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1.
Brucella canis is the causative agent of canine brucellosis and facultative intracellular pathogen. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods including agglutination and gel diffusion tests. In this study, crude antigens were extracted from B. canis using hot saline, coated on to latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. Mixing the antigen coated latex beads with the sera of dogs infected with B. canis produced clear agglutination, but this was not so for B. canis free dog sera. N-terminal amino acid sequence analysis of the crude hot saline extracts, showed that they contained copper-zinc superoxide dismutase, ribose ABC transporter and hypothetical protein of Brucella as antigens. A serological survey of canine serum samples conducted by means of an agglutination test using the antigen coated latex beads, showed that this method was more specific than the tube agglutination test using whole bacterial cell antigens. Although these results suggest that our method in which crude hot saline extracted antigens are coated on to latex beads would be useful in the serological diagnosis of canine brucellosis, we need further investigation using more serum samples to confirm the usefulness of our method.  相似文献   

2.
The performance of the rapid slide agglutination test, with and without 2-mercaptoethanol (RSAT and 2ME-RSAT) and agar gel immunodiffusion test (AGID) was evaluated for the diagnosis of brucellosis in naturally infected dogs. The microbiological culture, PCR and clinical parameters were used as reference. A total of 167 dogs were clinically examined and tested by blood culture, culture of semen/vaginal swab and PCR in blood and semen/vaginal swab. According to the results observed the 167 dogs were divided into three groups: Brucella canis infected dogs (Group 1), B. canis non-infected dogs (Group 2) and dogs with suspected brucellosis (Group 3). The dogs were then tested by RSAT, 2ME-RSAT and AGID. Groups 1 and 2 were used to calculate the diagnostic sensitivity and specificity of the serological tests and the results observed in Group 3 were also discussed. The diagnostic sensitivity of RSAT, 2ME-RSAT and AGID was respectively 70.58%, 31.76%, and 52.94%. The diagnostic specificity of RSAT, 2ME-RSAT and AGID was respectively 83.34%, 100%, and 100%. In dogs with suspected brucellosis 15% were RSAT positive, none was 2ME-RSAT positive and 5% were AGID positive. Although the serological tests are the most commonly used methods for brucellosis diagnosis, a significant proportion of false-negative results were observed highlighting the importance of the direct methods of diagnosis, like blood culture and PCR to improve the diagnosis of canine brucellosis.  相似文献   

3.
Brucella ovis causes a genital disease of sheep manifested by epididymitis in rams and placentitis in ewes producing reduced fertility in the flock. Clinical diagnosis is not sensitive enough and bacteriological testing is not feasible for detection of the disease in large numbers of animals. Indirect methods of serological testing are preferred for routine diagnosis, of which agar gel immunodiffusion (AGID), complement fixation (CF) and ELISA tests are recommended as the most efficient. Since B. ovis shares antigenic components with Brucella canis, it would seem that either strain could be used as antigen with the same results; however, the advantage of the B. canis (M-) strain variant is that it can be used to develop a satisfactory antigen for agglutination tests. We present data on AGID and IELISA tests using B. ovis antigen and rapid screening agglutination test (RSAT), 2-mercapto-ethanol RSAT (2ME-RSAT) and IELISA using B. canis antigen. We tested 225 animals. The cut-off values were adjusted by ROC analysis using 51 negative and 32 positive sera; the IELISA-B. canis cut-off value was 39 (%P) and IELISA-B. ovis, 51 (%P), with 100% sensitivity and specificity. Of the 32 positive sera from the infected flock RSAT detected 32 (100%), 2ME-RSAT 29 (91%) and AGID 31 (97%). Of the 142 sera from suspicious flocks, 46 were negative and 56 positive in all the tests; 16 were positive by RSAT, IELISA-B. canis and IELISA-B. ovis, 20 positive only with RSAT and 2 positive only by both IELISAs. RSAT is a very sensitive screening test that, because of its simplicity and easy interpretation, following a study in larger sample, could replace AGID as a screening test for diagnosis of ovine brucellosis caused by B. ovis. The IELISA-B. canis or IELISA-B. ovis could be used as confirmatory tests, since they show equal specificity and sensitivity.  相似文献   

