Application of Real-Time PCR Assays for Detection of Marine Brucellae in Teleost Fish
Marine Brucella spp., B. ceti and B. pinnipedialis, are documented in several marine mammal species1,2, and in three human cases3,4. However, transmission of Brucella in marine mammals or marine-origin zoonotic infection is not well-defined. Despite numerous reports of Brucella spp. in marine mammals, reports in fish are scarce. Nile catfish (Clarias gariepinus) experimentally infected with B. melitensis demonstrated antibodies and maintenance of positive titers several weeks post inoculation.5 Furthermore, natural infection of Nile catfish with B. melitensis has been demonstrated.6 Seroconversion and infection patterns in harbor seals (Phoca vitulina richardsi) in the US Pacific Northwest indicate exposure post-weaning, associated with prey consumption or lungworm infection, suggests fish and possibly invertebrate prey items play a role in the epizootiology of marine Brucella transmission.2,7 In light of this possible role, through direct consumption or parasites, and given the zoonotic potential, objectives of this study were to evaluate and validate a real-time (qPCR) assay to detect marine Brucella in extracted teleost fish DNA. Two qPCR assays were used, one targeting the IS711 gene8, the other, a ST27-specific IS711 chromosomal locus (BCETI_7000072) previously identified and evaluated for marine and terrestrial Brucella, to detect the genotype associated with human zoonotic infection.9,10 Strains B. ceti B92-1350 and B. pinnipedialis B04-0281 were used as positive controls to validate the qPCR assay in fish tissues and assess its sensitivity. Nine fish DNA samples were tested, each a 50/50 mix (by biomass) consisting of chub mackerel (Scomber colias) and one of the following: chum salmon (Oncorhynchus keta); coho salmon (O. kisutch); Pacific herring (Clupea pallasii); sardine (Sardinops sagax caerulea); Pacific hake (Merluccius productus); walleye pollock (Gadus chalcogrammus); or rock sole (Lepidopsetta bilineata), and included replicates of each of the salmon mixes. First, a mackerel/chum salmon sample was inoculated with decreasing amounts of B. ceti and pinnipedialis DNA, then analyzed using both primer/probe sets to determine that amplification, thus detection, occurs in fish DNA. A standard curve, created as previously described10, quantified the DNA and determined genome copy numbers of Brucella spp. in each sample. Lastly, DNA extracts from head kidney, liver, and spleen from Atlantic cod (Gadus morhua) experimentally infected with a B. pinnipedialis hooded seal (Cystophora cristata) strain were analyzed. Results showed the IS711 primer/probe detected different concentrations of control Brucella DNA (B. ceti and pinnipedialis) amongst the 50/50 mix samples. The ST27 (dolphin isolate, marine mammal-specific) primer/probe set only detected B. ceti, not B. pinnipedialis DNA, as expected. None of the 50/50 mix samples were positive. In the infected, culture-positive cod samples, the IS711 primer/probe level of detection was approximately one genome copy of B. pinnipedialis. This study demonstrates teleost fish DNA does not inhibit PCR amplification (and detection) of marine Brucella DNA. This qPCR assay will be useful in detecting low numbers of organisms within fish tissues, and in conjunction with culture surveys, can confirm survival of marine Brucella in fish, therefore elucidating their role in marine Brucella infections. Future surveillance of fish and invertebrates will greatly improve the understanding of Brucella transmission in the marine environment.
The authors wish to thank Ms. Dyanna Lambourn of the Washington Department of Fish and Wildlife, Marine Mammal Investigations and Dr. Joe Gaydos of the SeaDoc Society for helpful insights on Brucella occurrence in marine mammals
* Presenting author
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