Abstract

Since institution of the Marine Mammal Protection Act in 1972, there has been a significant increase in the number of harbour seals (Phoca vitulina richardsi) to historic high census levels within the Pacific Northwest. This expanding population, coupled with previous detection of circulating antibodies to, and isolation of Brucella spp from live capture and stranded wild animals, as well as recent reports of human infections and fatalities attributed to marine mammal variant brucellosis, prompted an investigation into the seroprevalence of antibody titres to Brucella spp in recovered and rehabilitated harbour seals.

During the 2005 pupping season (May to September), 174 neonatal Pacific harbour seals (Phoca vitulina richardsi) were admitted to the Vancouver Aquarium Marine Mammal Rescue and Rehabilitation Centre, Vancouver, British Columbia. On initial presentation, each animal was assigned a log number and stranding location and signalment documented. A physical examination was completed and serum drawn and banked. The seals were individually quarantined in 1m³ tote boxes for 1 month and maintained on a diet of commercial formula or fish formula with vitamin supplementation. To prevent cross contamination or pathogen exposure, strict biosecurity was enforced.

Each animal was assessed daily and clinically abnormal seals were brought to the attention of the head technician and if warranted, veterinarian. On completion of a 30 day quarantine, a second blood serum sample was obtained and archived. Once weaned and as space was available, animals were grouped together in 8 m diameter fresh water supplied, above surface pools with a haul out platform until released. This commingling varied from 2-12 weeks and a third and final serum sample was collected by staff immediately prior to release.

To determine antibody titres, sera were forwarded to the Animal Disease Research Institute, Canadian Food Inspection Agency, Nepean, Ontario. Each sample was screened by indirect enzyme immunoassay (iELISA), competitive enzyme immunoassay (cELISA), and Fluorescence Polarization Assay (FPA). Of 448 samples submitted, 7 animals had detectable titres (Table 1).

Table 1. All samples tested with C-ELISA, I-ELISA, and Florescent Polarization Assay (FPA) and had positive results are listed.

C-ELISA and I-ELISA values were expressed as a percentage of the optical density reading of the positive control.

Although these assays have previously been utilized to screen a variety of pinnipeds and cetaceans, it is important to note that these tests have been optimized and validated specifically for cattle and based on the sensitivity and specificity of these assays, only one animal (0.6% of animals tested) appeared to have detectable antibodies to Brucella spp (PV05141). Based on previous experiments with cattle, the cut-off value for a positive iELISA was set at >46% inhibition and the threshold level for a positive cELISA was set at >30%. The cut-off value for a positive FPA was set at >90mP. Previous studies on Hawaiian monk seals found strong positive results (i.e., >70-90% for iELISA and cELISA, and FPA>200mP) and concordance between tests. The results from this current study have less agreement, and because of low reactivity, may represent false positives.

Disagreement between tests when antibody levels are low is not uncommon and is usually attributable to different sensitivity capabilities of each test and to their measurement of different aspects of the antibody response. For instance, the iELISA measures all antibody, irregardless of affinity while cELISA and to some degree FPA tend to measure antibody of higher affinity values. This usually leads to a few discrepancies in low titered sera.

In the initial submission sera and 30 day samples of seals PV05149 and PV05152, there were no detectable titres; however, antibodies were detected in pre-release samples. Seroconversion prior to release cannot be entirely discounted. Nor can the possibility of a weak positive result due to passive transfer, as with PV05168 be entirely excluded.

In a previous survey of wild harbour seals encompassing all age classes, combinations of assays including Brucella buffered plate agglutination test antigen (BAPA), the Brucellosis Card test (BBA), Rivanol, and complement fixation (CF) test, or alternatively, iELISA, cELISA, and FPA, disclosed an overall seroprevalence of 15-20 percent of sampled animals in the Pacific Northwest. In 1 study, titres varied considerable between age classes with no detectable antibodies in neonates and juveniles and highest levels detected in yearlings and subadults. In this captive cohort, the lack of detectable antibodies is consistent with field observations; however, confounding factors may include in utero exposure, insufficient colostral consumption, in utero or post partum malnutrition, waning maternal immunity and other factors.

Although the sample size is small and biased, the risk of exposure has important public health and wildlife implication. Due to potential human infections, staff and volunteers at rehab facilities should be thoroughly instructed about potential zoonotic transmission and educated on appropriate hygienic practises. There are increasing numbers of pathogens identified in rehab animals that may pose a threat to human health and human health care should always be a prime consideration.