4.
An improved antigen is described for use in the serodiagnosis of canine brucellosis. The novel antigen employs a less mucoid (M-) variant of B. canis. The replacement of Brucella ovis with B. canis (M-) cells as antigen in slide agglutination tests would increase accuracy by reducing the rate of false positive results from ca. 50% to ca. 10%. Rose bengal stained B. canis (M-) cells are slightly more sensitive than the commercially available Brucella ovis as slide test antigens. Both test procedures require brief (less than 1 min) reaction with 2-mercaptoethanol in order to reduce the rate of false positive reactions.  相似文献   

5.
The diagnostic techniques most widely used for detecting brucellosis caused by Brucella ovis are serological tests such as complement fixation (CFT), agar gel immunodiffusion (AGID), and ELISAs. However, to our knowledge, milk tests, with the advantage that samples may be taken in a non invasive manner, have not been investigated as diagnostic tools. We studied 144 samples of milk and sera from lactating ewes, comparing bacteriological studies, serological and milk tests using Brucella canis and B. ovis antigens. A group of 75 ewes in an uninfected flock were serologically negative to rapid slide agglutination test (RSAT), indirect ELISA (IELISA)-B. canis, AGID and IELISA-B. ovis. The milk of these ewes had an IELISA-B. canis mean (%P) value of 16.18 (S.D. 5.63), while the IELISA-B. ovis had a mean (%P) value of 12.52 (S.D. 4.94). A cut-off value of (%P) 27.44 (+2 S.D.) or (%P) 33 (+3 S.D.) was determined by milk-ELISA-B. canis and (%P) 22.4 (+2 S.D.) and (%P) 27.34 (+3 S.D.) by milk-IELISA-B. ovis. These cut-off values were adjusted by receiver-operator characteristics (ROC) analysis using 23 positive samples from infected ewes, which indicated a milk-IELISA-B. canis cut-off value of (%P) 33 and milk-IELISA-B. ovis of (%P) 26 with 100% sensitivity and specificity. Based on our results, we propose that, following a study of a larger number of samples, the milk-IELISA-B. canis could be considered a suitable test for the diagnosis of B. ovis brucellosis in lactating ewes.  相似文献   

6.
The aim of this work was to compare the performance of 6 serological tests using outer or internal antigens from Brucella for the diagnosis of Brucella ovis infection in sheep in an endemic area. Outer membrane antigens included a hot saline extract (HS) and the rough lipopolysaccharide (R-LPS) from B. ovis. Internal antigens were LPS-free total cytosolic proteins (CP) and an 18-kDa cytosolic protein (p18) from Brucella spp. Sera from 200 sheep from naturally infected flocks were assayed by agar gel immunodiffusion test (AGID) and by complement fixation test (CFT), both using HS, and by 4 ELISA using HS, R-LPS, CP, and p18, respectively. The percentage of positive results was 45.5% for ELISA with HS, 42.0% for ELISA with p18, 39.5% for CFT, 33.5% for ELISA with R-LPS, 29.0% for ELISA with CP, and 18.0% for AGID. Taking CFT as the reference test for calculating relative test parameters, the ELISA with HS had the best sensitivity (96.2%), while AGID and the ELISA with R-LPS had the best specificity (96.6%). The ELISA with CP was not more sensitive than the ELISA with p18 (67.1% vs. 79.7%) in spite of the higher number of antigens in CP. The lower relative sensitivity of tests using internal antigens might reflect a lack of antibodies to cytosolic proteins in some infected animals or a shorter persistence of these antibodies relative to antibodies to outer membrane components after recovery from infection.  相似文献   

