Evidence of Brucella infection in marine mammals in the North Atlantic Ocean.

Between 1983 and 1996 a total of 1386 samples of serum were taken from four species of seal and three species of whale in the waters west of Iceland, the area of pack-ice north-west of Jan Mayen, the northern coast of Norway and the Kola Peninsula, the waters west of Svalbard, and the Barents Sea; they were tested for the presence of anti-Brucella antibodies with an indirect ELISA (protein G conjugate). The positive sera were re-tested with classical brucellosis serological tests, such as the serum agglutination test, the EDTA-modified serum agglutination test, the Rose Bengal test, and the complement fixation test, as well as an anti-complement ELISA. Anti-Brucella antibodies were detected in all the species investigated, except for the bearded seal (Erignathus barbatus), with the following prevalences: hooded seals (Cystophora cristata) 35 per cent; harp seals (Phoca groenlandica) 2 per cent; ringed seals (Phoca hispida) 10 per cent; minke whales (Balaenoptera acutorostrata) 8 per cent; fin whales (Balaenoptera physalus) 11 per cent; and sei whales (Balaenoptera borealis) 14 per cent. An isolate belonging to the genus Brucella was obtained from the liver and spleen of one of the seropositive minke whales. The findings suggest that antibodies against the surface lipopolysaccharide of Brucella species are widely distributed among marine mammals in the North Atlantic Ocean.     

IS711和omp2作为布氏杆菌种属及种株间分子鉴别诊断标记的研究

布氏杆菌为世界性具重要公共卫生意义的人兽共患疫病原，分6个种。建立种间及种株间安全敏感、经济有效的快速鉴别诊断方法对布病防制及分子流行病学研究具有重要意义。布氏杆菌IS711和omp2基因具有种属特异性，可用于布氏杆菌的PCR分子诊断。其中IS711为转座因子，在不同种布菌种存在插入位置的多态性，外膜蛋白OMP2编码基因则存在反向重复序列及种株间的多态性。为此，分别采用复式-PCR、PCR和限制性酶切片段长多态性（RFLP）分析，对分属于B．mclitcnsis、B．suis和B．abortus的不同种布氏杆菌的不同种株，M5、M16、S2、S6和S19进行分子鉴别诊断。结果显示，根据IS711基因特定PCR扩增片段长多态性，可进行布氏杆菌种间的快速鉴别；而omp2编码基因PCR扩增片段PsrⅠ、KpnⅠ、NcoⅠ和Eco47 Ⅲ等4种限制酶片段长多态性，则可成为布氏杆菌菌株间特异的分子鉴别诊断标记，甚至疫苗株M5和野毒株M16之间的分子诊断标记。     

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.     

A review of Brucella infection in marine mammals, with special emphasis on Brucella pinnipedialis in the hooded seal (Cystophora cristata)

ABSTRACT: Brucella spp. were isolated from marine mammals for the first time in 1994. Two novel species were later included in the genus; Brucella ceti and Brucella pinnipedialis, with cetaceans and seals as their preferred hosts, respectively. Brucella spp. have since been isolated from a variety of marine mammals. Pathological changes, including lesions of the reproductive organs and associated abortions, have only been registered in cetaceans. The zoonotic potential differs among the marine mammal Brucella strains. Many techniques, both classical typing and molecular microbiology, have been utilised for characterisation of the marine mammal Brucella spp. and the change from the band-based approaches to the sequence-based approaches has greatly increased our knowledge about these strains. Several clusters have been identified within the B. ceti and B. pinnipedialis species, and multiple studies have shown that the hooded seal isolates differ from other pinniped isolates. We describe how different molecular methods have contributed to species identification and differentiation of B. ceti and B. pinnipedialis, with special emphasis on the hooded seal isolates. We further discuss the potential role of B. pinnipedialis for the declining Northwest Atlantic hooded seal population.     

The muscles of mastication in the caspian seal (Phoca caspica)

The muscles of mastication and their related skull characters in the Caspian seal (Phoca caspica) were anatomically examined and compared with those of the Baikal (Phoca sibirica) and ringed (Phoca hispida) seals. A well-developed masseter muscle was observed in the Caspian seal, whereas the temporal muscle consisted of thin bundles. The skull of the Caspian seal possessed the same thin frontal bone and the dorso-ventrally developed zygomatic arch found in the Baikal seal that are required to install the enlarged eyeball into the orbit. The temporal bone was not robust, and the digastric muscle was well-developed in the ventral space of the auditory bulla. The present results suggest that the skull form of the Caspian seal has changed morphologically from its ringed seal-like ancestors, and suggest that the evolutionary strategy of the muscles of mastication in the Caspian seal is principally consistent with that of the Baikal seal.     

