First Isolation of Brucella canis from a breeding kennel in Italy.


Brucella canis

How to Cite

De Massis, F., Sacchini, F., Averaimo, D., Garofolo, G., Lecchini, P., Ruocco, L., Lomolino, R., Santucci, U., Sgariglia, E., Crotti, S., Petrini, A., Migliorati, G., D’Alterio, N., Gavaudan, S., & Tittarelli, M. (2021). First Isolation of Brucella canis from a breeding kennel in Italy. Veterinaria Italiana, 57(3). https://doi.org/10.12834/VetIt.2497.15848.1


Brucella canis has been isolated for the first time in Italy in a commercial breeding kennel. It was diagnosed after a deep investigation related to the onset of reproductive disorders. Animals were tested with direct and indirect techniques. The agent was first detected in two Chihuahua aborted foetuses by direct culture. Further, it was also isolated from blood samples of dogs hosted in the kennel, which also showed reaction to conventional serological tests (microplate serum agglutination test). The isolates were identified as B. canis by standard microbiological methods and a Bruce‑ladder multiplex PCR. To investigate the genomic diversity, whole genome sequencing was used, applying the core genome Multilocus Sequence Typing (cgMLST ). In a first round of serological testing performed on 598 animals, 269 (46.1%) tested positive. In the second round of laboratory testing carried out 4‑5 weeks apart, the number of serologically positive dogs was 241 out of 683 tested (35.3%), while the number of dogs positive to isolation was 68 out of 683 tested (10.0%). The PCR showed a lack of sensitivity when compared to direct isolation. The epidemiological investigation did not identify the source of the infection, given the time elapsed from the onset of abortions to the definitive diagnosis of B. canis infection in the kennel. The genomic analyses featured the strains as ST21 and, according to the cgMLST, revealed the presence of a tight cluster with a maximum diversity of four allelic differences. The observed limited genomic variation, largely within the known outbreak cut‑offs, suggests that the outbreak herein described was likely caused by a single introduction. Moreover, in a broader scale comparison using the public available genomes, we found that the closest genome, isolated in China, differed by more than 50 alleles making not possible to find out the likely origin of the outbreak. The lack of updated data on B. canis genome sequences in the public databases, together with the limited information retrieved from the epidemiological investigations on the outbreak, hampered identification of the source of B. canis infection.


Alton G.G., Jones L.M., Angus R.D. & Verger J.M. 1988. Techniques for the brucellosis laboratory. I.N.R.A., Paris, 169-174.

Auwera G.A., Carneiro M.O., Hartl C, Poplin R., Levy-Moonshine A., Jordan T., Shakir K., Roazen D., Thibault J., Banks E., Garimella K.V., Altshuler D., Gabriel S. & DePristo M.A. 2013. From FastQ data to high-confidence variant calls: the genome analysis toolkit best practices pipeline. Curr Protoc Bioinformatics, 43,11.10.1–11.10.33.

Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S., Lesin V.M., Nikolenko S.I., Pham S., Prjibelski A.D., Pyshkin A.V., Sirotkin A.V, Vyahhi N, Tesler G, Alekseyev MA & Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol ,19, 455– 477. https://doi.org/10.1089/cmb.2012.0021.

Blankenship R.M.& Sanford J.P. 1975. Brucella canis: a cause of undulant fever. Am J Med, 59, 424–426.

Brower A., Okwumabua O., Massengill C., Muenks Q., Vanderloo P., Duster M., Homb K. & Kurth K. 2007. Investigation of the spread of Brucella canis via the U. S. interstate dog trade. Int J Infect Dis, 11, 454–458. doi: 10.1016/j.ijid.2006.12.009

Cadmus S.I.B., Adesokan H.K., Ajala O.O., Odetokun W.O., Perrett L.L. & Stack J.A. 2011. Seroprevalence of Brucella abortus and B. canis in household dogs in southwestern Nigeria: a preliminary report. J S Afr Vet Assoc, 82, 56–57.

