Blood samples from MMP dolphins were initially collected by venipuncture from animals trained either to present their tail for sampling in the water or to rest on a foam mat during a routine physical examination out of the water. Samples were collected from the caudal peduncle vein by using a 20- or 21-gauge, 1.5-inch Vacutainer needle (Becton Dickinson Vacutainer Systems, Rutherford, NJ, USA) or from a fluke vein by using a 21-gauge, 1-inch butterfly needle. Blood was collected into a Vacutainer serum separator tube or a Vacutainer EDTA (K₃) tube for serum chemistries and complete blood counts, respectively.

Samples for chemistry analysis were centrifuged within 2 h of collection. Centrifugation was performed at 3,000 rpm at 21°C for 10 min. Fibrin clots were removed, and serum was transferred to a 5-mL plastic submission tube. Whole blood was collected in EDTA Vacutainer tubes. All samples were sent on wet ice by courier to Quest Diagnostic Laboratories in San Diego.

Automated hematologic analyses were conducted by Quest Diagnostic Laboratories with the Coulter LH 1500 Series (Beckman Coulter, Inc., Fullerton, CA, USA). The Fisherbrand Dispette 2 (Fisher Scientific, Pittsburgh, PA, USA), correlating with the Westergren method, was used in house to determine 60-min erythrocyte sedimentation rates (ESRs) from 1 mL EDTA whole blood. Remaining serum from these samples was archived at –80°C at the MMP facility at the time of initial blood collection.

Upon completed development of the PIV-antibody ELISA, the archived, frozen serum samples were shipped frozen overnight to the laboratory for PIV-antibody analysis. Total leukocyte count, absolute neutrophils, absolute lymphocytes, absolute monocytes, absolute eosinophils, and ESR results were incorporated into the retrospective PIV seroprevalence study and linked to animal age, sex, clinical signs, and PIV-antibody ELISA results.

Blood samples from free-ranging dolphins were obtained as part of a long-term health assessment conducted near Sarasota, involving a multigenerational resident population of ≈150 dolphins (8). Most of these dolphins are recognizable; are of known age, gender, and maternal lineage; and medical histories have been recorded for many years. Small groups of dolphins were encircled with a 5,000-m long × 5-m deep seine net in shallow water (<1.8 m). Each dolphin was shaded, kept cool and wet, and carefully monitored for signs of discomfort by 1 to 3 veterinarians experienced with cetaceans. The veterinary staff monitored the dolphin’s respiration rate and quality, responsiveness to external stimuli, mental alertness, skin temperature, and heart rate to evaluate the animal’s comfort level and immediate health status. Blood samples were drawn from the ventral fluke vasculature by a 19-gauge × ¾-inch butterfly catheter with a multisample adaptor (Becton Dickinson), which allowed the blood collection tubes to be filled directly from the venipuncture set.

Archived TtPIV-1 was propagated in BSC40 cells as previously described (15). Uninfected BSC40 cells were cultured as negative controls. When >90% of the infected monolayers showed cytopathic effect (CPE), all infected and uninfected cultures were harvested and centrifuged for 15 min at 1,500 rpm, and the supernatant media was removed. The remaining cell pellets were freeze-thawed 3× and centrifuged for 15 min at 3,000 rpm. The cell pellet was discarded, and the supernatant cell lysates were pooled for use as antigen in the ELISA. The protein concentration of the infected and uninfected cell lysates was determined by using a modified Bradford assay for ELISA, after which the cell lysates were diluted in phosphate-buffered saline (PBS) to the desired coating concentration.