A potential novel Brucella species isolated from mandibular lymph nodes of red foxes in Austria

The wild red fox (Vulpes vulpes) is a known indicator species for natural foci of brucellosis. Here, we describe phenotypic and molecular characteristics of two atypical Brucella strains isolated from two foxes hunted 2008 in Eastern Austria. Both strains agglutinated with monospecific anti-Brucella A serum and were positive in ELISA with monoclonal antibodies directed against various Brucella lipopolysaccharide epitopes. However, negative nitrate reductase- and negative oxidase-reaction were atypical traits. Affiliation to the genus Brucella was confirmed by 16S rRNA gene sequencing and by detection of the Brucella specific insertion element IS711 and gene bcsp31 using real-time PCR. Both fox strains showed identical IS711 Southern blot profiles but were distinct from known brucellae. The number of IS711 copies detected was as high as found in B. ovis or marine mammal Brucella strains. Molecular analyses of the recA and omp2a/b genes suggest that both strains possibly represent a novel Brucella species.     

Anti-Brucella antibodies in seals at coastal locations of Hokkaido,Japan, with focus on life stages

The enzyme-linked immunosorbent assay (ELISA) was applied to detect antibodies against Brucella abortus in serum samples from four seal species at nine coastal locations of Hokkaido, Japan. These antibodies were detected in 27% (32/118) of Western Pacific harbor seals (Phoca vitulina stejnegeri) at Cape Erimo. The antibodies were observed in spotted seals (P. largha) in one out of six at Nemuro, in two out of three at Rebun Island, in one out of two at Bakkai, and in examined one at Soya. They were also found in respective examined one ribbon seal (Histriophoca fasciata) and one ringed seal (Pusa hispida) at Akkeshi. Harbor seals that tested positive were mostly yearlings (35%, 20/57) and juveniles (45%, 10/22), while only one pup (1/13) and one subadult (1/5) tested positive with low titers of the antibody; no antibodies were observed in adults (n=21). These results suggest that Brucella mainly infected harbor seals from the environment while weaning, and the bacteria were cleared during the early life stage of the seals. In spotted seals, however, antibodies were also detected in adults, suggesting that spotted seals could become infected with Brucella even as adults. It is also possible that a different, more persistent strain of Brucella may have infected the spotted seals.     

Brucella pinnipedialis hooded seal (Cystophora cristata) strain in the mouse model with concurrent exposure to PCB 153

Brucellosis, a worldwide zoonosis, is linked to reproductive problems in primary hosts. A high proportion of Brucella-positive hooded seals (Cystophora cristata) have been detected in the declined Northeast Atlantic stock. High concentrations of polychlorinated biphenyls (PCBs) have also been discovered in top predators in the Arctic, including the hooded seal, PCB 153 being most abundant. The aim of this study was to assess the pathogenicity of Brucella pinnipedialis hooded seal strain in the mouse model and to evaluate the outcome of Brucella spp. infection after exposure of mice to PCB 153. BALB/c mice were infected with B. pinnipedialis hooded seal strain or Brucella suis 1330, and half from each group was exposed to PCB 153 through the diet. B. pinnipedialis showed a reduced pathogenicity in the mouse model as compared to B. suis 1330. Exposure to PCB 153 affected neither the immunological parameters, nor the outcome of the infection. Altogether this indicates that it is unlikely that B. pinnipedialis contribute to the decline of hooded seals in the Northeast Atlantic.     

Aerobic bacterial isolations from harbor seals (Phoca vitulina) stranded in Washington: 1992-2003.

