Brucella Multiplex Pcr

[Beronike Watkin]

Brucellacanis is a zoonotic bacterial pathogen of dogs that is notoriously difficult to diagnose and treat. Humans can become infected with B canis when an infected pet dog is brought into their home. Our objectives were to describe the clinical presentation and outcomes in dogs treated for B canis and evaluate the performance of the quantitative serologic canine Brucella multiplex (CBM) assay for monitoring treatment response.

While treatment protocols varied in the 30 treated dogs meeting the inclusion criteria, poly-antimicrobial therapy was prescribed in 97% (29/30) of cases. Gait abnormalities, spinal pain, and discospondylitis were the most common clinical abnormalities. A difference (P value = .0075) in the percent decrease in CBM assay PO1 antibody values was found in dogs with resolved clinical signs.

Young dogs presenting with recurring lameness or back pain should be screened for B canis infection. A 40% decline in CBM assay values 2 to 6 months posttreatment can be supportive of response to treatment. Further prospective studies are needed to determine the ideal B canis treatment regimen and the magnitude of public health risks associated with maintaining neutered B canis-infected animals as pets.

Brucella canis is a zoonotic bacterial pathogen of dogs that is notoriously difficult to diagnose and treat.1 Its intracellular nature and many mechanisms for evasion and multiplication within the host are implicated when dogs fail to respond to antimicrobial therapy or when relapse occurs following cessation of treatment.2 Due to their perceived risk to public health, dogs diagnosed with B canis are often euthanized. When treatment is attempted, there is no universally accepted antimicrobial protocol for veterinarians to follow. While combination antimicrobial therapy has been shown to be more effective than monotherapy protocols, the ideal combination of antimicrobials and duration of treatment required to mitigate clinical signs and reduce the risk of recrudescence has not been adequately defined, as addressed in the companion Currents in One Health by Pinn-Woodcock et al, JAVMA, April 2023.1

Evaluating the effectiveness of antibiotic treatment for a variety of different bacterial infections can be challenging. Quantitative or semiquantitative antibody values can be used to support effective treatment for other bacterial pathogens, where successfully treated individuals demonstrate a > 40% decline in antibody values by 6 months posttreatment.11,12 Utilizing a quantitative serologic assay to measure B canis antibody titers has been suggested as the best means of monitoring for successful treatment of B canis1; however, evaluation of the CBM assay for this purpose has not yet been performed.

Our objectives were to describe the diagnostic workup, treatment, and clinical outcome in dogs treated for B canis infection and to evaluate the performance of the CBM assay for monitoring response to treatment in these cases. We hypothesized that the resolution of clinical signs would correlate with a significant decline in CBM antibody values.

Diagnostic records from the laboratory management software at the Cornell University Animal Health Diagnostic Center (AHDC) in Ithaca, NY, were retrospectively reviewed for dogs that underwent repeat B canis serologic testing over a 5-year period, from January 1, 2017, through August 1, 2022. Dogs were considered for study inclusion only if they had at least 3 B canis serologic submissions, tested positive at least once on both the 2ME-RSAT/AGID II, and tested positive on either test on a follow-up submission. From this initial group of dogs, medical records were requested from the veterinary practice associated with the most recently requested B canis serologic testing. Furthermore, only dogs with medical records that included B canis treatment information were considered for further analysis.