Carmichael L.E Zoha S.J. & Flores-Castro R. 1984. Problems in the serodiagnosis of canine brucellosis: dog responses to cell wall and internal antigens of Brucella canis. Dev Biol Stand, 56, 371-383.

Carmichael L.E. & Greene G.E. 2013. Canine brucellosis. In Infectious Diseases of the Dog and Cat (Greene G.E. Ed.). 4 ed. Saunders, Philadelphia, 398–410.

Carmichael L.E. & Shin S. J. 1996. Canine brucellosis: a diagnostician’s dilemma. Semin Vet Med Surg (Small Anim), 11, 161–165.

Carmichael L.E. 1966. Abortion in 200 beagles. J Am Vet Med Assoc, 149, 1126.

Carmichael L.E. 1990. Brucella canis. In Animal Brucellosis (Nielsen K. & Duncan J. R., Eds). CRC Press, Boca Raton, Florida, 335-350.

Carmichael L.E., Flores-Castro R. & Zoha S. 1980. Brucellosis caused by Brucella canis: an Update of Infection in Animals and in Humans. Geneva, World Health Organization. Document WHO/BRUC./80.361 WHO/ZOON./80.135.

Carriço J.A., Silva-Costa C., Melo-Cristino J., Pinto F.R., de Lencastre H., Almeida J.S. & Ramirez M. 2006. Illustration of a common framework for relating multiple typing methods by application to macrolide-resistant Streptococcus pyogenes. J Clin Microbiol, 44, 2524–2532.

Center for Food Security and Public Health (CFSPH). 2020. The Canine Brucellosis. Brucella canis. Ames, Iowa. http://www.cfsph.iastate.edu/Factsheets/pdfs/brucellosis_canis.pdf .

Chinyoka S., Dhliwayo S., Marabini L., Dutlow K., Matope G. & Pfukenyi D.M. 2014. Serological survey of Brucella canis in dogs in urban Harare and selected rural communities in Zimbabwe. J S Afr Vet Assoc, 85, 1087.

Corrente M., Franchini D., Decaro N., Greco G., D’Abramo M., Greco M.F., Latronico F., Crovace A. & Martella V. 2010. Detection of Brucella canis in a dog in Italy. New Microbiol, 33, 337–341.

Cosford K.L. 2018. Brucella canis: an update on research and clinical management. Can Vet J, 59, 74–81.

Daly R., Willis K.C., Wood J., Brown K., Brown D., Beguin-Strong T., Smith R. & Ruesch H. 2020. Seroprevalence of Brucella canis in dogs rescued from South Dakota Indian reservations, 2015–2019. Prev Vet Med, 184, 105157.

Di D., Cui B., Wang H., Zhao H., Piao D., Tian L., Tian G., Kang J., Mao X., Zhang X., Du P., Zhu L., Zhao Z., Mao L., Yao W., Guan P., Fan W. & Jiang X. 2014. Genetic polymorphism characteristics of Brucella canis isolated in China. PLoS ONE, 9, 1– 7.

Dreer M.K.P., Dib Gonçalves D. D., Caetano I.C., Gerônimo E., Menegas P.H., Bergo D., Lopes-Mori F.M.R., Benitez A, de Freitas J.C., Evers F., Navarro T.I. & de Almeida Martins L. 2013. Toxoplasmosis, leptospirosis, and brucellosis in stray dogs housed at the shelter in Umuarama municipality, Paraná, Brazil. J Venom Anim Toxins Incl Trop Dis, 19, 23.

Egloff S., Schneeberger M. Gobeli Brawand S., Krudewig, C., Schmitt S., Reichler I. & Peterhans S. 2018. Brucella canis infection in a young dog with epididymitis and orchitis. Schweiz Arch Tierheilkd, 160(12), 743-748.

