neospora caninum, showed no cross-reactivity by the ELISA with rBgTRAP. A total of 107 blood samples collected from dogs that had been diagnosed as having babesiosis at veterinary hospitals in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA and PCR. Ninety-six (89.7%) and 89 (83.2%) of the tested samples were positive by the ELISA and PCR, respectively, while 11 (10.3%) and 4 (3.7%) were ELISA+/PCR- and ELISA-/PCR+, respectively. In addition, the sensitivity of the ELISA with rBgTRAP was much higher than that of previously established ELISAs with rBgP50, rBgSA1, and rBgP32. These results indicate that the rBgTRAP is the most promising diagnostic antigen for the detection of an antibody to B. gibsoni in dogs and that the combined ELISA/PCR approach could provide the most reliable diagnosis for clinical sites.     

Serological diagnosis of brucellosis caused by Brucella canis

Blood serum samples from 2,328 dogs were tested to detect antibodies against Brucella canis with the agar gel immunodiffusion (AGID) and 2-mercaptoethanol slide agglutination test (ME-SAT) using Brucella ovis as the antigen. All blood serum samples were also evaluated for antibodies against Brucella abortus and Brucella melitensis using the Rose Bengal test. Twentyfive (1.07%) of the sera evaluated were considered positive with AGID test. Only 4 (16%) of these blood serum samples were positive when evaluated with ME-SAT. The 25 AGID positive samples and 25 AGID negative serum samples were also examined by: the complement fixation test (CFT) using B. ovis hot saline extract (HSE) as the antigen, indirect enzyme linked immunosorbent assay (ELISA) and immunoblotting (IB) using B. canis and B. ovis HSE antigens. Two positive canine sera from culture positive dogs and the serum of an experimentally RM6/66 B. canis-infected rabbit were employed as positive controls and one serum from a known uninfected dog as a negative control. ELISA with B. canis antigen gave 9 (18%) positive results (6 AGID-positive and 3 AGID-negative sera). ELISA performed with B. ovis antigen detected 15 (30%) positive samples (10 AGID-positive, 5 AGID-negative and 8 B. canis ELISA positive sera). IB analysis of known positive controls sera employing B. canis antigen detected bands with molecular weights of 94-80, 64-50, 35, 32-30, 28, 23, 20-18, 15-12 kDa. The same sera tested with B. ovis antigen revealed bands of 35, 32-30, 25, 23, 20-18, 15-12 kDa. No bands were observed with the negative control serum and the 50 canine tested sera.     

Comparison of different PCR methods for detection of Brucella spp. in human blood samples

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitive method showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.     

Complete Genome Sequence of Brucella canis Strain HSK A52141, Isolated from the Blood of an Infected Dog

Brucella canis infection can be clinically inapparent in dogs, and when infection goes unnoticed, there is a chance for dog-to-human transmission. A new strain of B. canis was isolated from the blood of an infected dog in order to analyze the pathogenic mechanism, compare genetic properties, and develop new genetic tools for early diagnosis of canine brucellosis. Herein, we report the complete genome sequence of the strain B. canis HSK A52141. This is the second complete genome sequence and biological annotation available for a member of B. canis.     

Diversity of Babesia and Theileria species in symptomatic and asymptomatic dogs in Croatia

Babesiosis, the disease caused by tick-borne hematozoan parasites of the genus Babesia, is particularly common in dogs, and is caused by several “large” species of Babesia, as well as by an increasing number of “small” species of Babesia, some of which appear to be more closely related to members of the genus Theileria. In this work, blood samples were collected from 848 randomly selected, asymptomatic dogs and from 81 symptomatic dogs, microscopically positive for Babesia, and characterised by PCR and sequence analysis of a fragment of the ssrRNA gene. A prevalence of 3.42% (29 of 848) was found in asymptomatic dogs and sequence analysis revealed the presence of Babesia canis canis in 20 dogs (69%), Babesia gibsoni in six dogs (21%), Babesia canis vogeli in two dogs (7%) and Theileria annae in one dog (3%). In the group of symptomatic dogs, which were all positive by PCR, B. canis canis was the predominant species (78 dogs, or 96%), followed by single infections with B. canis vogeli, Babesia caballi and Theileria equi. Our study has confirmed that dogs are infected with a wide range of both large and small piroplasm species and subspecies, including B. caballi and T. equi, two parasites usually found in horses. The detection of the pathogenic species B. canis canis and B. gibsoni in asymptomatic dogs indicates that the relationship between parasite species/subspecies and clinical signs of infection in dogs deserves further investigation. Finally, the identities of the tick vectors transmitting T. annae and B. caballi remain to be elucidated.     

