Embryonic sex steroid hormones accumulate in the eggshell of loggerhead sea turtle (Caretta caretta).

Steroids hormones such as estradiol-17ß (E2) and testosterone (T) are involved in gonadal differentiation of oviparous animals with temperature-dependent sex determination (TSD), and are greatly distributed. This hypothesizes that these embryonic steroid hormones probably accumulate in the eggshell throughout blood or/and chorioallantoic fluid in sea turtle species with TSD, producing females at higher temperature. To demonstrate this hypothesis, concentrations of E2 and T in the blood plasma from the hatchling loggerhead sea turtle (Caretta caretta) and in their eggshells were measured by radioimmunoassay. In the present study we propose that both concentrations of E2 and T in the blood plasma are correlated with amounts of these sex steroids in the eggshell. Moreover, contents of E2 in the eggshell showed a significant positive correlation with mean incubation temperatures during a thermosensitive period in the experimental nests, whereas T contents in the eggshell did not. Taken together, these findings indicated that embryonic E2 and T that accumulated in the eggshell can be extracted and measured. Furthermore, the present study suggested that contents of E2 in the eggshell may differ between male and female, and monitoring of these steroids is a useful method to identify the sex of loggerhead sea turtle hatchling.

Japanese Encephalitis Virus and Schizophyllum commune Co-Infection in a Harbor Seal in Japan.

The Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has a wide host range, extending from pigs and ardeid birds to opportunistic dead-end hosts, such as humans and horses. However, JEV encephalitis infections in aquatic mammals are rare, with only two cases in seals reported to date. Here, we report a lethal case of JEV and Schizophyllum commune co-infection in an aquarium-housed harbor seal in Japan. We isolated JEV from the brain of the dead seal and characterized its phylogeny and pathogenicity in mice. The virus isolate from the seal was classified as genotype GIb, which aligns with recent Japanese human and mosquito isolates as well as other seal viruses detected in China and Korea, and does not exhibit a unique sequence trait distinct from that of human and mosquito strains. We demonstrated that the seal isolate is pathogenic to mice and causes neuronal symptoms. These data suggest that seals should be considered a susceptible dead-end host for circulating JEV in natural settings.

Characterization of alpha-fetoprotein levels in three dolphin species: development of sensitive immunoassays for analysis of the pregnancy-associated variations.

A single radial immunodiffusion (SRID) assay and a chemiluminescent immunoassay (CLIA) were initially developed for alpha-fetoprotein (AFP) of the striped dolphin. Utilizing these developed assays, we investigated pregnancy-associated changes in the levels of AFP in the sera of fetuses and pregnant females of three dolphin species; samples were either collected from captive individuals or obtained as fishery by-products. The concentrations of AFP in the fetal serum ranged from 419.0 to 2026.3 µg/ml in the striped dolphin, 12.6 to 1218.7 µg/ml (for an AFP equivalent; eqAFP) in the common bottlenose dolphin and 770.6 to 3129.1 µg eqAFP/ml in the Risso's dolphin. AFP levels decreased with increased fetal size in fetuses over 20 cm in length. The concentrations of AFP in sera of pregnant females ranged from 7.18 to 8068.7 ng/ml in the striped dolphin, 6.6 to 1241.1 ng eqAFP/ml in the common bottlenose dolphin and 3.4 to 2868.7 ng eqAFP/ml in the Risso's dolphin. The levels in most pregnant females were equal to or lower than those found in males and nonpregnant individuals, although a few pregnant females exhibited extremely high levels (in the range of hundreds to thousands of nanograms per milliliter). Such high levels of AFP were not observed during pseudopregnancy. To our knowledge, this is the first report on basal profiles for serum AFP levels in small odontocetes. The profiles indicated that AFP may play a significant role during embryonic development, although maternal levels do not appear to be a diagnostic biomarker for monitoring pregnancy.

Multi-tissue DNA methylation aging clocks for sea lions, walruses and seals.