A serum sample collected postmortem from the case dolphin was used as the positive reference serum. A negative reference serum sample was collected from an immunologically naive neonate bottlenose dolphin that had not yet nursed. The positive and negative reference serum samples were used to optimize the ELISA conditions. All assay parameters were varied (working volume 50–100 μL; coating concentration 1–20 μg/mL; serum dilution 1:50–1:400; developing time 15–60 min), and the assay conditions with the highest ratio of the optical density at 405 nm (OD₄₀₅) of positive reference serum sample to the OD₄₀₅ of negative reference serum sample were selected. The conditions of the optimized ELISA protocol were as follows. Wells of a high protein-binding microplate (Nunc Maxisorp, Fisher Scientific) were coated with 50 μL of infected or uninfected cell lysates at 5 μg/mL in PBS and were left to adsorb overnight at 4°C. After this and each subsequent step, all wells were washed 3× with PBS with 0.05% Tween by using an automated EL 404 microplate washer (Biotek Instruments, Winooski, VT, USA). After washing, all wells were blocked with 300 μL of Superblock blocking buffer (Pierce, Rockford, IL, USA) in PBS with sodium azide (PBS/Az), after which the dolphin serum samples were applied (1:500 in 1% bovine serum albumin [BSA] in PBS/Az). All sera were applied in triplicate to wells that were coated with either infected or uninfected cell lysate antigen. The positive and negative control sera were included on each plate. A biotinylated monoclonal antibody specific for bottlenose dolphin IgG (15) was used at a concentration of 5 µg/mL (in 1% BSA in PBS/Az) as the secondary reagent for the detection of bound antibodies. Each step of the ELISA was left to incubate with gentle agitation (Nutator; Adams, Fisher Scientific) for 1 h at ≈22°C. Finally, 1.0 mg/mL p-nitrophenyl phosphate (Sigma, St. Louis, MO, USA) substrate was added. The OD₄₀₅ was recorded 60 min after addition of the substrate by using a Spectramax 250 microplate reader (Molecular Devices, Sunnyvale, CA, USA). For analysis, the mean OD₄₀₅ of the triplicate readings on the uninfected antigen was subtracted from the mean OD₄₀₅ of the triplicate readings by using the infected antigen. All results were presented as OD₄₀₅ ratios, defined as the OD₄₀₅ reading of the unknown samples divided by the OD₄₀₅ reading of the positive control sample for that plate.

Archived hematologic and clinical observation data (1999–2006) were mined to identify dolphins with hemograms similar to that of the positive control animal, including a neutrophilic, monocytic leukocytosis or a high ESR, as defined by MMP reference ranges (16). Archived serum samples collected within 60 days before, during, and <90 days after an inflammatory hemogram or clinical illness from suspected PIV cases were subsequently analyzed for PIV antibodies with the dolphin-specific ELISA. PIV seroconversion was defined as a >4-fold increase in PIV antibody OD₄₀₅ level within a 3-month period.

Analyses were conducted to describe frequencies of abnormal clinicopathologic values and clinical signs among PIV seroconversion cases from 60 days before the highest OD₄₀₅ ratio to 30 days after the highest OD₄₀₅ ratio. Median clinicopathologic blood values were calculated among PIV seroconversion cases by using the serum sample with the highest leukocyte count from each identified animal (e.g., the blood sample most likely representing the most severe phase of disease).

Serum samples collected during July to December 2006 from 58 MMP bottlenose dolphins in San Diego and samples collected during 2003–2005 from 56 free-ranging bottlenose dolphins living near Sarasota were analyzed for PIV antibodies. Because only 1 positive control animal had been identified, a conservative interpretation of ELISA results was adopted. Samples with an OD₄₀₅ ratio >1.0 (seropositive) contained an antibody level at least as high as the positive control’s highest antibody level during the time of PIV infection. Samples with an OD₄₀₅ ratio of 0.0 (seronegative) contained an anti-PIV antibody level that was less than or equal to the negative control. OD₄₀₅ ratios >0 and <1 were categorized as inconclusive. Midrange values were used to compare mean OD₄₀₅ ratios by population location, age, and sex. Midrange values were also used to assess changes within 1 animal over time (see Methods, PIV Seroconversion).

Descriptive statistics were used to determine the prevalence of PIV-seropositive, -seronegative, and -inconclusive animals among the 2 study populations. To assess the clinical relevance of PIV exposure among presumably healthy dolphins, mean values of hematologic and serum biochemical inflammatory indicators (leukocyte counts and ESR) were subsequently compared between PIV-seropositive and -seronegative animals in the MMP 2006 population by using analysis of covariance (ANCOVA) with a general linear model to control for varying numbers of samples, age, and sex of animals. Age and sex were controlled covariates because of the previously documented effects of age and sex on healthy, normal reference ranges in dolphins (16). A type I sum of squares p value was used to determine significance. Mean comparisons were reported by using least squares means when controlling for covariates.

