Rapid Canine Brucella Ab Test kit

Canine brucellosis is a zoonosis whose etiologic agent is Brucella canis, although other Brucella species can infect dogs, including B. melitensis, B. abortus, and B. suisThe disease is transmitted mainly through the oronasal route or contact with a fetus, fetal fluids, placenta, and vaginal discharge after abortions. Venereal and intrauterine routes are also important for the transmission of B. canis. Canine brucellosis Genprice Gentaur is associated with metritis, placentitis, and abortion in females, and epididymitis, orchitis, and prostatitis in males. In addition, B. canis infection is associated with birth of weak pups and high neonatal mortality rates.

Therefore, the disease is responsible for economic losses in commercial kennels.

  • Despite being a zoonosis, few cases of human infection have been reported, which supports the notion that B. canis has low zoonotic potential.
  • However, the real importance of B. canis infection in human patients may be underestimated because, in most cases, this pathogen causes subclinical infection, which makes the diagnosis challenging. In addition, most serologic tests used in the diagnosis of human brucellosis employ smooth Brucella antigens, which are ineffective for detecting B. canis.2
  • Similar to other Brucella species, B. canis causes an occupational disease that affects mostly veterinarians or kennel workers who have close contact with dogs and their secretions, particularly fetal tissues or fluids, placenta, or vaginal discharge, and also laboratory personnel who may be exposed to large amounts of bacteria.
  • Infections of dog owners have been reported, highlighting the relevance of canine brucellosis as an emerging urban zoonosis. Given the demand for companion dogs, and the deficiency in detection of canine and human B. canis infection, this pathogen may become a serious public health issue.

The diagnosis of canine brucellosis is problematic, given that the available serologic tests have low sensitivity and specificity.

Definitive diagnosis is supported by bacterial isolation followed by PCR, but culture is not widely available because it is time-consuming and requires a laboratory with proper biosafety conditions given that this pathogen poses a potential health risk to laboratory workers as a result of aerosol transmission. Therefore, serologic methods are commonly used in the diagnosis of canine brucellosis.

The most common serologic tests include: agar gel immunodiffusion (AGID), rapid slide agglutination test (RSAT), tube agglutination test (TAT), microagglutination test (MAT), complement fixation test (CFT), and several ELISA protocols with different antigens. Despite being simple to perform, serologic tests have limitations such as nonspecific reactions, especially in agglutination tests, which are more likely with hemolyzed sera, samples with high lipid content, or because of cross-reaction with other pathogens, including Pseudomonas spp., Bordetella bronchiseptica, Streptococcus spp., Staphylococcus spp., Salmonella spp., Yersinia enterocolitica, and Escherichia coli. Cross-reaction and false-positive results are eliminated when B. canis cytoplasmic antigens are employed in AGID. AGID sensitivity is 27.9–52.9% and is influenced by the type of antigen employed. MAT has sensitivity of 66.7–88.9% and specificity of 100%. False-positive reactions as a result of hemolysis do not occur with MAT, as are often observed with AGID.

Serum treated with 2-mercaptoethanol (2-ME) prior to AGID or immunochromatographic tests increases specificity, but significantly lowers the sensitivity of these tests. Indirect immunofluorescence has also been employed for the detection of B. canis infection.

Previous studies, mostly based on serology, have demonstrated the presence of B. canis in various Brazilian regions, affecting dogs in commercial kennels, households, or street dogs in urban or rural areas. However, marked variations in B. canis infection frequency or prevalence among different surveys have been observed, possibly as a result of the use of different tests. Our goal was to compare different serologic methods and PCR used in the detection of B. canis infection in dogs.

Kennels and sampling

We evaluated samples from 254 dogs (161 females and 93 males) belonging to 5 breeding kennels located in Belo Horizonte, State of Minas Gerais (MG), Brazil (n = 129 from 3 kennels) or Vila Velha, State of Espírito Santo (ES), Brazil (n = 125 from 2 kennels). Our study was advertised at the breeders’ association, and enrollment was voluntary.