7.
Brucella spp. are Gram-negative, facultative, intracellular coccobacilli that are pathogenic to a variety of mammals, including ruminants and humans. The conventional serological test for diagnosing brucellosis in cattle in Korea is the standard tube agglutination test. However, agglutination tests sometimes give false-positive results due to cross-reactions with other pathogens. The outer membrane proteins of Brucella species have been extensively studied for their immunogenicity and serodiagnostic applications. However, an application of B. abortus OMPs for serodiagnosis has not been successfully established. In this study, cloning and expression of B. abortus Omp28, a group 3 antigen, were accomplished by PCR amplification cloning into a pMAL expression system, and purification of a recombinant Omp28 (rOmp28). The immunogenicity of rOmp28 was confirmed by Western blot analysis with Brucella-positive bovine serum. To determine whether rOmp2 has a potential benefit for use in the serodiagnosis of bovine brucellosis, rOmp28-based ELISA and latex bead agglutination test were performed. B. abortus positive (n=122) or negative (n=88) from TAT were positive (118/122, 96.7%) or negative (84/88, 95.4%) in ELISA and were positive (94/122, 77%) or negative (71/88, 81.7%) in that the latex bead agglutination test, respectively. The sensitivity, specificity and accuracy were 96.7, 95.4, 96.2% in ELISA and 77, 80.6, 78.5% in latex bead agglutination test, respectively. These findings suggest that the rOmp28 of B. abortus might be a good candidate for developing serological diagnostic tools for bovine brucellosis.  相似文献   

8.
A focus of B. canis infection was identified in Moreno, Argentina, among the stray dog population by serologic methods and confirmed in a second survey which included cultural isolations. A counterimmunoelectrophoresis technique using a specific rough Brucella surface antigen was applied to the serodiagnosis of canine brucellosis. This method was found to be as effective as the mercaptoethanol tube agglutination test and the gel diffusion test in detecting B. canis antibodies in natural and experimentally infected animals. The results are discussed in terms of the diagnostic significance of the three tests employed.  相似文献   

9.
Objective  To describe historical, clinical and diagnostic features of dogs with Brucella canis endophthalmitis and the response to medical therapy.
Animals studied  Three dogs with naturally acquired B. canis endophthalmitis.
Procedure  Dogs were treated symptomatically with topical ophthalmic anti-inflammatories and a novel antimicrobial protocol that included doxycycline, enrofloxacin, rifampin and streptomycin.
Results  All dogs presented with chronic or recurrent uveitis in the absence of overt systemic disease. Clinical ophthalmologic abnormalities were unilateral in each dog and included mild-to-moderate anterior uveitis, iris hyperpigmentation, marked vitreal infiltrates, and multifocal chorioretinitis. Dogs were diagnosed with canine brucellosis serologically and by blood culture ( n  = 2 dogs) or polymerase chain reaction of aqueous humor and blood ( n  = 1 dog). Active ocular inflammation resolved in all dogs during treatment, with preservation of vision in 2 dogs. Following treatment, B. canis could not be cultured from blood samples and serological values declined with seronegativity achieved in all dogs after a median of 96 weeks (range: 36–112 weeks) of therapy.
Conclusions  Brucella canis infection should be included in the differential diagnosis for dogs with intraocular inflammation, regardless of previous history or neuter status. This is the first report of apparently successful medical therapy of canine brucellosis with ocular involvement.  相似文献   

10.
An Irish Setter with scrotal enlargement had an agglutination titer to Brucella canis of greater than or equal to 1:200, but B canis was not cultured from the blood. Unusual findings included draining ulcers in the scrotum from which B canis was cultured, a necrotic testicle, and inflammation of the entire scotum. Other clinical and postmortem findings were as previously described in other cases of naturally occurring and experimentally induced canine brucellosis.  相似文献   

