The transmission of phocine herpesvirus-1 in rehabilitating and free-ranging Pacific harbor seals (Phoca vitulina) in California

Phocine herpesvirus-1 (PhHV-1) causes regular outbreaks of disease in neonatal harbor seals (Phoca vitulina) at rehabilitation centers in Europe and in the U.S. To investigate transmission of this virus samples were collected from harbor seal pups during exposure studies at a Californian rehabilitation center from 1999 to 2002 and from free-ranging harbor seals off central California during the same period. The exposure studies provided evidence that PhHV-1 can be transmitted horizontally between animals most likely through direct contact with oro-nasal secretions. However vertical transmission may also occur, as adult female harbor seals were found to be shedding the virus in vaginal and nasal secretions, and premature newborn pups had evidence of early infection. Results also indicated that PhHV-1 infections were common in both free-ranging (40%, 49/121) and rehabilitating (54%, 46/85) young harbor seals, during the spring and early summer. This timing, which correlated with pupping and weaning, suggested that the majority of animals were infected and infective with PhHV-1 between pupping and breeding.     

Mass mortality in seals caused by a newly discovered morbillivirus

During a recent disease outbreak among harbour seals (Phoca vitulina) in the North and Baltic seas, more than 17,000 animals have died. The clinical symptoms and pathological findings were similar to those of distemper in dogs. Based on a seroepizootiological study, using a canine distemper virus (CDV) neutralization assay, it was shown that CDV or a closely related morbillivirus (phocid distemper virus-PDV) was the primary cause of the disease. The virus was isolated in cell culture from the organs of dead seals and characterized as a morbillivirus by serology (immunofluorescence neutralization and enzyme-linked immunosorbent assays) and by negative contrast electron microscopy. Experimental infection of SPF dogs resulted in the development of mild clinical signs of distemper and CDV-neutralizing antibodies. The disease was reproduced in seals by experimental inoculation of organ material from animals that had died during the outbreak. However, seals that had been vaccinated with experimental inactivated CDV vaccines were protected against this challenge. This fulfilled the last of Koch's postulates, confirming that the morbillivirus isolated from the seal organs, was the primary cause of the disease outbreak. The recent demonstration of the presence of a similar virus in Lake Baikal seals (Phoca sibirica), which infected these Siberian seals 1 year before the northwestern European seals were infected, raises new questions about the origin of this infectious disease in pinnipeds.     

Serum chemistry values for free-ranging ringed seals (Pusa hispida) in Svalbard

BACKGROUND: Diseases and abnormal physiologic conditions can alter the concentrations of enzymes, metabolites, minerals, and hormones in the blood of animals. The ringed seal (Pusa hispida) has been selected as a key species for environmental monitoring, but information on disease and health parameters for this species is scarce. OBJECTIVES: The aim of the study reported here was to obtain serum chemistry reference intervals for free-ranging ringed seals in Svalbard, and then to evaluate serum chemistry values in relation to age, body condition, and sex. METHODS: Blood samples were collected after death from ringed seals in Wijdefjorden and Billefjorden, Svalbard (2002-2003; n = 75). Serum was analyzed for 24 selected serum chemistry parameters (enzymes, protein, metabolites, minerals, and cortisol). RESULTS: Compared with younger or older animals, seals between 7 and 16 years of age had larger variations in the activities of alanine transaminase, aspartate transaminase, lactate dehydrogenase, and creatine kinase (CK). Animals classified as having low body condition status had more variation in the serum activity of these enzymes, compared with that in animals with higher condition scores. Serum cortisol concentration was higher in young animals (1-5 years) than in older animals. Serum CK activity was higher in males than in females. CONCLUSION: The data reported here may be useful in monitoring the health of ringed seals and for tracking the impact of environmental changes in the Arctic.     

Herpesvirus in harbour seals (Phoca vitulina): isolation, partial characterization and distribution

From post-mortem material (liver and lung) and leucocytes of four (3.6%) out of 112 examined harbour seals during the seal epizootic in 1988 six cytopathogenic viral isolates were obtained which were provisionally classified as herpes-like viruses. Results of physico-chemical and electron microscopic investigations suggested their relationship to the herpesvirus family. Serological examinations were carried out with sera from wildlife as well as captive animals using herpesvirus isolates from four different seals. The neutralization tests revealed as only moderate distribution of seropositive reagents up to 53% of the wildlife seal population. Amongst the seals in the orphanage of Norddeich a very small number of seropositive animals was found. The results obtained indicated a minor role of herpesviruses as primary cause of seal mortality in the North Sea during the 1988 season.     