Bacterial cultures collected over 12 yr from stranded harbor seal (Phoca vitulina) pups and weanlings located in the North Puget Sound and San Juan Islands region of Washington were analyzed retrospectively to determine the most common pathogenic isolates and to describe their antimicrobial resistance patterns. Culture attempts (n = 58) from wounds, umbilici, ears, conjunctiva, nares, oral lesions, and feces yielded 134 pathogenic isolates that represented 17 genera. The majority of isolates were Gram-negative (n = 87; 65%) and of the tested isolates were most susceptible to amikacin (n = 76; 99%) and gentamicin (n = 76; 97%) and least susceptible to ampicillin (n = 76; 26%). Of the Gram-positive isolates tested (n = 29), all were susceptible to amoxicillin/clavulanic acid. The most frequent isolates were Escherichia coli (17%), beta-hemolytic Streptococcus spp. (15%), Enterococcus spp. (11%), and Pseudomonas aeruginosa (11%), with all four exhibiting resistance to more than 50% of the antimicrobials tested. The variety of organisms isolated, the variation in either Gram-negative or Gram-positive predominance, and the multiple drug resistance patterns observed suggest that when treating stranded harbor seals, culture and sensitivity testing are warranted and that antibiotic therapy should be based on results.     

Macroscopic anatomy of the heart of the ringed seal (Phoca hispida)

Anatomical properties of the ringed seal (Phoca hispida) heart and associated blood vessels reveal adaptations related to requirements for diving. Seven adult ringed seals were embalmed and dissected to document the gross anatomical features of the heart. Computed tomography images of the thoracic cavity were taken on one seal prior to dissection. The shape and position of the heart is different from the typical carnivore heart. The most notable difference is its dorsoventral flattened appearance with its right and left sides positioned, respectively, within the thoracic cavity. The long axis of the heart is positioned horizontally, parallel to the sternum. The right ventricle is spacious with thin walls which extend caudally to the apex of the heart such that the apex is comprised of both right and left ventricles. The cusps of the left atrioventricular valve of the ringed seal heart resemble an uninterrupted, circular curtain making it challenging to distinguish the divisions into parietal and septal cusps.     

Morbillivirus infections of seals during the 1988 epidemic in the Bay of Heligoland: III. Transmission studies of cell culture-propagated phocine distemper virus in harbour seals (Phoca vitulina) and a grey seal (Halichoerus grypus): clinical, virological and serological results

Eight harbour seals (Phoca vitulina), two of them seronegative, six seropositive against PDV and a seronegative grey seal (Halichoerus grypus) were exposed to a low doses of a cell culture-propagated phocine distemper virus isolate (PDV 2558/Han 88). An intranasal route of inoculation was chosen. Clinical signs, resembling those of 1988's seal disease and seroconversion were observed in both seronegative harbour seals. One of them succumbed to the infection. The virus was not transmitted to another susceptible harbour seal which served as in-contact animal. Virus could be recovered from leucocytes of the diseased seals. Viremia was also present in a seropositive harbour seal that developed mild clinical signs; other seropositive seals were protected from clinical disease. The grey seal showed seroconversion upon inoculation, but did not develop any signs of disease. The humoral immune response of the seals plainly discriminated between homologous (PDV) and heterologous (canine distemper virus, CDV) virus as shown by virus neutralization tests and an antibody-binding assay (PLA).     

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.     

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.     

Development of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar 3

One hundred twenty-nine Brucella field strains isolated from cattle in Cantabria, Spain, from March 1999 to February 2003, were analysed by using the AMOS-ERY PCR assay and by Southern blot hybridisation with a probe from insertion sequence IS711. Most of the field isolates produced only the ery band in the AMOS-ERY assay and showed a hybridisation pattern identical to that exhibited by reference strains of biovars 5, 6 and 9 of Brucella abortus, but different from strain Tulya, belonging to biovar 3 of B. abortus. However, typing of these strains by standard methods demonstrated that they belonged to biovar 3 of B. abortus. These results indicated that B. abortus biovar 3 was not genetically homogeneous and at least could be divided in two. In one class, that we called biovar 3a, would be the Tulya strain, while the local field strains would belong to biovar 3b. Cloning and nucleotide sequencing of a DNA fragment containing an IS711 copy exclusive of the B. abortus field strains from biovar 3b and reference strains from biovars 5, 6 and 9, revealed the existence of a 5.4 kb deletion close to an IS711 copy. Based on these data, we designed a new primer, which together with the IS711 AMOS primer produced a PCR fragment of 1.7 kb only from the isolates of biovars 3b, 5, 6 and 9 of B. abortus. No amplification products were produced with these primers from strains of the rest of species and biovars of Brucella and from bacteria phylogenetically close to Brucella analysed in this work. Addition of this primer to the AMOS-ERY PCR primer cocktail allows the positive distinction of B. abortus biovars 3b, 5, 6 and 9 from the rest of Brucella species and biovars.     

