Performance characteristics of three Brucella canis serological assays in the United States

Tessa E. LeCuyer ^1* Rebecca Franklin-Guild ² Cassandra Guarino ² Alexandra Fox ³ Kelli Maddock ⁴ Rebecca Barber ⁵ David Baum ⁶ Felipe Bustamante ⁷ Joshua Daniels ⁸ David M De Avila ⁹ Dubraska Diaz-Campos ¹⁰ Lisa G Glick ⁵ Jessica Haley ³ Sheila Heinen ⁶ Laura Leger ¹¹ John Dustin Loy ¹¹ Deepti Pillai ¹² Korakrit Poonsuk ⁹ Michael M Russell ⁸ Mithila Shukla ¹² Erika R Schwarz ¹³ Matthew Stempien ¹⁰ Deepanker Tewari ⁷ Joany C Van Balen ¹⁰ Stephen Werre ¹⁴ Julie T Cecere ¹⁵

• ¹ Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, Davis, California, United States
• ² Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States
• ³ Virginia Tech Animal Laboratory Services, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, United States
• ⁴ North Dakota State University Veterinary Diagnostic Laboratory, North Dakota State University, Fargo, United States
• ⁵ Diagnostic Laboratories, College of Veterinary Medicine, University of Florida, Gainesville, Florida, United States
• ⁶ Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Iowa State University, Ames, Iowa, United States
• ⁷ Pennsylvania Veterinary Laboratory, Pennsylvania Department of Agriculture, Harrisburg, United States
• ⁸ Veterinary Diagnostic Laboratory, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States
• ⁹ Washington Animal Disease Diagnostic Laboratory, Washington State University, Pullman, United States
• ¹⁰ OSU Veterinary Medical Center – Clinical Microbiology Laboratory, The Ohio State University, Columbus, United States
• ¹¹ Nebraska Veterinary Diagnostic Center, School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States
• ¹² Animal Disease Diagnostic Laboratory, College of Veterinary Medicine, Purdue University, West Lafayette, Indiana, United States
• ¹³ Montana Veterinary Diagnostic Laboratory, Montana Department of Livestock, Bozeman, United States
• ¹⁴ Department of Population Health Sciences, Virginia Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, United States
• ¹⁵ Department of Small Animal Clinical Sciences, Virginia Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, United States

Brucella canis is a zoonotic pathogen of dogs that poses diagnostic challenges. While direct detection of B. canis by PCR or culture is ideal, serologic diagnosis is necessary for identification of carrier animals and can support a clinical diagnosis of brucellosis. Prior to 2022, B. canis seroscreening in the United States was primarily performed using a commercially available rapid slide agglutination test. However, the kit was discontinued by the manufacturer in early 2022, leaving a gap in the availability of commercial B. canis seroassays. The goal of this study was to compare the performance of three B. canis serologic tests that are currently available: VMRD Brucella ovis ELISA, Bionote Anigen Rapid C.Brucella Ab immunochromatographic lateral flow assay, and VMRD B. canis indirect fluorescent antibody (IFA) assay. A panel of 56 banked serum specimens originally submitted to the Cornell University Animal Health Diagnostic Center (the study reference laboratory) for B. canis seroscreening was distributed to 12 testing laboratories. Each sample was run on three assays developed at the reference lab: rapid slide agglutination test with 2-mercaptoethanol (2-ME RSAT), agar gel immunodiffusion test using cytoplasmic antigen (AGID II), and Canine Brucella Multiplex. Five testing labs ran the ELISA, six ran the lateral flow, and six ran the IFA. When evaluated as a screening assay, we compared the assays to the 2ME-RSAT. The ELISA had the highest sensitivity (96.8%, 95%CI 83.8-99.9) but the lowest specificity (79.3%, 95%CI 57.9-92.9). The sensitivity of the lateral flow was 90.6% (95%CI 75-98%) and the IFA was 87.5% (95%CI 71-96.5). Specificity for the lateral flow was 95.8% (95%CI 78.9-99.9) and IFA was 97.5% . When compared to AGID II and Canine Brucella Multiplex, the test assays were all highly sensitive, but specificity was <90%. Interrater reliability was highest for IFA (K=0.92) and lowest for the lateral flow (K=0.82). Serial testing of positive samples with a more specific test, such as AGID II, will continue to be necessary when using any of the three assays tested in this study.