Brucella canis: Fatty acid variability as a potential indicator of strain origin and biologic behavior Alexandra Brower, DVM, DACVP Clinical Associate Professor of Diagnostic Pathology University of Wisconsin School of Veterinary Medicine

Canine Brucellosis Relatively new bacterial disease of dogs - B. canis was first discovered in 1966. The major symptoms of infection in dogs are abortion and infertility. Canine infections are through contact with placental fluids and aborted tissues. Other mechanisms of transmission: mating (venereal infections) contact with urine Risk as a zoonosis is considered low

Where are we likely to find B. canis in the US? “Dozens and dozens of farms have signs up advertising “puppies for sale” or have placed ads in the area papers listing many different breeds at the same address. Many of them say, “No Sunday Sales” which is a good bet the seller is Amish or Mennonite. I think it’s just such a shame that Clark County has become the hotbed for puppy mills in Wisconsin so quickly and it is expanding so rapidly. What a black eye for us. How humiliating and sad.” Clark county humane society, article titled A dog auction experience

Where else do outbreaks occur? May 6, 2009. A Puppy Farm in Ireland at Centre of Canine Brucellosis Outbreak: Has 700 breeding bitches and there are currently up to 300 puppies on the premises. K9 Magazine News Editor What about human infections? Argentina and Mexico – Multiple published and soon to be published case reports and serologic data on human infection . N. Lucero et al Brucellosis Laboratory, Buenos Aires, Argentina

Diagnostic pathology interface Veterinary diagnostic laboratories receive aborted fetuses, other tissues and blood samples from kennel outbreaks of canine brucellosis. In Wisconsin, by late 2005 submission of samples and positive tests was clearly on the rise. Follow up with kennel owners and veterinarians led to recognition of the following important issues regarding B. canis:

B. canis is a common, unregulated, potentially zoonotic disease in commercial and private canine breeding facilities in Wisconsin. Wisconsin outbreaks are due to trade practices that link kennels throughout the country. There is an under appreciation of the disease among dog breeders, veterinarians, and diagnostic and government agencies.

Outbreaks from 2002- 2008 and total number of dogs in each kennel A – 10 B – 1,000 C – 60-70 D – 30-50 E – 100-120 F – 60-80 G – 2 H – 30-40 I – 30-40 J – 100-120 G C B E A, H, I, J D F

Discrimination of B. canis isolates Identify a way to trace B. canis outbreaks. Initial investigations used samples from three canine brucellosis outbreaks in Wisconsin dog kennels, and B. canis isolates from Missouri and Arkansas. Brucella species have minimal genetic diversity…

Molecular techniques unable to discriminate isolates include: Ribotyping with multiple restriction endonucleases, outer membrane protein analysis and pulsed-field gel electrophoresis (PFGE) *Gas chromatography was found to be a technique that could discriminate between isolates.

Dendrogram analysis of fatty acid profiles Dendrogram analysis of fatty acid profiles. Two major groups were found, described as southern and northern strains. Brower et al. Investigation of the Spread of Brucella canis via the U.S. Interstate Dog Trade. International Journal of Infectious Diseases, Volume 11, Issue 5, Pages 454-458. 2007.

Next question, why are human case reports coming from Argentina, when we have so much exposure in the US? Compared lipid profiles of 1 human B. canis isolate, and 6 canine isolates from Argentina with 36 canine isolates from 8 other countries including the US.

Review of the profiles showed that only the Argentinean and Mexican isolates had19:O cyclopropane (lactobacillic acid), cis-11,12-methylene octadecanoic acid.

Retention time (minutes) Fatty acid Percent of total fatty acids in sample 2.871 16:0 10.16 3.190 17:0 0.38 3.458 18:1 w7c 82.10 3.505 18:0 2.02 Features of a typical U.S. B. canis isolate profile and corresponding chromatograph (Isolate 19-50).

Retention time (minutes) in Fatty acid Percent of total fatty acids in sample 2.870 16:0 11.42 3.189 17:0 0.51 3.457 18:1 w7c 39.44 3.505 18:0 5.20 3.530 11 methyl 18:1 w7c 1.02 3.793 19:0 CYCLO w8c 37.50 4.035 20:2 w6,9c .78 Features of a typical B. canis isolate profile from Argentina and corresponding chromatograph (Isolate 5-46).

Brucella melitensis, suis and abortus index profile. Reference index number Fatty acid Percent of total fatty acids 63 16:0 13.00 82 17:0 0.67 92 18:1 w7c 19.69 95 18:0 9.04 96 11 methyl 18:1 w7c 2.17 105 19:0 CYCLO w8c 50.23 113 20:2 w6,9c 3.17 Brucella melitensis, suis and abortus index profile. This index includes the species of Brucella classically considered to be pathogenic to humans.

Argentina Isolate US Isolate Index profile Fatty acid Percent of total fatty acids in sample 16:0 11.42 17:0 0.51 18:1 w7c 39.44 18:0 5.20 11 methyl 18:1 w7c 1.02 19:0 CYCLO w8c 37.50 20:2 w6,9c .78 Fatty acid Percent of total fatty acids 16:0 13.00 17:0 0.67 18:1 w7c 19.69 18:0 9.04 11 methyl 18:1 w7c 2.17 19:0 CYCLO w8c 50.23 20:2 w6,9c 3.17 Argentina Isolate Fatty acid Percent of total fatty acids in sample 16:0 10.16 17:0 0.38 18:1 w7c 82.10 18:0 2.02 US Isolate Index profile

Dendrogram of 44 B. canis isolates

Brucella species lack most functional gene sequences encoding virulence – a critical component of virulence is likely the LPS surface antigen. Sanchez et al. 2001. Gene Discovery through Genomic Sequencing of Brucella abortus. Infect. Immun. 69:865-868. Heterogeneity of the Lipid A domain of Brucella LPS may primarily depend on fatty acid substitutions Gomes Cardoso et al. 2006. Review: Brucella spp noncanonical LPS: structure, biosynthesis and interaction with host immune system. Microbial Cell Factories 5: [Online.] Might the fatty acid difference found in the Argentinean and Mexican isolates be related to increased virulence in humans?

UK – Comparative study between the lipid profiling technique used by our laboratory, and a variable tandem repeat sequencing technique that the Whatmore laboratory uses to discriminate Brucella isolates. University of Iowa – Seroepidemiologic study of B. canis that focuses on the human populations in the U.S. that are most likely to be exposed to the bacterium.

I am grateful to the following people for their wonderful collaboration on this and other Brucella canis projects. Dr. Ogi Okwumabua, University of Wisconsin Dr. Nidia Lucero, ANILIS Argentina Dr. Adrian Whatmore, VLA United Kingdom Dr. Chuck Massingil, Missouri Department of Agriculture Dr. Greg Gray and Whitney Baker, University of Iowa, College of Public Health

Questions?