European Union (EU). 2003. Directive 2003/99/EC of the European Parliament and of the Council of 17 November 2003 on the monitoring of zoonoses and zoonotic agents, amending Council Decision 90/424/EEC and repealing Council Directive 92/117/EEC. OJ, L 325, 12/12/2003.

Flores-Castro R., Suarez F., Ramirez-Pfeiffer C. & Carmichael L.E. 1977. Canine brucellosis: bacteriological and serological investigation of naturally infected dogs in Mexico City. J Clin Microbiol, 6, 591–597.

Gardner D. & Reichel M. 1997. No evidence of Brucella canis infection in New Zealand dogs. Surveillance, 24, 17–18.

Garofolo G., Ancora M. & Di Giannatale E. 2013. MLVA-16 loci panel on Brucella spp. using multiplex PCR and multicolor capillary electrophoresis. J Microbiol Methods, 92, 103–107.

George L.W Duncan J.R. & Carmichael L.E. 1979. Semen examination in dogs with canine brucellosis. Am J Vet Res, 40(11), 1589-1595.

Georgia Department of Agriculture (GDA). 2020. Canine Brucellosis (Brucella canis). Atlanta, Georgia. http://agr.georgia.gov/Data/Sites/1/media/ag_animalindustry/animal_health/files/caninebrucellosis.pdf

Gyuranecz M., Szeredi L., Rónai Z., Dénes B., Dencso L., Dán Á., Pálmai N., Hauser Z., Lami E., Makrai L., Erdélyi K. & Jánosi S. 2011. Detection of Brucella canis-induced reproductive diseases in a Kennel. J Vet Diagnostic Investig, 23, 143–147.

Hayashi T.T.A & Ysayama Y. 1977. Detection of Brucella canis infection in dogs in Hokkaido. Microbiol Immunol, 21(5), 295-298.

Hensel M.E., Negron M. & Arenas-Gamboa A.M. 2018. Brucellosis in dogs and public health risk. Emerg Infect Dis, 24, 1401–1406.

Hofer E., Bagó Z., Revilla-Fernández S., Melzer F., Tomaso H., López-goñi I., Fasching G. & Schmoll F. 2012. First detection of Brucella canis infections in a breeding kennel in Austria. New Microbiol, 35, 507–510.

Holst B.S., Löfqvist K., Ernholm L., Eld K., Cedersmyg M. & Hallgren G. 2012. The first case of Brucella canis in Sweden: background, case report and recommendations from a northern European perspective. Acta Vet Scand, 54, 18.

Janowicz A., De Massis F., Ancora M., Cammà C., Patavino C., Battisti A., Prior K., Harmsen D., Scholz H., Zilli K., Sacchini L., Di Giannatale E. & Garofolo G. 2018. Core genome multilocus sequence typing and single nucleotide polymorphism analysis in the epidemiology of Brucella melitensis infections. J Clin Microbiol, 56(9), e00517-18.

Junemann S., Sedlazeck F.J., Prior K., Albersmeier A., John U., Kalinowski J., Mellmann A., Goesmann A., von Haeseler A., Stoye J. & Harmsen D. 2013. Updating benchtop sequencing performance comparison. Nat Biotechnol, 31, 294 –296.

Kaden R., Ågren J., Båverud V., Hallgren G., Ferrari S., Börjesson J., Lindberg M., Bäckman S. & Wahab T. 2014. Brucellosis outbreak in a Swedish kennel in 2013: determination of genetic markers for source tracing. Vet Microbiol, 174, 523–530.

Kauffman L. K., Bjork J. K., Gallup J. M., Boggiatto P. M., Bellaire B. H. & Petersen C. A. 2014. Early Detection of Brucella canis via Quantitative Polymerase Chain Reaction Analysis. Zoonoses Public Health, 61, 48-54.

Kaufmann L.K. & Petersen C.A. 2019. Canine brucellosis: old foe and reemerging scourge. Vet Clin North Am Small Anim Pract,49(4), 763-779.