Comparison of culture and PCR for the detection of Brucella melitensis in blood and lymphoid tissues of serologically positive and negative slaughtered sheep

Aims: To compare the culture and PCR methods for detection of Brucella melitensis in blood and lymphoid tissue samples obtained from slaughtered sheep (n = 162) testing positive/negative in serological tests (Rose Bengal test and serum agglutination test). Methods and Results: Of 162 sheep examined, 45 were positive and 117 negative in serological tests. A PCR assay based on a pair of Br. melitensis‐specific primers was used to detect DNA in blood and lymphoid tissue. Brucella melitensis was isolated from 1·2% (2/162) and 17·2% (28/162) of the blood and lymphoid tissue samples respectively. Positive PCR products with a molecular size of 731 bp were obtained from 27·7% (45/162) of blood and 29·0% (47/162) of lymphoid tissue samples. Conclusions: The species‐specific PCR assay detected a higher number of Br. melitensis DNA both from serologically positive (P < 0·01 in blood PCR, P < 0·001 in tissue PCR) and serologically negative (P < 0·001 in both blood PCR and tissue PCR) sheep compared with classical bacteriological culture methods. Significance and Impact of the Study: The results emphasize the importance of using more than one type of diagnostic technique for the detection of animals positive for brucellosis, especially with epidemiological purposes.     

Detection of Neospora caninum in the semen and blood of naturally infected bulls

A prospective study was designed to investigate the presence of Neospora caninum in semen and blood of eight bulls seropositive to N. caninum using nested-PCR procedures. Positive semen and blood samples were bioassayed in a BALB/c nu/nu mouse model. Specific anti-N. caninum serological and interferon-gamma (IFN-gamma) responses were also studied. In parallel, five seronegative bulls acted as non-infected controls. All bulls were located in a collaborating AI centre and monitored for 22 weeks. Six of eight seropositive bulls showed N. caninum DNA in their semen and/or blood samples at some time during the course of the study. In all positive semen samples, we consistently found Neospora-DNA in the cell fraction and not in seminal plasma. Parasite load, as determined by a real-time PCR in nested-PCR positive semen samples, ranged from 1 to 10 parasites/ml. We found no association between the presence of N. caninum DNA in semen and blood. N. caninum could not be detected in the BALB/c nu/nu mice inoculated with PCR-positive semen or blood samples. Specific IgG antibody levels in seropositive bulls fluctuated over time, at times falling below cut-off level. The response was predominantly IgG2, with significant differences compared to control bulls (P < 0.05). The overall mean specific IFN-gamma response in seropositive bulls was also higher than those observed in the control group (P < 0.05), although extensive variation in individual responses was observed among bulls and over time. No significant association was found between bulls showing Neospora DNA in semen, blood, or both, and specific IgG, IgG1, IgG2, IgM and IgA levels or IFN-gamma response. This study is the first to report the presence of Neospora DNA in semen and blood of naturally-infected bulls. Our observations indicate intermittent presence of N. caninum in blood and semen and shedding in semen in low numbers.     

Rapid β-lactoglobulin genotyping of cattle using the polymerase chain reaction

A polymerase chain reaction (PCR) assay has been developed for genotyping beta-lactoglobulin A and B variants in dairy cattle. Either blood or semen samples can be used as a source of DNA. The method is accurate, faster than Southern blot analyses and should prove a useful tool in breeding programmes.     

Evaluation of Micro Complement Fixation Tests for Antibodies Against Group A Streptococcal M and M-Associated Antigens in Rabbit and Human Sera

The microslide gel-diffusion and macro-tube agglutination techniques to detect Brucella canis antibodies in dogs were compared. Sera from dogs experimentally infected with B. canis and a random sample of dog sera with unknown histories of exposure to this organism were examined. The results of the gel-diffusion method employing specific rough Brucella saline-extract antigens of B. canis and Brucella ovis were comparable to those obtained by the tube agglutination test. The easily prepared, stable R antigen in the freeze-dried form offers a convenient, simple, and reliable diagnostic method for the serological detection of canine brucellosis by the gel-diffusion test.     