PIV-antibody levels were compared between healthy MMP dolphins in San Diego and free-ranging dolphins living near Sarasota. Differences in age and sex between the 2 study populations were analyzed by using a general linear model (PROC GLM; CLASS population; MODEL age = population; MEANS population) and a Mantel-Haenszel χ2 test, respectively. Mean OD₄₀₅ levels were compared by population, age, and sex (controlling for age due to identified differences in ages between the 2 populations) by using ANCOVA with a general linear model to control for varying numbers of samples by animal (PROC GLM Overview, SAS Online Doc, Version 8, SAS Institute, Inc.).

PIV serum antibody OD₄₀₅ levels were determined in a dolphin from which TtPIV-1 was successfully isolated from antemortem and postmortem lung samples. Low PIV antibody levels appeared to be present 300 days before illness, and rising antibody levels were detected during the course of TtPIV-1–associated respiratory illness (Figure 1).

PIV antibody levels were determined in 588 serum samples collected during 1999–2006 from 58 selected MMP bottlenose dolphins before, during, and after an inflammatory hemogram similar to that of the positive control. Within this sample set, 22 dolphins were identified that seroconverted within a 3-month period (examples, Figure 2, panels A–C). Eleven (50%) of the dolphins that seroconverted were female; median age was 22.2 years (range 0.3–43 years).

As expected because of the selection criteria for suspected cases, dolphins with PIV seroconversion had a neutrophilic leukocytosis (18, 81.8%) or high ESR (16, 72.7%). Other clinicopathologic abnormalities that were present in at least half of the case dolphins included hyperglobulinemia, monocytosis, thrombocytosis, and high alanine aminotransferase (ALT) levels (Tables 1, 2).

Of 22 dolphins that seroconverted, 5 (22.7%) maintained normal behavior and appetite, and 17 (77.3%) had at least 1 clinical sign. The most commonly reported behavioral abnormalities were decreased appetite (12, 54.5%) and lethargy (10, 45.5%). Veterinary observations also included respiratory (7, 31.8%) and ocular (5, 22.7%) clinical signs (Table 3). Respiratory signs included ventral left lung consolidation, pleuritis, tachypnea, coughing, and abnormal blowhole fluid. Ocular clinical signs were reported as bilateral or unilateral blepharospasm, intermittent squinting, or corneal opacities. Other clinical signs were diffuse, miliary skin or mucosal lesions (2 dolphins) and orange-yellow liquid feces and excessive flatulence (2 dolphins).

Of 13 dolphins that had clinical signs and recovered, the average duration of clinical illness was 9.8 days (range 1–40 days). Four dolphins had PIV antibody seroconversion within 30 days of death, but the presence of TtPIV-1 was not confirmed on virus culture from any animal tissues upon necropsy except from the positive control dolphin. Of these 4 dolphins, 2 died from bacterial pneumonia confirmed by laboratory culture and histologic examination, and 1 had mild to moderate growth of Candida glabrata from lung samples. All 4 animals had a mild to moderate tracheitis or laryngitis not explained by finding intralesional bacterial or fungal infection upon histopathologic examination.

Comparisons of age and sex distribution among the 2 dolphin populations are provided in Table 4. The median age of dolphins was 15.5 years (range 0.2–49.2). Sex was determined for 110 dolphins; of these, 50% were female.

Of 114 clinically healthy dolphins tested for PIV antibodies, 13 (11.4%) were positive, 34 (29.8%) were negative, and 67 (58.8%) were inconclusive (0 <OD₄₀₅ ratio <1.0). Mean and median PIV OD₄₀₅ ratios were 0.4 (standard deviation = 0.4) and 0.2 (range 0.0–2.8), respectively. There were no significant differences in OD₄₀₅ ratio by age (p = 0.2) or sex (females = 0.33, males = 0.43, p = 0.3). When Florida free-ranging healthy dolphins were compared with San Diego managed healthy dolphins, there were no significant differences in mean PIV OD₄₀₅ ratios (p = 0.6) or percentages of animals categorized as negative, positive, or inconclusive (Table 4; p = 0.5). Among the 2006 MMP healthy dolphin serosurvey population, no significant differences were identified when hematologic indicators of inflammation among healthy PIV antibody–positive dolphins were compared with PIV antibody–negative dolphins (Table 5).