Keid L. B., Soares R. M., Vasconcellos S. A., Salgado V. R., Megid J. & Richtzenhain L. J. 2010. Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis. Vet Rec, 167, 96-99.

Keid L.B., Chiebao D.P., Batinga M.C.A., Faita T.,Diniz J.A., Oliveira T.M.F. de S., Ferreira H.L. & Soares R.M. 2017. Brucella canis infection in dogs from commercial breeding kennels in Brazil. Transbound Emerg Dis, 64, 691–697.

Keid L.B., Soares R.M., Vasconcellos S.A., Megid J., Salgado V.R. & Richtzenhain L.J. 2009. Comparison of agar gel immunodiffusion test, rapid slide agglutination test, microbiological culture and PCR for the diagnosis of canine brucellosis. Res Vet Sci, 86, 22–26.

Krueger W.S., Lucero N.E., Brower A., Heil G.L. & Gray G.C. 2014. Evidence for unapparent Brucella canis infections among adults with occupational exposure to dogs. Zoonoses Public Health, 61, 509–518.

Landis J.R. & Koch G.G. 1977. The Measurement of Observer Agreement for Categorical Data. Biometrics, 33, 159-174.

Le Flèche P., Jacques I., Grayon M., Al Dahouk S., Bouchon P., Denoeud F., Nockler K., Neubauer H., Guilloteau L.A. & Vergnaud G. 2006. Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay. BMC Microbiol, 6, 9.

Letunic I. & Bork P. 2019. Interactive Tree Of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res, 47(W1, W256-W259.

Li H. 2013. Aligning sequence reads, clone sequences, and assembly contigs with BWA-MEM. arXiv, 13033997v1. https://arxiv.org/abs/1303.3997.

Marrodan T., Nenova-Poliakova R., Rubio M., Ariza J., Clavijo E., Smits H.L. & Diaz R. 2001. Evaluation of three methods to measure anti-Brucella IgM antibodies and interference of IgA in the interpretation of mercaptan-based tests. J Med Microbiol, 50(8), 663-666.

Mellmann A., Harmsen D., Cummings C.A., Zentz E.B., Leopold S.R., Rico A., Prior K., Szczepanowski R., Ji Y., Zhang W., McLaughlin S.F., Henkhaus J.K., Leopold B., Bielaszewska M., Prager R., Brzoska P.M., Moore R.L., Guenther S., Rothberg J.M. & Karch H. 2011. Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology. PLoS One, 6, e22751.

Michaux-Charachon S., Jumas-Bilak E., Allardetservent A., Bourg G., Boschirolim L., O’Callaghan D. 2002. The Brucella genome at the beginning of the post-genomic era. Vet Microbiol, 90, 581-585.

Mol J.P.S., Guedes A.C.B., Eckstein C., Quintal A.P.N., Souza T.D., Mathias L.A., Haddad J.P.A., Paixao T.A. & Santos R.L. 2020. Diagnosis of canine brucellosis: comparison of various serologic tests and PCR. J Vet Diagn Invest, 32, 77-86.

Mor S.M., Wiethoelter A.K., Lee A., Moloney B., James D.R. & Malik R. 2016. Emergence of Brucella suis in dogs in New South Wales, Australia: clinical findings and implications for zoonotic transmission. BMC Vet Res, 12, 199.

Nascimento M., Sousa A., Ramirez M., Francisco A.P., Carrico J.A., Vaz C. 2017. PHYLOViZ 2.0: providing scalable data integration and visualization for multiple phylogenetic inference methods. Bioinformatics, 33, 128 –129.

National Association of State Public Health Veterinarians (NASPHV). 2012. Public Health Implications of Brucella canis Infections in Humans. http://nasphv.org/Documents/BrucellaCanisInHumans.pdf

Nicoletti P. 1980. The epidemiology of bovine brucellosis. Adv Vet Sci Comp Med, 24, 69-98.