Contraceptive effect of proligestone on spotted seals and crossbreeds of spotted seals and harbor seals

Spotted seals (Phoca largha) and related crossbreeds maintained at Kamogawa Sea World breed seasonally from the end of January to middle of March. For contraception in these animals, the effect of a single administration of a contraceptive synthetic luteal hormone for dogs, proligestone (PRG), was investigated. The animals tested were 10 seals aged 4-24 years old, and a total of 35 trials were performed over five years. PRG was administered in 23 trials during January, which was one month before the estimated estrus, and in 12 trials during December of the previous year, which was two months before the estimated estrus. The dose of PRG was 5 mg/kg in 32 trials and 10 mg/kg in 3 trials. The effect of the contraception was judged by the presence or absence of delivery. Among 23 animals treated in January, 2 animals treated with 5 mg/kg PRG became pregnant, but the contraception was successful in the other 21 animals. Contraception was successful in all 12 trials treated with 5 mg/kg PRG in December. Overall, contraception was successful in 94.3% (33/35). Therefore, a single administration of 5 mg/kg PRG in December may be an effective method of contraception for seals.     

Morbillivirus infection of seals (Phoca vitulina) during the 1988 epidemic in the Bay of Heligoland. I. Mode, frequency and significance of cultural virus isolation and neutralizing antibody detection

From 16 (14%) out of 112 dead or euthanized seals originating from wildlife and seal orphanages phocine morbillivirus was isolated. The majority of viral isolates in cell cultures was obtained from lung homogenates of 15 out of 71 free-ranging seals (21%). The virus was isolated by longterm cultivation in roller cultures of seal kidney cells. The phocine morbillivirus was detected by typical cytopathogenic alteration and by peroxidase-linked antibody (PLA) assay, respectively. A neutralization test based on PLA was used for antibody detection in seals using a canine distemper virus (CDV) strain and in parallel one of the phocine morbillivirus isolates. All sera tested were proven to contain neutralizing antibodies of higher titres against the latter virus than against the CDV strain. Several seals furnished morbillivirus isolates and at the same time exhibited neutralizing antibodies of low to medium titres. No viral isolates were obtained from the majority of sick animals with moderate to high neutralizing titres (greater than 1/1,000). The significance of these findings is discussed in relation to the cause of the mass mortality amongst seals observed in 1988 in the Bay of Heligoland.     

Reference values for serum biochemical parameters in free-ranging harp seals

Background — The harp seal (Phoca groenlandica) is one of the most important predators in the Northeastern Atlantic ecosystem. Establishing biochemical reference intervals is important for evaluating the health status of harp seals kept in captivity and for evaluating the effects of environmental changes on the health of populations in the wild. Objective — The purpose of this study was to determine reference values for serum biochemical parameters in wild adult harp seals using readily available current methods. Methods — Blood samples were obtained from 14 adult female harp seals and 9 suckling pups on the pack ice of the Greenland Sea in early March 1998. Seven seals were humanely killed on the ice by permission of the Norwegian Directory for Fisheries and in conjunction with several other research projects. The seals were sampled within 15 minutes postmortem. Remaining seals were captured alive and sampled via the extradural intravertebral vein. Serum biochemical parameters were measured using a Technicon Axon analyzer and included electrolytes (sodium, chloride, potassium, magnesium, and calcium), substrates (free fatty acids, triglycerides, fructosamine, and glucose), end products (urea and uric acid), and proteins (total protein, globulins, and albumin). Serum protein electrophoresis also was done. Data were tested for normality and reference limits were calculated as mean ±1.96 × SD. Results between groups were compared using 2‐tailed t‐tests. Results — Serum levels of glucose and triglycerides were lower, but serum levels of urea were higher in dead animals than in animals that were captured alive. Serum levels for 7 of 17 parameters were significantly different in pups compared with adults. Separate reference intervals were calculated for adult seals and seal pups. Conclusion — Both sampling method and age should be considered when evaluating the results of analysis of serum parameters in wild and captive harp seals.     