11.
In the present study, the validation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of canine brucellosis is described. Two different antigenic extracts, obtained by heat or ultrasonic homogenization of microbial antigens from a wild isolate of Brucella canis bacteria, were compared by ELISA and Western blot (WB). A total of 145 canine sera were used to define sensitivity, specificity and accuracy of the ELISA as follows: (1) sera from 34 animals with natural B. canis infection, confirmed by blood culture and PCR, as well as 51 sera samples from healthy dogs with negative results by the agar–gel immunodiffusion (AGID) test for canine brucellosis, were used as the control panel for B. canis infection; and (2) to scrutinize the possibility of cross reactions with other common dog infections in the same geographical area in Brazil, 60 sera samples from dogs harboring known infections by Leptospira sp., Ehrlichia canis, canine distemper virus (CDV), Neospora caninum, Babesia canis and Leishmania chagasi (10 in each group) were included in the study. The ELISA using heat soluble bacterial extract (HE-antigen) as antigen showed the best values of sensitivity (91.18%), specificity (100%) and accuracy (96.47%). In the WB analyses, the HE-antigen showed no cross-reactivity with sera from dogs with different infections, while the B. canis sonicate had various protein bands identified by those sera. The performance of the ELISA standardized with the heat soluble B. canis antigen indicates that this assay can be used as a reliable and practical method to confirm infection by this microorganism, as well as a tool for seroepidemiological studies.  相似文献   

12.
Twenty mammary lymph node samples were collected from cattle on a farm in the Republic of Korea. These cattle were serologically negative for Brucella by tube agglutination test (≤ 1:50) and serum agglutination test (≤ 1:50). Out of 20 lymph node samples, two samples were positive for Brucella growth on Brucella agar as well as blood agar. Tests for urease, hydrogen sulphide and reactions against monospecific sera A and M indicated that these two isolates (No. 15 and 16) belong to the genus Brucella. Genus specific, AMOS (abortus, melitensis, ovis, suis) and Bruce-ladder multiplex polymerase chain reaction (PCR) assays confirmed the Brucella isolates as either a B. abortus or a B. canis strain. This is the first report of the occurrence of a B. canis infection in cattle in Korea. More survey data are needed to determine whether B. canis is a significant aetiology in the cases of cattle brucellosis in Korea.  相似文献   

13.
1000 random serum samples of pet dogs were examined in the serum tube agglutination test for antibodies to Brucella canis. Agglutinating antibodies to Br. canis antigen were detected in 18 cases in a serum dilution of 1 : 50, in 29 cases in a serum dilution of 1 : 100, in 13 cases in a serum dilution of 1 : 200 and in a serum dilution 1 : 400. But the positive results of the agglutination tests were confirmed by complement fixation, agargel-precipitation and indirect immunofluorescence only in 2 cases (0,2%) with titers of 1 :200 and 1 :400. These serological findings indicate that in the Federal Republic of Germany Br. canis infections are rarely in pet dogs as compared with dogs (Beagles) held in research kennels.  相似文献   

14.
In a serologic survey of stray and pet dog populations of Georgia, serums were screened for Brucella canis antibodies, using the slide agglutination test. If results were positive, B canis antibody titers were determined, using the standard tube agglutination test. The stray dogs had significantly (P less than 0.01) higher titers than did the pet dogs. The reactor rate was 58% higher for the slide agglutination test than for the tube agglutination test. The manufacturer's evaluation of the slide agglutination test was based on a comparison of the serologic results of that test with those of the tube agglutination test, using a comparative method that permitted the results to be interpreted as 99% agreement between the 2 tests. Reevaluation of the manufacturer's data by a different method indicated that the slide agglutination test is very accurate when the results are negative (99.7% specific) but less so when the results are positive (62.5% sensitive).  相似文献   

15.
A preliminary serological study of 366 household dogs in Lagos and Ibadan, southwestern Nigeria, was carried out to determine antibodies due to exposure to Brucella abortus and B. canis, using the rose bengal test (RBT) and the rapid slide agglutination (RSA) test, respectively. Results showed that 5.46 % (20/366) and 0.27 % (1/366) of the dogs screened were seropositive to B. abortus and B. canis, respectively. Of all dogs, 36 had a history of being fed foetuses from cows and 11 (30.6 %) of these tested positive in the RBT. Our findings, although based on a limited sample size and a dearth of clinical details, revealed that dogs in Nigeria may be infected with Brucella spp. given the wide range of risk factors. Further studies are recommended to elucidate the epidemiology of brucellosis in dogs and its possible zoonotic consequences in the country.  相似文献   