犬布鲁氏菌病

目前,犬布鲁氏菌病在全球多个国家和地区都有发生,而且发病数量逐年上升,由犬布鲁氏菌病感染人的病例报道也日益增多。因此,犬布鲁氏菌病的控制和根除对人类健康和公共卫生安全具有重要的意义。据报道,我国多个省份存在犬布鲁氏菌病,而且感染和发病率有上升的趋势。由于我国养犬数量巨大,犬与人类接触密切,病犬作为人类布鲁氏菌病的传染源不容忽视。犬布鲁氏菌病的研究起步较晚,流行病学资料欠完善,公共卫生学意义有待阐明,诊断、免疫预防等诸多问题还没完全解决,加强犬布鲁氏菌病的研究和防控应引起足够的重视。本文就犬布鲁氏菌病的病原学、流行病学、病理学、临床症状、诊断、治疗、预防及公共卫生安全等分别做一阐述。     

The occurrence of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis and Anaplasma phagocytophium in dogs in China

A survey of the occurrence of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis and Anaplasma phagocytophium in dogs was undertaken in the People's Republic of China between October 2008 and October 2009. A total of 600 blood samples were taken from dogs in four cities in China: 300 in Beijing, 150 in Shenzhen, 30 in Shanghai and 120 in Zhengzhou. All samples were tested for the heartworm antigen and antibodies of canine B. burgdorferi, E. canis and A. phagocytophium by using the canine SNAP? 4Dx? test kit. The occurrence of D. immitis, B. burgdorferi, E. canis and A. phagocytophium was 1.17% (7/600), 0.17% (1/600), 2.17% (13/600) and 0.5% (3/600), respectively. In Shenzhen city 2% (3/150), 8.67% (13/150) and 2% (3/150) of samples were positive for D. immitis, E. canis and A. phagocytophium, respectively. The occurrence of heartworm antigen was 0.33% (1/300) in Beijing, 2.00% (3/150) in Shenzhen, 3.33% (1/30) in Shanghai and 1.67% (2/120) in Zhengzhou. We found E. canis and A. phagocytophium only at one site, Shenzhen, while the only occurrence of B. burgdorferi was at Beijing. In conclusion, the dog population in China is at potential risk for D. immitis, B. burgdorferi, E. canis and A. phagocytophium infection, the risk being especially high in southern China.     

Detection of Brucella canis in a dog in Italy

Brucella spp. is a worldwide zoonotic pathogen. Infection by Brucella canis in dogs is endemic in the Southern USA and in Central and South America, but it appears sporadically in other parts of the world, including Europe. Tissue samples from a dog with chronic prostatitis, discospondylitis and locomotor problems were subjected to clinical and laboratory examinations. B. canis was detected by PCR in biological fluids and tissues of the animal, while antibodies to B. canis were found in the serum, providing additional strong evidence for the circulation of B. canis in Italy.     

A combined approach for the enhanced detection and isolation of Bartonella species in dog blood samples: pre-enrichment liquid culture followed by PCR and subculture onto agar plates

Historically, direct plating, lysis centrifugation, or freeze-thaw approaches have proven to be highly insensitive methods for confirming Bartonella species infection in dogs. A prospective study was designed to compare diagnostic methods for the detection of Bartonella using samples submitted to the Vector-Borne Disease Diagnostic Laboratory at North Carolina State University. Methods included indirect immunofluorescence assay, PCR, direct inoculation of a blood agar plate (trypticase soy agar with 5% rabbit blood), and inoculation into a novel pre-enrichment liquid medium, Bartonella/alpha-Proteobacteria growth medium (BAPGM). Sequential research efforts resulted in the development of a combinational approach consisting of pre-enrichment culture of Bartonella species in BAPGM, sub-inoculation of the liquid culture onto agar plates, followed by DNA amplification using PCR. The multi-faceted approach resulted in substantial improvement in the microbiological detection and isolation of Bartonella when compared to direct inoculation of a blood agar plate. Importantly, this approach facilitated the detection and subsequent isolation of both single and co-infections with two Bartonella species in the blood of naturally infected dogs. The use of a combinational approach of pre-enrichment culture and PCR may assist in the diagnostic confirmation of bartonellosis in dogs and other animals.     