Age determination of wild animals, including pinnipeds, is critical for accurate population assessment and management. For most pinnipeds, current age estimation methodologies utilize tooth or bone sectioning which makes antemortem estimations problematic. We leveraged recent advances in the development of epigenetic age estimators (epigenetic clocks) to develop highly accurate pinniped epigenetic clocks. For clock development, we applied the mammalian methylation array to profile 37,492 cytosine-guanine sites (CpGs) across highly conserved stretches of DNA in blood and skin samples (n = 171) from primarily three pinniped species representing the three phylogenetic families: Otariidae, Phocidae and Odobenidae. We built an elastic net model with Leave-One-Out-Cross Validation (LOOCV) and one with a Leave-One-Species-Out-Cross-Validation (LOSOCV). After identifying the top 30 CpGs, the LOOCV produced a highly correlated (r = 0.95) and accurate (median absolute error = 1.7 years) age estimation clock. The LOSOCV elastic net results indicated that blood and skin clock (r = 0.84) and blood (r = 0.88) pinniped clocks could predict age of animals from pinniped species not used for clock development to within 3.6 and 4.4 years, respectively. These epigenetic clocks provide an improved and relatively non-invasive tool to determine age in skin or blood samples from all pinniped species.

Growth, maturity, reproduction, and life expectancy in ex-situ pacific walruses (Odobenus rosmarus divergens).

BACKGROUND: Pacific walruses are found in Arctic regions of the Chukchi and Bering Sea where rapid changes in environmental conditions resulting in loss of sea ice are occurring. Therefore, accurate life history data are crucial for species management plans and longitudinal data collected over the lives of individual walruses housed in zoos and aquaria provide otherwise difficult to obtain biological information. RESULTS: While similar at birth, Gompertz regression curves indicated that males grew faster than females (p < 0.0001) in weight (99 kg vs 57.6 kg/y) and length (26.9 cm vs 26.3 cm/y) with physical differences being detected by age 3 for weight and age 7 for length. Males reached adult weight at 13.5 ± 3.3 y and females by age 12.3 ± 2.3 y. The mean age at first ovulation and at first conception occurred at 8.8 y and 9.6 y. Greater than 75% of all conceptions and calving occurred between February and March and from May to June, respectively. Mean gestation lasted 423 d and false pregnancies lasted at least 169 d with a decrease (p < 0.05) in serum progesterone concentration between false pregnancy and pregnancy occurring within 6 months after ovulation. Based on these results, we estimated embryonic diapause to last from 120 to 139 days, and fetal growth last ~ 284 days. All males older than 8 y had an increase in serum testosterone and body weight that was highest in February and lowest in July. Overall, no differences were observed between male and female survival, with a mean (± SEM) life expectancy of 19.5 ± 1.5 y, respectively. Currently, the oldest male and female captive walruses are 40 and 43 y, respectively. CONCLUSIONS: Data provided herein include details of life history characteristics of zoo and aquaria housed walruses that are useful for wild population recovery models. In particular, results on survivorship and the identification of the most vulnerable period for calf survival can help with model development and suggests that for recovery to occur birthing locations for this species must be protected.

Amyloid ß and tau pathology in brains of aged pinniped species (sea lion, seal, and walrus).

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-ß (Aß) as senile plaques and cerebral amyloid angiopathy, and hyperphosphorylated tau (hp-tau) as neurofibrillary tangles in the brain. The AD-related pathology has been reported in several non-human animals, and most animals develop only the Aß or tau pathology. We herein describe the Aß and hp-tau pathology in the brains of aged pinniped species (seal, sea lion, and walrus). Molecular analyses revealed that the sequence of pinniped Aß was identical to that of human Aß. Histopathological examinations detected argyrophilic plaques composed of Aß associated with dystrophic neurites in the cerebral cortex of aged pinnipeds. Astrogliosis and microglial infiltration were identified around Aß plaques. Aß deposits were observed in the blood vessel walls of the meninges and cerebrum. Pinniped tau protein was physiologically subjected to alternative splicing at exons 2, 3, and 10, and presented as five isoforms: two 3-repeat tau isoforms (1N3R, 2N3R) and three 4-repeat tau isoforms (0N4R, 1N4R, 2N4R); 0N3R tau isoform was absent. Histopathological examinations revealed argyrophilic fibrillar aggregates composed of hp-tau in the neuronal somata and neurites of aged pinniped brains. Few hp-tau aggregates were found in oligodendrocytes and microglia. Biochemically, hp-tau of the 3-repeat and 4-repeat isoforms was detected in brain sarkosyl-insoluble fractions. Aß and hp-tau both predominantly accumulated in the neocortex, particularly the frontal cortex. Furthermore, the activation of GSK-3ß was detected within cells containing hp-tau aggregates, and activated GSK-3ß was strongly expressed in cases with severe hp-tau pathologies. The present results suggest that, in association with Aß deposition, the activation of GSK-3ß contributes to hp-tau accumulation in pinniped brains. Here, we report that pinniped species naturally accumulate Aß and tau with aging, similar to the human AD pathology.

