Understanding the zoonotic pathogen, Coxiella burnetii in Australian fur seal breeding colonies through environmental DNA and genotyping

Abstract

Coxiella burnetii is suspected as a novel pathogen contributing to decreased pup production in Australian fur seals (Arctocephalus pusillus doriferus). It has recently been described from a single breeding colony in Bass Strait, has previously been associated with two decreasing populations of northern hemisphere pinnipeds and is a known reproductive pathogen. Data around its disease ecology in marine mammals are sparse. Aims. To determine whether environmental DNA (eDNA) can be used to survey for C. burnetii in Australian fur seal breeding colonies. To determine whether C. burnetii in Australian fur seals is the same genotype as terrestrial Australian C. burnetii. Methods. Soil samples were collected from Kanowna Island and Seal Rocks. Placental samples were collected from Kanowna Island. Soil was evaluated for eDNA using a quantitative polymerase chain reaction (qPCR) for com1 gene. Placental samples were evaluated with com1, htpAB and IS1111 markers. Multiple-locus variable number of tandem repeats analysis for three microsatellite loci (ms-24, ms-28 and ms-33) was used to determine relatedness to Australian C. burnetii genotypes. Key results. eDNA results varied between preand post-pupping at Seal Rocks. When targeting the com1 gene, the post-pupping prevalence at Kanowna Island and Seal Rocks was 59.6% and 90%, respectively. eDNA PCR inhibition of samples was low at 1.9%. There was very poor, sporadic to absent IS1111 amplification in placental samples. The com1 and htpAB qPCRs had an overall prevalence across placental samples of 39.2% and 56.7% respectively. In 90.1% of placental samples (n = 11), the ms-28 locus amplified. Neither ms-24 nor ms-33 amplified. Conclusions. eDNA is an effective tool to survey Australian fur seal breeding colonies in the post-pupping period for C. burnetii. The prevalence appears to be much higher in the Seal Rocks colony than in the Kanowna Island colony. It appears that this is not a terrestrial Australian genotype but rather closely related to genotypes detected in marine mammals in the northern hemisphere. Implications. This research significantly expands our ability to survey for C. burnetii in Australian fur seals and other marine mammals. It highlights knowledge gaps in our understanding of the disease ecology and phylogeny of C. burnetii in marine mammals.

Pacific Gulls (Larus pacificus) as Potential Vectors of Coxiella burnetii in an Australian Fur Seal Breeding Colony

“…Kanowna Island is the third largest breeding colony for AuFS, having approximately 15,000 animals present on the island [ 6 ] and more than 2200 pups born annually [ 16 ]. A prevalence of up to 56.7% for C. burnetii in AusFS placentas has been reported from KI during the peak of pupping [ 7 ]. Therefore, a high density of potentially infected placental material is produced over a very concentrated pupping season.…”

“…The complete lack of IS1111 amplification in all samples from both PG colonies further supports the hypothesis that C. burnetii in AusFS is potentially not related to terrestrial Australian genotypes of the organism but rather closer in relation to marine mammal strains detected in the northern hemisphere [ 5 , 12 ]. In two extensive studies of marine mammals, it has been found that placental tissue fails to show or shows poor amplification of IS1111 [ 7 , 12 ]. As this is a multicopy with high sensitivity [ 18 ], it has been proposed as a potential tool to distinguish between terrestrial and marine mammal-derived strains [ 7 , 12 ].…”

“…In two extensive studies of marine mammals, it has been found that placental tissue fails to show or shows poor amplification of IS1111 [ 7 , 12 ]. As this is a multicopy with high sensitivity [ 18 ], it has been proposed as a potential tool to distinguish between terrestrial and marine mammal-derived strains [ 7 , 12 ].…”

“…The same qPCR techniques were used as employed by researchers in the initial detection of C. burnetii in AusFS [ 5 ], apart from the addition of the IS1111 insertion. The IS1111 marker is presumed to be absent from C. burnetti in AusFS [ 7 , 12 ]. The IS1111 markers were based on previously described techniques [ 18 ].…”

“…Australian fur seals have recently had decreased pup production, more obviously in their largest breeding colony at Seal Rocks [ 6 ]. A post-pupping environmental DNA prevalence of over 90% for C. burnetii has been detected in this breeding colony [ 7 ].…”