Evaluation of three polymerase chain reaction techniques for detection of Brucella DNA in peripheral human blood

Brucellosis is a widespread zoonosis. Currently the diagnosis of this zoonosis is based on microbiological and serological laboratory tests. Polymerase chain reaction (PCR) has been used to detect DNA from Brucella. Different target genes, primer pairs, PCR techniques, and extraction procedures have previously been published for Brucella detection. But only a few of these primers have been used in human samples, and only one study has been carried out to compare sensitivity between them. In the present study, 3 sets of primers and 3 different PCR protocols amplifying 3 different regions of the Brucella genome were compared for detection of Brucella DNA in a peripheral-blood PCR assay to conclude which is most suitable for the clinical diagnostic laboratory. These 3 pairs of primers amplify 3 different fragments included in (i) a gene encoding a 31 kDa Brucella abortus antigen (B4/B5), (ii) a sequence 16S rRNA of B. abortus (F4/R2), and (iii) a gene encoding an outer membrane protein (omp-2) (JPF/JPR). Some modifications on the reported techniques were applied during the present work to improve the outcome. The results showed that the B4/B5 primer pair had the highest sensitivity for detection of positive samples (98%), the JPF/JPR primer pair detected 88.4% of positive samples, whereas F4/R2 primer pair was the least sensitive, being able to detect only 53.1% of positive samples. The specificity of the 3 techniques was 100%. The B4/B5 primer pair was also able to detect the smallest number of bacteria (700 cfu/mL), whereas JPF/JPR was able to detect 7 x 105 cfu/mL and F4/R2 was able to detect 7 x 107 cfu/mL. It is thus concluded that using the B4/B5 primer PCR with the suggested modifications is a robust assay, which meets the sensitivity requirements to be used for testing of human blood samples for brucellosis in the diagnostic laboratory.     

Comparison of three different PCR methods for detection of Brucella spp in human blood samples

In the diagnosis of human brucellosis, PCR could be a more sensitive technique than blood cultures and more specific than conventional serological tests. We compared three different PCR methods for the detection of Brucella spp. and we studied whether human genomic DNA affect the sensitivity of three primer pairs for the detection of Brucella DNA in a peripheral-blood PCR assay. These three pairs of primers amplified three different fragments included in: (i). a gene encoding a 31-kDa Brucella abortus antigen (primers B4/B5), (ii). a sequence 16S rRNA of B. abortus (primers F4/R2), and (iii). a gene encoding an outer membrane protein (omp-2) (primers JPF/JPR). The three primers assayed showed a difference in sensitivity for detecting purified Brucella DNA, ranging between 8 fg and 20 pg. However, the sensitivity of the primers F4/R2 and B4/B5 was affected by the presence of human DNA while the primers JPF/JPR were not. Therefore, although the sensitivity of PCR using primers F4/R2 is affected by human DNA, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis.     

Serologic and molecular evidence of Ehrlichia spp. in coyotes in California

In order to determine the role of coyotes in the epidemiology of granulocytic and monocytic ehrlichial agents in California (USA), we tested 149 serum samples for antibodies against Ehrlichia equi, E. risticii, and E. canis, using an indirect immunofluorescent antibody test. Polymerase chain reaction (PCR) assay was used to survey for the presence of members of the E. phagocytophila genogroup, E. risticii and E. canis in blood samples of 95 coyotes. Sixty-eight (46%) samples were seropositive for E. equi, two (1%) for E. risticii and none of the samples had antibodies reactive to E. canis. Two and one coyote were positive for E. risticii and members of the E. phagocytophila genogroup by PCR assay, respectively. In contrast, the 95 samples were negative for E. canis by PCR. Ninety-five percent of the 68 E. equi seropositive coyotes and the one coyote PCR positive for members of the E. phagocytophila genogroup originated from a coastal area. However, the two E. risticii seropositive coyotes and the two coyotes PCR positive for E. risticii were from northern California. Sequence analysis of the three amplified PCR products revealed the agent to be similar in two coyotes to the sequences of E. risticii from horses originating from northern California and identical in one coyote to the agent of human granulocytic ehrlichiosis and E. equi from California. Thus, coyotes are exposed to granulocytic ehrlichiae and E. risticii and may play a role in the epidemiology of these ehrlichial agents in California.