16.
Male Beagles infected with Brucella canis for greater than or equal to 3 months developed serum antibodies that agglutinated normal canine spermatozoa. Titers were highest in dogs that had been infected for 4 to 6 months. Lower spermagglutinin titers were detected in sera collected 10 months after inoculation. Antibodies were also observed in seminal plasma of chronically infected dogs. Seminal plasma from infected, but not from clinically normal dogs, caused head-to-head agglutination of normal sperm. In contrast to macroagglutination of sperm by serum antibodies, agglutination by seminal plasma antibodies was detected only by microscopic examination. Seminal plasma agglutinins were not inactivated by heat (56 C, 1 hour) or by reduction with 2-mercaptoethanol. When seminal plasma and sperm were mixed with 2 hemolytic units of guinea pig complement, spermatozoa were not inactivated. Spermagglutinin activity was present in the first 2 spectral absorption peaks of Sephadex G-200 fractionated seminal plasma. Fractions that had the highest spermagglutinin titers contained mostly immunoglobulin A. Seminal plasma from infected dogs also contained cytophilic factors for normal splenic macrophages that caused sperm adherence to macrophages. Dogs with a bacteremia lasting greater than 4 months had cutaneous delayed-type hypersensitivity reactions when tested with soluble canine testicular extracts. Reactions did not occur in normal dogs. Dogs with testicular atrophy had the most severe skin test responses. Seemingly, isoimmune responses to sperm antigens are involved in infertility caused by B canis infection of male dogs.  相似文献   

17.
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.  相似文献   

18.
Brucella, a causative agent of brucellosis and facultative intracellular pathogen, has been isolated recently from a variety of wild mammals. In this study, serum samples from 115 Japanese wild boar (Sus Scrofa leucomystax) killed by hunters in the 4 Prefectures of Shikoku, Japan were tested for antibodies to Brucella spp. by means of the tube agglutination test (TAT) and enzyme-linked immunosorbent assay (ELISA) using antigens extracted with n-lauroylsarcosine. In 9 of the 115 samples (7.8%) antibodies to Brucella spp. were detected by TAT and ELISA. These results suggest that wild boar in Shikoku may be exposed to Brucella spp. or other cross-reactive pathogen infection.  相似文献   

19.
A seroepidemiological survey was conducted to investigate the prevalence of antibodies reactive with Ehrlichia canis and Hepatozoon canis antigens in free-ranging red foxes (Vulpes vulpes) in Israel. Of 84 fox sera assayed, 36% were seropositive for E. canis by the indirect fluorescent antibody (IFA) test and 24% were positive for H. canis using an enzyme-linked immunosrbent assay (ELISA). Canine ehrlichiosis and hepatozoonosis appear to be endemic in the wild red fox populations in Israel, and foxes may serve as a reservoir for infection of domestic dogs and other wild canine species.  相似文献   

20.
用培养的布氏杆菌菌体，通过超声波裂解、反复离心制备出布氏杆菌细胞壁抗原。将细胞壁抗原作1：128稀释，用作牛种布氏杆菌酶联免疫吸附试验（ELISA)的抗原；将布氏杆菌细胞壁抗原作1：32稀释，用作平板凝集试验抗原；把细胞壁抗原作1：16稀释用作试管凝集试验抗原，分别建立了牛布氏杆菌ELISA试验、平板凝集试验和试管凝集试验。用这3种方法，检测已知200份平板凝集试验阴性血清，5份平板凝集试验阳性血清。结果5份阳性血清在平板凝集试验、试管凝集试验和ELISA试验中均为阳性；200份阴性血清在平板凝集试验和试管凝集试验中均为阴性，在ELISA试验中有1份为阳性?试验证明，细胞壁抗原，既能用于传统的平板凝集试验和试管凝集试验，又能用于ELISA试验。  相似文献   

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