from august 2004 through april 2007 , 10 live striped dolphins , stenella coeruleoalba ( 3 female adults , 2 female juveniles , 1 female calf , 4 juvenile males ) , were found stranded in populated areas at the pacific shoreline of the puntarenas province of costa rica . all animals had swimming problems compatible with neurologic disorders and died within 48 hours of being found . corpses were kept on ice and transported to the pathology unit , veterinary school , national university , costa rica , for sampling ; necropsy ; and histopathologic , immunohistochemical , and serologic studies . with exception of 1 dwarf sperm whale , kogia sima , these 10 dolphins were the only cetaceans we were able to examine during this 32-month period . because marine brucella spp . have been reported to cause intracerebral infections ( 3 ) , we decided to perform immunohistochemical and serologic tests . abortus lipopolysaccharide ( lps ) was produced and isolated as described elsewhere ( 5 ) . antibodies against dolphin steno bredanensis igg were produced in rabbits , purified according to described protocols ( 6 ) . both rabbit antibodies were linked to fluorescein isothiocyanate and peroxidase and were assayed by using immunofluorescent and western blot techniques , respectively ( 5,7 ) . rose bengal agglutination test , immunofluorescent assays , and competitive elisa were designed and used as described ( 8,9 ) . blood was collected from the live dolphins in situ , serum was obtained , and physical and chemical examinations were performed , followed after death by necropsies and gross pathologic and histopathologic studies . tissues were fixed in formalin , embedded in paraffin wax , sectioned , and stained with hematoxylin and eosin ( 10 ) . organs and tissues of 5 adult females and 1 juvenile male were analyzed for bacteria ( 11 ) ; however , no samples for bacteriologic studies were available from the other dolphins that were stranded before july 2006 . fresh tissue impressions or pellets from supernatants of macerated tissues were fixed with cold 3% paraformaldehyde for 15 min on ice and subjected to immunofluorescence for detection of brucella spp . genotyping of brucella isolates was performed by pcr , using 5-ggctgatctcgcaaagat-3 and 5-ccaggtccttggcttccttgag-3 primers ( invitrogen corporation , carlsbad , ca , usa ) for the amplification of ribosomal protein l12 and pcr - styi restriction fragment length polymorphism of the omp2b locus , as described ( 11,12 ) . all animals displayed neurologic disorders characterized by the inability to maintain buoyancy and by opisthotonus , tremors , and seizures ( figure 1 , panel a ) . in all animals , parasitosis was evident and included gastric , intestinal , and pulmonary nematodes as well as subblubber and retroperitoneal cestode larvae . the pregnant female had milk in her mammary glands , a 66-cm fetus , and small placental abscesses ( figure 1 , panels b , c ) . hyperemic meninges , congested brain , and altered cerebrospinal fluid ( augmented in volume and cellularity ) were evident in all dolphins ( figure 1 , panels e , f ) . clinical , pathologic , and immunofluorescence findings in stranded striped dolphin , stenella coeruleoalba . a ) striped dolphin displaying swimming disorders being assisted by local persons ; b ) dolphin fetus within placenta ; c ) punctuated placental abscesses ( arrows ) ; d ) immunofluorescent brucellae in impressions of placenta tissues ; e ) congested and hyperemic brain and cerebellum ; f ) wright - giemsa  stained mononuclear cell infiltrate in cerebrospinal fluid ; g ) immunofluorescent green brucella spp . and brucella debris within phagocytic cells infiltrating cerebrospinal fluid ; the inset corresponds to an amplified phagocytic cell with fluorescent brucella spp . and debris . all dolphins had antibodies against brucella lps , as determined by immunofluorescence ( > 1:150 dilution ) , competitive elisa ( > 60% positivity ) , and western blot . with the exception of the female calf were demonstrated by immunofluorescence of cerebrospinal fluids and thereafter in tissue impressions of the brain , spinal cord , lymph nodes , spleen , liver , and kidneys . the pregnant animal had bacteria in the placenta ( figure 1 , panel d ) , umbilical cord , milk , allantoic and amniotic fluids , and fetal tissues . a large number of phagocytes infiltrating the cerebrospinal fluid of these 6 dolphins contained intracellular brucella organisms ( figure 1 , panel g ) . histopathologic examination of the central nervous system was performed on only the 6 dolphins from which brucella organisms were