Multi-species and multi-tissue methylation clocks for age estimation in toothed whales and dolphins.

The development of a precise blood or skin tissue DNA Epigenetic Aging Clock for Odontocete (OEAC) would solve current age estimation inaccuracies for wild odontocetes. Therefore, we determined genome-wide DNA methylation profiles using a custom array (HorvathMammalMethyl40) across skin and blood samples (n = 446) from known age animals representing nine odontocete species within 4 phylogenetic families to identify age associated CG dinucleotides (CpGs). The top CpGs were used to create a cross-validated OEAC clock which was highly correlated for individuals (r = 0.94) and for unique species (median r = 0.93). Finally, we applied the OEAC for estimating the age and sex of 22 wild Norwegian killer whales. DNA methylation patterns of age associated CpGs are highly conserved across odontocetes. These similarities allowed us to develop an odontocete epigenetic aging clock (OEAC) which can be used for species conservation efforts by provide a mechanism for estimating the age of free ranging odontocetes from either blood or skin samples.

Detection of serum antibodies to Brucella in Russian aquatic mammals.

A serologic survey of Brucella infection was performed in Caspian seals (Pusa caspica, n=71), Baikal seals (P. sibirica, n=7), ringed seals (P. hispida hispida, n=6), and beluga whales (Delphinapterus leucas, n=4) inhabiting Russian waters, by enzyme-linked immunosorbent assay (ELISA) using Brucella abortus and B. canis as antigens. The sera of 4 Caspian seals (4%) tested positive for B. abortus. The same sera samples demonstrated weaker yet detectable affinity for B. canis antigens. Several discrete bands against B. abortus and B. canis antigens were detected on Western blot analysis of the ELISA-positive seal sera; the bands against B. canis were weaker than those against B. abortus. The sera of 3 beluga whales (75%) were positive for B. abortus antigens but showed no binding to B. canis antigens in the ELISA. The positive whale sera showed a strong band appearance only against B. abortus antigens in the Western blot analysis. Many detected bands were discrete, while some of them had a smeared appearance. The present results indicate that Brucella infection occurred in Caspian seals and beluga whales inhabiting Russian waters, and that the Brucella strains infecting the seals and the whales were antigenetically distinct.

Natriuretic peptides in cetaceans: identification, molecular characterization and changes in plasma concentration after landing.

Dolphins are aquatic animals free from gravity, and this may have imposed significant changes in their cardiovascular status and its hormonal regulation compared with terrestrial animals. This study molecularly characterized two major cardiovascular hormones, atrial and B-type natriuretic peptides (ANP and BNP) and measured their changes in dolphin plasma concentrations in relation to the cardiovascular status of the animal. We initially identified ANP and BNP in three species of dolphins (Lagenorhynchus obliquidens, Phocoenoides dalli and Tursiops truncatus). ANP precursors are highly conserved in most mammals, but dolphin BNP precursors were more variable. In molecular phylogenetic analyses, dolphin ANP and BNP precursors grouped with those of artiodactyls, particularly to the camel peptides. The chromatographic characterization of tissue and plasma molecular forms using specific radioimmunoassays showed that the predominant ANP and BNP in the atrium are prohormone and mature peptide, respectively, whereas mature ANP and BNP are circulating in the dolphin blood. A mass spectrometric analysis showed that atrial BNP consists of 26 amino acids, rather than the 32-amino-acid form detected in other mammals. Finally, changes in plasma ANP and BNP concentrations were examined in captive bottlenose dolphins (Tursiops truncatus) after their pool was drained. Plasma ANP and BNP concentrations did not change after landing, unlike terrestrial mammals. Plasma angiotensin II and cortisol concentrations did not change either, showing minor stress after landing. Since landed dolphins show a different cardiovascular status on land than terrestrial mammals, plasma ANP and BNP concentrations seem to reflect the cardiovascular status characteristic of dolphins.