Nöckler K., Kutzer P., Reif S., Rosenberger N., Draeger A., Bahn P., Göllner C. & Erlbeck C. 2003. Canine brucellosis–a case report. Berl Munch Tierarztl Wochenschr, 116, 368–372.

Nurk S., Bankevich A., Antipov D., Gurevich A.A., Korobeynikov A., Lapidus A., Prjibelski A.D., Pyshkin A., Sirotkin A., Sirotkin Y., Stepanauskas R., Clingenpeel S.R., Woyke T., McLean J.S., Lasken R., Tesler G., Alekseyev M.A. & Pevzner PA. 2013. Assembling single-cell genomes and minimetagenomes from chimeric MDA products. J Comput Biol, 20, 714 –737.

Rovid Spickler A. 2018. Brucellosis: Brucella canis. United States Department of Agriculture Animal and Plant Health Inspection Service, 1–10. http://www.cfsph.iastate.edu/Factsheets/pdfs/brucellosis_canis.pdf

Sahl J.W., Beckstrom-Sternberg S.M., Babic-Sternberg J., Gillece J.D., Hepp C.M., Auerbach R.K., Tembe W., Wagner D.M., Keim P.S. & Pearson T. 2015. The In Silico Genotyper (ISG): an open-source pipeline to rapidly identify and annotate nucleotide variants for comparative genomics applications. bioRxiv , 1-7. https://doi.org/10.1101/015578.

Severiano A., Pinto F.R., Ramirez M. & Carriço J.A. 2011. Adjusted Wallace coefficient as a measure of congruence between typing methods. J Clin Microbiol, 49, 3997– 4000.

Shin S. & Carmichael L.E. 1999. Canine brucellosis caused by Brucella canis. In Recent advances in canine infectious disease (Carmichael L.E., ed). New York International Veterinary Information Service, Ithaca NY, 1-4.

Sidstedt M., Hedman J., Romsos E.L., Waitara L., Wadsö L., Steffen C.R., Vallone P.M. & Rådström P. 2018. Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR Anal Bioanal Chem, 410, 2569–2583.

Sivia D.S. 1996. Data Analysis: A Bayesian Tutorial. Oxford University Press, Oxford, UK.

Taylor D.J. 1980. Serological evidence for the presence of Brucella canis infection in dogs in Britain. Vet Rec, 106, 102–104.

United States Department of Agriculture Animal and Plant Health Inspection Service (USDA). 2015. Best Practices for Brucella canis Prevention and Control in Dog Breeding Facilities. U. S. Department of Agriculture. https://www.aphis.usda.gov/animal_welfare/downloads/brucella_canis_prevention.pdf

Vicente A.F., Girault G., Corde Y., Mioni M.S.R., Keid L.B., Jay, M., Megid J. & Mick V. 2018. New insights into phylogeography of worldwide Brucella canis isolates by comparative genomics-based approaches: focus on Brazil. BMC genomics, 19(1), 636.

Von Kruedener R.B. 1976. Outbreak of a Brucella canis infection in a beagle colony in West Germany. Dev Biol Stand, 31, 251–253.

Wanke M.M. 2004. Canine brucellosis. Anim Reprod Sci, 82-83, 195–207.

Whatmore A., Perret L. & Friggens M. 2017. Second UK isolation of Brucella canis. Vet Rec, 180 (25), 617.

Whatmore A.M., Perrett L.L. & MacMillan A.P. 2007. Characterisation of the genetic diversity of Brucella by multilocus sequencing. BMC Microbiol, 7, 34.

World Organization for Animal Health (OIE). 2019. Brucellosis (Brucella abortus, B. melitensis and B. suis) (infection with B. abortus, B. melitensis and B. suis) In Manual of Diagnostic Tests and Vaccines far Terrestrial Animals. Paris, Office International des Epizooties, 1–44.

Yoak A.J., Reece J.F., Gehrt S.D. & Hamilton I.M. 2014. Disease control through fertility control: secondary benefits of animal birth control in Indian street dogs. Prev Vet Med, 113, 152–156.