An outbreak of leptospirosis in seals (Phoca vitulina) in captivity

An outbreak of leptospirosis in seals (Phoca vitulina) in captivity is described. In a zoo in The Netherlands 5 adult seals died within 12 days. At necropsy all animals showed signs of acute septicaemia, consistent with acute leptospirosis. Serological examination of one animal was positive for antibodies against Leptospira interrogans serovar Icterohaemorrhagiae and the serologically closely related serovar Copenhageni. Polymerase chain reaction was positive in one other animal. 8 nutria (Myocastor coypus) were examined, serologically, through bacteriological culture and PCR. 81,8% (9/11) were serologically positive for Leptospira. The seals and nutria were housed in the same water system.     

The seal death in Danish waters 1988. 2. Virological studies.

Mass abortions and high mortality were observed in harbour seals in Danish waters during 1988. Severe pneumonia and emphysema were typical clinical and post-mortem findings. Virological studies were carried out to identify the cause of the epidemic. Although seal herpesvirus (SeHV) was isolated in 23 of 114 animals this virus was subsequently found not to be the primary cause of the disease. Following the observation of seroconversion against canine distemper virus (CDV) in diseased seals (Osterhaus & Vedder 1988) a CDV-like morbillivirus (phocine distemper virus, PDV) was identified in organs of diseased animals. It is concluded that the epidemic was caused by introduction of PDV into a highly susceptible population presumably free from morbillivirus infection. The origin of PDV remains unknown but evidence of prior morbillivirus infection has been found in arctic and antarctic seal populations.     

Quantitative analysis of the 2002 phocine distemper epidemic in the Netherlands

Phocine distemper virus (PDV) caused thousands of deaths among harbor seals (Phoca vitulina) from the North Sea in 1988 and 2002. To examine the effects of different factors on the pathology of phocine distemper, we performed necropsies and laboratory analyses on 369 harbor seals that stranded along the Dutch coast during the 2002 PDV epidemic. Diagnostic tests for morbillivirus infection indicated a differential temporal presence of morbillivirus in lung and brain. Seals of 3 years or older were significantly more often IgG positive than younger seals. The most frequent lesions in PDV cases were bronchopneumonia, broncho-interstitial pneumonia, and interstitial emphysema. Extra-thoracic emphysema was rare in <1-year-olds compared with older seals, even though severe pneumonia was more common. PDV cases generally had empty stomachs and less blubber than by-caught seals from before the epidemic. In PDV cases involving older animals, lung, kidney, and adrenal weights were significantly increased. Bordetella bronchiseptica was isolated from lungs in two thirds of the PDV cases examined. Our results indicate that brain should be included among the tissues tested for PDV by RT-PCR; that either phocine distemper has a longer duration in older seals or that there are age-related differences in immunity and organ development; that dehydration could play a role in the course and outcome of phocine distemper; and that bacterial coinfections in lungs are more frequent in PDV cases than gross lesions suggest. These results illustrate how quantitative analysis of pathology data from such epidemics can improve understanding of the causative disease.     

Fecal shedding of Helicobacter spp. by co-housed Australian sea lions (Neophoca cinerea) and Australian fur seals (Arctocephalus pusillus doriferus)

With the emergence of Helicobacter species as agents of gastrointestinal disease within a broad range of animal hosts, there is growing awareness of the need to identify such species and the potential role(s) they play within the intestine. Of interest in this study are captive seals and sea lions, where close proximity to one another may enhance the transmission of pathogens, in particular Helicobacter. The feces of several captive Australian sea lions and Australian fur seals were assessed for the occurrence of Helicobacter over 31 days. The presence of Helicobacter, detected by the polymerase chain reaction (PCR) varied over time and at times could not be detected. Helicobacter species were detected in five of the six animals examined of which two species were identified. This is the first report of Helicobacter species in captive seals and demonstrates the diversity and potential role(s) they may play in the gut of these animals.     

Ultrasonographic monitoring of the fetal growth of common seals (Phoca vitulina vitulina)

Three captive female common seals (Phoca vitulina vitulina) were monitored ultrasonographically during five pregnancies. Pregnancy was diagnosed in the third month and fetal monitoring was continued until parturition. The ultrasound measurements were made with the active collaboration of the animals by training them to cooperate with the procedure.     