Circulating gonadotropins and testicular hormones during sexual maturation and annual changes in male bottlenose dolphins (Tursiops truncatus).

To reveal the reproductive biology in male bottlenose dolphins (Tursiops truncatus), circulating gonadotropins (follicle stimulating hormone [FSH] and luteinizing hormone [LH]) and testicular hormones (testosterone and inhibin) were monitored for 8-12 years in 2 captive bottlenose dolphins (Mars and Regulus). During the study period, Mars was undergoing sexual maturation, whereas Regulus was already mature at the beginning of the study. Assuming that Mars had reached sexual maturity when the significant increase in circulating testosterone levels was observed, serum concentration of inhibin was higher in the sexually immature stage than in the mature stage, whereas the serum concentration of FSH was higher in the sexually mature stage than in the immature stage. No difference was observed in the LH levels between pre- and post-sexual maturation. There was a significant increase in serum concentration of testosterone during spring in both animals. These results suggest that the mechanism responsible for regulating FSH secretion by inhibin functions during the sexually immature stage in this species.

Microanatomy of the terminal air spaces of Baird's beaked whale (Berardius bairdii) lungs.

The terminal airways and microvasculature of five adult Baird's beaked whales (Berardius bairdii) lungs have been examined by means of light and scanning electron microscopy of corrosion casts. The respiratory system of the Baird's beaked whale has various anatomical features which allow them to attain great depths and remain submerged for long periods. The whale lung has components including hyaline cartilage and smooth muscle throughout, reaching as far as the peripheral bronchi, sphincters surrounding the terminal bronchioles, the thick alveolar septa with a connective tissue core and a bi-layer capillary bed, and a distinctive venous plexus of the pulmonary veins. The well-developed venous plexuses of the pulmonary vein are found in the interlobular connective tissue, and around the airways and pulmonary arteries with close apposition. The hyaline cartilage throughout the airways may increase the effective dead air space that accommodates most of the air forced from the collapsed alveoli during a dive. The sphincter might serve as a cock for regulating buoyancy and for trapping air in the alveoli to prevent their complete collapse and a sucking in of alveolar tissue as the dive becomes deeper. The venous plexuses might be for pooling the large volume of blood in the lung to conserve oxygen for deep and prolonged diving.

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.

Circulating testosterone and inhibin levels at different ages in the male beluga (Delphinapterus leucas).

This study is the first report on circulating testosterone and inhibin levels in a species of whales, the beluga. Circulating testosterone and immunoreactive (ir-) inhibin levels in two captive male belugas ("Nack", originally from Canada and "Duke", from the Okhotsk Sea) were measured every month for 9 years between 1995 and 2003. Assuming that clearly increased testosterone levels in the circulation indicates that the belugas had reached sexual maturity, at the ages of 10 ("Nack") and 11 years old ("Duke"). Their testosterone levels before the significant increase (pre-pubertal) were 0.42 ± 0.07 ng/ml (n=18) and 0.35 ± 0.10 ng/ml (n=18) and, those of after the increase (maturity) were 1.65 ± 0.14 ng/m l (n=74) and 2.06 ± 0.14 ng/ml (n=74). Circulating ir-inhibin levels before sexual maturity were 0.78 ± 0.04 ng/ml (n=18) and 0.64 ± 0.04 ng/ml (n=15) and, after sexual maturity were 0.52 ± 0.02 ng/ml (n=56) and 0.43 ± 0.02 ng/ml (n=67). Seasonal changes were observed in the testosterone levels after sexual maturity and the levels increased during March and April in Canadian origin "Nack", and peaked in February in Okhotsk origin "Duke". Circulating ir-inhibin level gradually decreased as they aged. A negative correlation between the circulating testosterone and ir-inhibin was observed. No seasonal changes were observed in the ir-inhibin levels after sexual maturity. These data will surely correspond to clarification of endocrinology and the successful reproduction of the beluga.

Study on reproduction of captive marine mammals.