isolated ; tissues examined were spinal cord , medulla oblongata , cerebellum , pons , thalamic area , and the occipital and frontal cortices of the cerebrum . the most common and relevant histopathologic findings demonstrated meningoencephalitis ( figure 2 ) and were similar to those previously described ( 13 ) . the predominant feature was nonsuppurative meningitis , which was more severe in the spinal cord , medulla oblongata , and cerebellum and somewhat more moderate and mild in the cerebral cortices . mild encephalitis was evidenced by a perivascular mononuclear infiltrate in the white and gray matter of the cerebrum , cerebellum , and brainstem and by a periventricular encephalitis that was widespread surrounding the third ventricle with the same cellular infiltrate found in the meninges . a major loss of ependyma was evident ; a moderate to severe mononuclear choroiditis was also present . . little or no involvement of the neural tissue was noted in areas other than the periventricular zone . a ) mononuclear infiltrates in the meninges ( arrow ) surrounding the spinal cord ; b ) mononuclear infiltrate around vessels ( arrow ) of the cerebellum ; c ) mononuclear infiltrate ( arrow ) in the meninges around the brain ; d ) hyperemic vessels and mononuclear cell infiltrate in the meninges around the cerebellum ( arrow ) . organisms compatible with brucella ( nonmotile ; urease- , catalase- , and oxidase positive ; gram - negative bacteria not requiring co2 for growth ) were isolated in blood agar or selective media from different organs , brain , and fluids from the 6 dolphins and the fetus subjected to detailed analysis . standard bacteriologic , immunochemical , biochemical , and molecular typing indicated that the causative agent in all cases was b. ceti type i dolphin strain ( table ) . * rtd , routine test dilution of phages tbilisi ( tb ) , weybridge ( wb ) , izatnagar ( iz ) , and rough type wb derivative ( r / c ) . dye concentrations expressed in g / ml of culture medium with 10% co2 or , within parenthesis , raw incubation without co2 . all reference and isolated strains were sh2 negative , urease positive and amplified brucella spp l12 rrna by pcr . this report documents the presence of marine brucellosis along latin american shorelines . the dolphins neurologic lesions were similar to those described previously in an infected s. coeruleoalba dolphin ( 13 ) . this finding calls attention to possible increased susceptibility of this species to neurobrucellosis . indeed , from a total of 46,826 individuals , corresponding to 28 cetacean species counted in the costa rican littorals , striped dolphins ( inhabiting deep waters ) represent only 13% of the animals sighted ( 14 ) . from january 2004 through december 2006 , the number of cetaceans reportedly stranded along the pacific coast of costa rica was 31 ( 15 ) . of these , 14 were s. coeruleoalba ; the other 16 comprised 11 species of odontocetes . during the 32-month period , we were able to collect samples : 10 ( this study ) from s. coeruleoalba and 1 from k. sima . endemic and migrating groups of delphinus delphis and stenella attenuate are found more frequently along the costa rican shorelines ; however , few strandings of these species have been reported ( 14 ) . the relatively high number of stranded s. coeruleoalba dolphins along the costa rican shorelines is in agreement with records of strandings along the european littorals ( 11 ) . the isolation of b. ceti from milk , fetal tissues , and secretions of a pregnant dolphin , and a similar discovery in european littorals , suggests that b. ceti is able to display tropism for placental and fetal tissues and that the bacteria may be shed , as it is with brucella - infected livestock . moreover , the localization of the bacteria in particular organs suggests the possibility of transmission through sexual intercourse and may ensure the prevalence of both clinical and latent infections . in terms of zoonotic transmission , we noticed that people handled and touched all these infected dolphins , mainly as an attempt to return them to the ocean . other stranded dolphins have been transferred to privately owned swimming pools or to slaughter for use as a food source for humans and domestic animals . in this
ten striped dolphins , stenella coeruleoalba , stranded along the costa rican pacific coast , had meningoencephalitis and antibodies against brucella spp . 
 brucella ceti was isolated from cerebrospinal fluid of 6 dolphins and 1 fetus . 
 s. coeruleoalba constitutes a highly susceptible host and a potential reservoir for b. ceti transmission .