Comparison of the somatotropic axis in free-ranging and rehabilitated harbor seal pups (Phoca vitulina)

The somatotropic axis, including growth hormone (GH), insulin-like growth factor (IGF)-I, and IGF binding proteins (IGFBP), is a bridge between growth physiology, developmental age, and nutritional status in domestic animals. However, the importance of the somatotropic axis in nutrition, growth, and development of harbor seals has not been previously explored. Given the difficulty of conducting longitudinal studies in free-ranging harbor seals, this study focused on the potential use of harbor seals in rehabilitation facilities as a model for free-ranging seals. The purpose of this research was to compare concentrations of components of the somatotropic axis in free-ranging versus rehabilitated harbor seal pups. The hypothesis was that measurements of the somatotropic axis will be similar between individuals of comparable age and nutritional status (fasting versus feeding). To investigate this hypothesis, harbor seal pups (n=8) brought to The Marine Mammal Center (Sausalito, California, USA) or Mystic Aquarium (Mystic, Connecticut, U.S.A.) were initially assessed and determined to be healthy but abandoned. All pups were less than 2 wk of age upon arrival at rehabilitation facilities. Standard length was assessed at the time of arrival and again at release. Body mass was measured every week and blood samples were collected from each pup at 0, 4, and 8 wk of rehabilitation. Blood was collected and morphometrics assessed in free-ranging harbor seal pups (n=8) from the Gulf of Maine. Sera were analyzed for GH, IGF-I, and IGFBP concentrations. Concentrations of GH, IGF-I, and IGFBP-2 and -3 in rehabilitated pups were within a similar range compared with free-ranging pups when considered in the context of presumed nutrient intake. These data suggest that rehabilitated harbor seals may provide a useful model to investigate the effects of nutrient intake on growth and development of harbor seals, and will provide insight into phocid endocrinology and metabolism.     

The prevention of distemper in zoo animals

The distemper virus infection of different non-domestic carnivorous zoo animals is known since long. All species involved belonged to the suborder Fissipedia. In 1988 a distemper or morbillivirus-like infection occurred in harbour seals, a member of the suborder pinnipedia. For the prophylaxis of distemper in dogs attenuated live vaccines are commonly used. In zoo animals, however, these vaccines caused distemper several times. In contrast, an inactivated virus vaccine proved both its safety and efficacy in more than hundred zoo animals of various species.     

Apparent Seroprevalence of Salmonella spp. in Harp Seals in the Greenland Sea as Determined by Enzyme-linked Immunosorbent Assay

An indirect ELISA was developed as a possible tool for surveillance of the seroprevalence of Salmonella spp. in harp seals. This species is hunted for human consumption and thus transmission of disease to humans cannot be excluded. To cover a broad spectrum of serogroups, a mixture of the lipopolysaccharides (LPS) of S. typhimurium and S. choleraesuis was used as the antigen in this pilot study. Chicken anti-harp-seal immunoglobulin horseradish peroxidase conjugate served as the immunoconjugate. Sera from four captive harp seals, which were Salmonella culture-negative and had no clinical or historical evidence of salmonellosis, were used as negative controls. After immunization with an inactivated S. typhimurium vaccine, further sera from these seals were used as positive controls, as no serum from naturally infected animals was available. Serum samples from 93 harp seals caught in the Greenland sea in 1999 were examined, and anti-Salmonella antibodies were found in the samples from two individuals (seroprevalence 2.2%). Although Salmonella has been isolated from other pinniped species, this is the first documentation of Salmonella-seropositive harp seals. This study contributes to the evaluation of the importance of salmonellosis in arctic marine mammals and thus to the prevention of potential outbreaks of this important zoonosis.     

Clostridium perfringens toxin types in hooded seals in the Greenland Sea, determined by PCR and ELISA

Very little is known about the occurrence of Clostridium perfringens and of diseases caused by this anaerobic bacterium in marine mammals, especially those that are free-living. During a scientific expedition to the Greenland Sea (West Ice) in spring 1999, faeces samples from 70 hooded seals (Cystophora cristata) were taken to isolate C. perfiringens. Subsequently, PCR analysis of the isolates was performed with oligonucleotide primers of the genes encoding the four major lethal toxins (alpha, beta, epsilon and iota) for classification of toxin type and of the genes encoding C. perfringens beta2-toxin and enterotoxin for further subclassification. In addition, a commercial ELISA kit for detection of C. perfringens alpha, beta- and epsilon-toxin was used. C. perfingens was isolated in samples from 38 (54.3%) hooded seals. All isolates were C. perfringens toxin type A (alpha-toxin positive). This is the first report on the occurrence of C. perfringens in this arctic marine mammal species. Myositis and enterotoxemia caused by C. perfrigens were described in other marine mammals and it may be assumed that the pathogenesis of an outbreak of disease is similar to that encountered in terrestrial animals. Although there is some controversy surrounding the enteropathogenicity and virulence of alpha-toxin (concerning enterotoxemia), this study suggests that a possible outbreak of enterotoxemia caused by C. perfringens type A in hooded seals may, however, not be excluded.     