History of abortion and previous diagnosis of brucellosis were investigated. Samples were collected between February and July 2016. Serum and whole blood samples were collected from all dogs for serologic tests (AGID, rose Bengal plate test [RBPT], CFT, MAT, and dot-ELISA) and PCR. Vaginal swabs from 52 bitches and placentas from 2 puppies (all that were made available from February to July 2016) were processed for bacterial isolation to confirm B. canis circulation in the kennel. Experimental procedures were approved by the Ethics Committee of the Federal University of Minas Gerais (CEUA/UFMG, protocol 56/2016).

Isolation and identification of Brucella spp

Samples of placentas from 2 puppies from kennel A, and vaginal swabs from 52 bitches from kennels B (n = 9), D (n = 35), and E (n = 8) were used for bacterial isolation. Tissue samples were stored at −80°C, and swabs with Stuart medium were kept frozen at −20°C until further processed. For bacterial isolation, placentas were thawed and homogenized with a scalpel blade in 500 µL of sterile PBS; samples contained in swabs were resuspended in 300 μL of sterile PBS and homogenized.

Placenta and vaginal swab homogenates were inoculated (100 μL) on tryptose agar (BD Difco, Franklin Lakes, NJ) without or with selective supplementation (2,500 IU of polymyxin B; 12,500 IU bacitracin; 50,000 IU of nystatin; 50 mg of cycloheximide; 2.5 mg of nalidixic acid; and 10 mg of vancomycin; MilliporeSigma, St. Louis, MO), and in 10 mL of tryptose broth (BD Difco) containing selective supplement. Plates were incubated at 37°C with 5% CO2, and bacterial colony growth was checked every 48 h. Broth tubes were incubated at 37°C with 5% CO2 for 7 d and then 100 μL was inoculated on antibiotic tryptose agar. Plates were kept incubated for 21 d and considered negative in the absence of bacterial growth in that period. Colonies with morphology compatible with the genus Brucella were identified based on an acriflavine test, PCR for the bcsp31 gene, and B. ovis–specific PCR.

For the detection of Brucella sp. genomic sequences in canine whole blood samples, 250 μL of blood were processed for DNA extraction. After extraction, DNA concentration was measured and adjusted to 250 ng/μL. Bacterial colonies were collected from agar plates into 200 µL of sterile PBS, and heat-killed (100°C for 1 h) for DNA extraction as described previously.

For B. ovis–specific PCR, 22 μL of SuperMix PCR (22 mM Tris-HCl pH 8.4, 55 mM KCl, 1.65 mM MgCl2, 220 μM dGTP, 220 μM dATP, 220 μM dTTP, 220 μM dCTP, and 22 U/mL of recombinant Taq DNA polymerase; Invitrogen, Carlsbad, CA), 0.5 μL of each primer (25 μM; 5’-GCCTACGCTGAAACTTGCTTTTG-3’ and 5’-ATCCCCCCATCACCATAACCGAAG-3’), and 2.0 μL of DNA sample were used. Amplification was performed at 95°C for 5 min, 95°C for 1 min, 57°C for 1 min and 72°C for 1 min for 35 consecutive cycles, followed by final extension at 72°C for 5 min, with an expected product of 228 bp, which was analyzed in 1.5% agarose gel stained with SYBR Safe DNA gel stain (Invitrogen). PCR targeted the bcsp31 gene with primers 5′-

Canine brucellosis is an infectious and contagious disease associated with reproductive losses in breeding kennels. As a zoonotic disease, it poses a risk to human health, especially for veterinarians and breeders who handle materials potentially contaminated with Brucella canis . However, canine brucellosis is a neglected and underestimated disease given the difficulties in establishing a definitive diagnosis.

We evaluated the frequency of detection of B. canis in 5 breeding kennels by using various serologic methods and PCR. Circulation of B. canis in these kennels was confirmed by bacterial isolation. The frequency of positive serologic results varied from 6.3% by AGID to 16.5% by dot-ELISA. There was no positive serology for smooth Brucella. PCR testing was positive in 13.9% of samples.

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The only detection tests with reasonable agreement were PCR and 2ME-MAT. The diagnosis of canine brucellosis  remains challenging. The use of a single laboratory method, or even the use of different laboratory methods, may not be sufficient to reach a definitive diagnosis.

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