The signalment, clinical signs, treatment, and outcome characteristics were analyzed using descriptive statistics. Statistical analysis was performed in GraphPad Prism version 6.07. Changes in CBM antibody values, measured as percent decrease from the initial diagnosis to a time point 2 to 6 months postinitiation of treatment, were compared between groups using an unpaired t test with 2-tailed P value; P The mean time lapsed from the onset of relevant clinical signs to diagnosis was 191 days (median = 92.5 days, minimum = 0 days, and maximum = 982 days). A radiographic diagnosis of discospondylitis (n = 27) was the most common impetus for B canis testing. Other catalysts for B canis testing included abortion (n = 1) or B canis diagnosis in a housemate (1) or littermate (1). Testing to diagnose B canis was not immediately performed in many cases, including for 2 dogs with a history of discospondylitis; dog 4 was diagnosed with discospondylitis and was not tested for B canis until 6 months later, and dog 13 was diagnosed with severe spondylosis at the lumbosacral junction but was not tested for B canis until 2.25 years later. The initial radiographs of dog 1 and dog 6 failed to reveal discospondylitis, delaying diagnosis for 2 months and 9 months, respectively. The delay in diagnosis for dog 7 was related to a delay in seroconversion, where an in-clinic RSAT was positive initially, but the 2ME-RSAT in-clinic test did not produce a positive result for nearly 7 months. Dog 30 was not conclusively diagnosed for several months because of repeated inconclusive 2ME-RSAT/AGID II results. Dog 12 was tested following B canis diagnosis of another dog in the home, after having presented with persistent hind-end lameness in the preceding months. Dog 2 was tested because of an alert from the state veterinarian that a littermate tested positive, after having presented with lameness or difficulty walking at 4 clinic visits over the preceding year.

All dogs included in this study received antibiotic treatment following a serologic diagnosis of B canis infection. Combination antimicrobial therapy was prescribed at the time of B canis diagnosis in 93% (28/30) of dogs in this study, with delayed initiation in 1 additional dog. Doxycycline was a component of the antimicrobial therapy for 90% (27/30) of dogs. The antibiotic combinations administered are listed (Table 2). The aminoglycosides used were gentamicin (n = 5), amikacin (3), or streptomycin (1) and were administered SQ for 7 days on weeks 1 and 4 (8) or every 3 to 4 weeks (1); for 4 dogs, aminoglycoside therapy was repeated after the first month. Twelve dogs were administered long-term (> 4 mo duration) antibiotic treatment, as indicated, despite the resolution of clinical signs in 6 of these dogs. Antibiotic treatment was terminated in 2 dogs because of negative serology, 1 of which subsequently tested positive at a 3-month follow-up, and the other was lost to follow-up.

The 2ME-RSAT/AGID II assay had been used to monitor the serologic response in all dogs included in this study. We aimed to investigate whether the CBM assay could be used to monitor the response to treatment more effectively, given the quantitative nature of the assay. Stored serum samples obtained from the time of B canis diagnosis were available for the 26 dogs in this study diagnosed after January 1, 2017, all of which were nonnegative on the CBM assay. Serum from all 26 dogs contained antibodies directed against the PO1 antigen, while only 50% (15/30) were found to have antibodies directed against the BP26 antigen.

Effective antimicrobial treatment should be associated with a substantial decline in antibody values. We found that the quantity of antibodies directed against the PO1 antigen decreased by > 40% in 12 of these dogs by 3 to 6 months posttreatment and in an additional 3 dogs by 8 to 12 months posttreatment. Of note, 1 of the dogs, dog 26, had an initial decrease of > 40% in PO1 antibody values at 3 months, but antibody values rebounded 2 months later, suggestive of recrudescence, but the dog was subsequently lost to follow-up.

It has previously been proposed that 2 sequential negative results on the AGID II assay are supportive of elimination of infection.14 Only 1 dog, dog 3, seroconverted to negative on 2 sequential 2ME-RSAT/AGID II assays, while 4 dogs returned to negative on the CBM assay (Figure 1). Dog 1 and dog 29 achieved a negative CBM result approximately 3 months postinitiation of treatment, while dog 28 and dog 3 did not achieve a negative result until 1 and 3 years posttreatment, respectively. Clinical signs had resolved in all 4 of these dogs at the time of the last serologic sample. Dog 28 was reported to have had recrudescence of clinical signs 1.75 years later; however, serologic samples from that period were not available. Three of these dogs were maintained on antibiotics indefinitely (dog 1, dog 28, and dog 29), while dog 3 received 2 finite courses of enrofloxacin, with the first 104-day course concluding after a negative AGID II results and the second 180-day course beginning after a positive AGID II result was received 4 months later.

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