Seroconversion and abortion in cattle experimentally infected with Brucella sp. isolated from a Pacific harbor seal (Phoca vitulina richardsi).

Previously unrecognized Brucella species have been isolated from a number of marine mammals, including harbor seals (Phoca vitulina richardsi) in the Puget Sound area of the state of Washington. Because of the presence of dairy herds in proximity to the harbor seal populations, a study was conducted to determine the effects of the harbor seal Brucella isolate in experimentally inoculated cattle. Six pregnant cattle were exposed by intravenous injection (n = 3) or intraconjunctival inoculation (n = 3). Two pregnant cows were intravenously injected with saline and served as controls. All of the cows receiving the Brucella seroconverted on 1 or more tests commonly used for the detection of Brucella abortus infection. Two of the cattle receiving the intravenous inoculation aborted, and brucellae were demonstrated in the fetuses and dams immediately following abortion. The remaining 4 Brucella-inoculated animals and their fetuses were culture negative for the organism at 14 weeks postinoculation. Results of this study indicate the marine mammal Brucella is capable of producing seroconversion and abortion in cattle but is less pathogenic in that species than B. abortus.     

Serological survey of pre-weaned New Zealand fur seals (Arctocephalus forsteri) for brucellosis and leptospirosis

AIM: To conduct a longitudinal serological survey for evidence of Brucella spp and Leptospira spp infection of pre-weaned New Zealand fur seals in a colony on the Otago Peninsula. METHODS: Seal pups were repeatedly captured on a monthly basis from February through to July 2001. Pups were tagged at first capture and a blood sample was taken at each capture event. A total of 163 sera were collected from 118 seal pups. Where sufficient volume was collected, the sera were tested for leptospirosis using the microscopic agglutination test (MAT), and for brucellosis using the competitive enzyme-linked immunosorbent assay (ELISA) for Brucella abortus. RESULTS: None of 128 sera from 101 seals tested positive to the ELISA for B. abortus. All tests for Leptospira interrogans serovars Grippotyphosa, Copenhageni, Bratislava and Leptospira borgpetersenii serovar Ballum were negative at a cut-off of <1/100 dilution. Positive or suspicious titres were found to L. interrogans serovars Canicola and Pomona and L. borgpetersenii serovar Hardjo. The highest titres (12,800) were found to serovar Pomona. The titre to serovar Pomona in one seal rose from <1/50 in March to 12,800 in April and was <1/50 when re-sampled in July. The titre to serovar Pomona in another seal dropped from 12,800 in May to <1/50 in June. These seals also had titres to serovar Hardjo, which rose or fell in the same manner. All suspicious or positive titres occurred in late April and early May, when the pups were approximately 4-5 months old. In June and July, all seals tested were negative. CONCLUSIONS: There was no serological evidence of Brucella infection in the pre-weaned fur seals at the colony. Positive titres to serovars Pomona, Hardjo, or Canicola suggest that a Leptospira species was present at the colony, however isolation or visualisation of the organism is required to confirm this. Care should be exercised when handling New Zealand fur seals to prevent human infection or inadvertent transfer of leptospirosis to another marine mammal species.     

Fatal enterocolitis in harbour seals (Phoca vitulina) caused by infection with Eimeria phocae

Coccidiosis due to Eimeria phocae infection has been described in harbour seals (Phoca vitulina) from the western Atlantic population, but not in any detail in seals from the eastern Atlantic population. This paper describes fatal enterocolitis due to E phocae infection in three juvenile harbour seals at a rehabilitation centre in the Netherlands in July 2003. The clinical signs were lethargy, bloody faeces, and intermittent convulsions and muscle tremors just before they died; the nervous signs resembled those of nervous coccidiosis in calves. The main pathological finding was severe, diffuse, haemorrhagic enterocolitis; there were diffuse inflammatory changes in the lamina propria of the jejunal, ileal, caecal and colonic mucosa that were associated with the presence of the sexual stages and oocysts of a coccidian species identified as E phocae. A retrospective microscopical examination of intestinal tissues from 113 harbour seals that had died between 1999 and 2004 revealed one seal that was positive for E phocae.     

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.