The reproductive endocrinological characteristics of beluga, killer whale, spotted seal and bottlenose dolphin were evaluated and used in conjunction with applied reproductive research to enhance captive breeding programs. Results from 8 y of biweekly serum progesterone determination in a female beluga indicated that sexual maturity occured at approximately age 13, two to seven estrous cycles, lasting 37 +/- 3.9 days, per yr began in April-May every year. Rectal temperature was positively correlated with serum progesterone levels and negatively associated with behavioral estrus. In five cases of pregnancy of two female killer whale, positive relationship was found between serum progesterone concentration and temperature during the first period of 18 month-gestation. In the normal parturitions (n=4), rectal temperature decreased 0.8 C lower than average rectal temperature during pregnancy. Sexual maturity of female killer whales occurred at age nine. Yearly contraception in the mono-estrus captive spotted seals (n=10) using a single dose of the progestagen (proligestone(TM); 5 or 10 mg/kg s.c.) was achieved in 94% (33/35) of the attempts over 5 yr when the hormone was administered two months prior to the breeding season. Artificial insemination trials (n=4) were conducted in female bottlenose dolphin (n=3) using fresh and frozen-thawed semen. Estrus synchronization using regumate (27 days) resulted in ovulation occurring 19 to 24 days post withdrawal. Conception was confirmed in 75% of the attempts, with two females successfully delivering calves.

Cetacean Toll-like receptor 4 and myeloid differentiation factor 2, and possible cetacean-specific responses against Gram-negative bacteria.

Toll-like receptor 4 (TLR4) and myeloid differentiation factor 2 (MD-2) are essential for recognizing the lipopolysaccharides (LPS) of Gram-negative bacteria. We determined the sequences of cDNAs encoding TLR4 and MD-2 from cetaceans and generated three-dimensional (3D) models for a better understanding of their modes of interaction and LPS recognition. The 3D reconstructions showed that cetacean TLR4 and MD-2 formed a horseshoe-like structure comprised of parallel ß-strands and a ß-cup structure consisting of two anti-parallel ß-sheets, respectively. The (TLR4-MD-2)(2) duplex-heterodimer was shown to form a symmetrical structure. Comparison with the interfaces of the complexes in other mammals revealed that cetacean TLR4s have some amino acid residue substitutions involved in duplex-heterodimer formation and in species variation for LPS recognition. These substitutions in the changed amino acid residues may alter the interaction among TLR4, MD-2, and LPS and modify the TLR4/MD-2 immunological responses.

Host-virus specificity of morbilliviruses predicted by structural modeling of the marine mammal SLAM, a receptor.

Signaling lymphocyte activation molecule (SLAM) is thought to be a major cellular receptor for high-host specificity morbilliviruses, which cause devastating and highly infectious diseases in mammals. We determined the sequences of SLAM cDNA from five species of marine mammal, including two cetaceans, two pinnipeds and one sirenian, and generated three-dimensional models to understand the receptor-virus interaction. Twenty-one amino acid residues in the immunoglobulin-like V domains of the SLAMs were shown to bind the viral protein. Notably, the sequences from pinnipeds and dogs were highly homologous, which is consistent with the fact that canine distemper virus was previously shown to cause a mass die-off of seals. Among these twenty-one residues, eight (63, 66, 68, 72, 84, 119, 121 and 130) were shared by animal groups susceptible to a particular morbillivirus species. This set of residues appears to determine host-virus specificity and may be useful for risk estimation for morbilliviruses.

Basal body temperature method for detecting ovarian cycle in the Captive Beluga (Delphinapterus leucas).

The relationship between basal body temperature and circulating progesterone levels were investigated in a female beluga. Body temperature and serum concentrations of progesterone were measured daily and at 2-4 week intervals respectively, in a female beluga that was in captivity for 7 years between 1996 and 2003. The beluga first ovulated in April, 2000 (13 years old). Thereafter, serum concentrations of progesterone showed cyclic changes, indicating that the ovulatory cycle had started. Serum concentrations of progesterone ranged from 0.1 ng/ml to 15.7 ng/ml. Body temperature also showed cyclic changes during the estrous cycle. Body temperature ranged from 34.9 to 35.9 C, and tended to reach the peak during the high progesterone phase. Mating behavior was observed during the low body temperature phase. The changes in body temperature positively correlated with the circulating progesterone levels. The length of the estrous cycle was 36.7 +/- 3.9 (mean +/- SEM) days based on the intervals between the days of mating behavior. This is the first report demonstrating that body temperature clearly changes during the estrous cycle in a captive female beluga. The present finding suggests that measurement of body temperature is a useful method for detecting the ovarian cycle of the beluga in captivity.