Efficacy of ivermectin and moxidectin against Otostrongylus circumlitus and Parafilaroides gymnurus in harbour seals (Phoca vitulina)

Verminous bronchopneumonia caused by infection with Otostrongylus circumlitus and Parafilaroides gymnurus is an important cause of death during the rehabilitation of harbour seals (Phoca vitulina). During the winter of 2000/01, 35 juvenile harbour seals with severe clinical signs of verminous bronchopneumonia were treated with either 0.2 mg/kg ivermectin orally or 0.2 mg/kg moxidectin subcutaneously, and monitored for 30 days. The efficacy of the anthelmintics was determined by the pattern of larval excretion (Baermannisation) and the progress of the clinical signs. Both anthelmintics had reduced larval excretion by at least 99 per cent after 10 days, but the seals' rapid breathing rate and and dyspnoea returned to normal more quickly in the animals treated with moxidectin. The pharmacokinetics of the anthelmintics were determined by solid-phase extraction, and high-performance liquid chromatography with fluorescence detection. Moxidectin had a mean (sd) residence time of 9.04 (2.12) days compared with 4.83 (1.14) days for ivermectin.     

Evidence of Brucella sp. infection in marine mammals stranded along the coast of southern New England.

After recent isolations of Brucella sp. from pinnipeds and cetaceans, a survey was initiated to investigate the prevalence of Brucella sp. infections and serologic evidence of exposure in marine mammals stranded along the coasts of Connecticut and Rhode Island. One hundred and nineteen serum samples from four species of cetaceans and four species of pinnipeds were collected from 1985 to 2000 and tested for antibodies to Brucella sp. using the brucellosis card test, buffered acidified plate antigen test, and rivanol test. In addition, 20 of these were necropsied between 1998 and 2000, with lymphoid and visceral tissues cultured for Brucella sp. Three of 21 (14%) harbor seals (Phoca vitulina) and four of 53 (8%) harp seals (Phoca groenlandica) were seropositive. Brucella sp. was isolated from two of four (50%) harbor seals and three of nine (33%) harp seals. Of the five animals with positive cultures, two were seropositive and three seronegative. Brucella sp. was most frequently cultured from the lung and axillary, inguinal, and prescapular lymph nodes. Tissues from which Brucella sp. was isolated showed no gross or histopathologic changes. These results indicate that marine mammals stranded along the coast of southern New England can be exposed to and infected with Brucella sp.     

The effect of rehabilitation of northern elephant seals (Mirounga angustirostris) on antimicrobial resistance of commensal Escherichia coli

The aim of this study was to determine if antimicrobial drug use increases resistance of commensal gastrointensinal Escherichia coli of wild northern elephant seals (Mirounga angustirostris) treated in rehabilitation, and, if so, identify the risk factors involved. Minimum inhibitory concentration (MIC) levels of twelve antimicrobial drugs were determined for 289 E. coli isolates from 99 seals sampled at admission and 277 isolates obtained at release from rehabilitation using broth microdilution. Prevalence of E. coli antimicrobial resistance, MIC(50), MIC(90), and clustering of MIC values were determined for seals and the data were analyzed using Fisher's exact test, ordinal logistic regression and negative binomial regression. At release from rehabilitation 77.8% of the seals had antimicrobial resistant E. coli compared to 38.4% of the seals at admission. The MIC(90) for amoxicillin-clavulanic acid, chloramphenicol, enrofloxacin, ticarcillin-clavulanic acid, and trimethoprim-saulfamethoxazole were at levels considered to be sensitive at admission but they increased to levels of resistance at release. E. coli were grouped into four clusters by their MIC values, with increasing levels of resistance going from Cluster 1 to 4. A primary risk factor associated with the probability of a seal having E. coli in Clusters 3 and 4 was time in rehabilitation, regardless of whether the animal received treatment with antimicrobial drugs, suggesting nosocomial infection. The results of this study provide evidence that increased levels of hygiene and appropriate use of antimicrobial therapy might be important in the rehabilitation of wild animals to prevent rise in the prevalence of antimicrobial resistant bacteria.