Body temperature and circulating progesterone levels before and after parturition in killer whales (Orcinus orca).

Killer whales were used to evaluate peripartum changes and interactions between body temperature and circulating progesterone concentration. Daily body temperature was measured between 0800 h and 0830 h before activity. Body temperature clearly increased during the first phase of pregnancy, and this was followed by a gradual decline until full term. The initial increase was paralleled by circulating levels of progesterone. A decrease in body temperature was first detected on day 5 (p < 0.01) before parturition, and body temperature decreased significantly every 2 days until delivery. Five days before delivery, body temperature was 0.3 C lower than the mean value during the pregnancy period, and the decrease was more marked on day 1 before delivery (0.8 C). Serum progesterone levels during pregnancy showed a general pattern of initial rapid elevation (increasing phase), followed by a gradual decline (decreasing phase) throughout the remainder of pregnancy. However, statistically significant correlations between body temperature and progesterone pattern were only found only during the increasing phase. These results suggest that monitoring body temperature variation can be considered valuable for predicting impending parturition in killer whales.

Diurnal and annual changes in serum cortisol concentrations in Indo-Pacific bottlenose dolphins Tursiops aduncus and killer whales Orcinus orca.

Until present, fundamental studies on cortisol secretory patterns have not been conducted in cetaceans. The objectives of this study were: (1) to examine diurnal changes in serum cortisol concentrations in Indo-Pacific bottlenose dolphins Tursiops aduncus and killer whales Orcinus orca, (2) to investigate annual cortisol changes in killer whales, and (3) to investigate the relationship between cortisol and sex steroids (testosterone and progesterone) concentrations in killer whales. Diurnal changes in serum cortisol concentrations were investigated at various intervals in the two species. In Indo-Pacific bottlenose dolphins, serum cortisol levels exhibited the same episodic fluctuations for 24 h as did diurnal terrestrial mammals: cortisol levels were lower at 18:00 h and higher in the early morning. In killer whales, cortisol concentrations continued to decrease until 18:00 h, after which they fluctuated, and then increased in the next morning. Annual changes in cortisol levels were investigated by collecting blood samples every two weeks from two male killer whales and a pregnant female one twice per day (during 09:00-10:00 and 16:00-17:00 h) throughout a one-year period. Regarding sera collected during 09:00-10:00 h from the female, cortisol concentrations showed cyclic changes having about 4-month intervals. In males, cortisol showed higher concentrations in winter and lower concentrations during the summer season. There was a negative correlation between cortisol and progesterone levels in the female and a negative correlation was also observed between cortisol and testosterone in male no. 2. In the female and male no. 1, cortisol levels during 09:00-10:00 h were significantly higher than those during 16:00-17:00 h, and their data are considered to support observations regarding diurnal changes in cortisol levels in the two cetacean species.

Serological evidence of transmission of human influenza A and B viruses to Caspian seals (Phoca caspica).

Seroepidemiological surveillance of influenza in Caspian seals (Phoca caspica) was conducted. Antibodies to influenza A virus were detected in 54% (7/13), 57% (4/7), 40% (6/15) and 26% (11/42) of the serum samples collected in 1993, 1997, 1998 and 2000 by enzyme-linked immunosorbent assay (ELISA). In an hemagglutination-inhibition (HI) test using H1-H15 reference influenza A viruses as antigens, more than half of the examined ELISA-positive sera reacted with an H3N2 prototype strain A/Aichi/2/68. These sera were then examined by HI test with a series of naturally occurring antigenic variants of human H3N2 virus, and H3 viruses of swine, duck, and equine origin. The sera reacted strongly with the A/Bangkok/1/79 (H3N2) strain, which was prevalent in humans in 1979-1981. The present results indicate that human A/Bangkok/1/79-like virus was transmitted to Caspian seals probably in the early 1980s, and was circulated in the population. Antibodies to influenza B virus were detected by ELISA in 14% (1/7) and 10% (4/42) serum samples collected from Caspian seals in 1997 and 2000, respectively. Our findings indicate that seal might be a reservoir of both influenza A and B viruses originated from humans.