Abstract Juvenile salmon sharks beach yearly along the California coast during late summer and early fall. To determine the epidemiological condition of beached animals, frozen and formalin fixed beached specimens were collected from various Fish and Game facilities for examination and freshly beached specimens were also examined when possible. Histology revealed meningoencephalitis with intralesional bacteria in almost all of the specimens, including the freshly beached sharks, suggesting that this infection plays a role in this unusual behavior. The pathogenic bacteria was cultured from four separate shark specimens, and each isolate was characterized serologically and biochemically as belonging to the genus Carnobacteria. In order to identify the species, we sequenced -540 bp of the 16s ribosomal DNA and a similarly sized portion of the large ribosomal DNA intergenic spacer (ITS or ISR). The 16s sequences obtained from all samples were identical and 99% similar to C. piscicola. The ISR sequences obtained from all samples were also identical to each other, but were only 92% homologous to C. piscicola, and did not match closely with any other sequence in GenBank. This is the first report of Carnobacterium infection in any shark species, though the identity of the pathogenic bacteria remains uncertain. Introduction During the summer and fall of every year, young salmon sharks (£ Im in length) are found beached along the coast of California. To determine the cause of this unusual behavior, eleven beached salmon sharks representing the past three years' beachings were collected and analyzed histologically. Bacterial meningitis was noted in almost all of the brains by pathologist Dr. Corrine Davis at the Research Animal Facility (RAF) of the Stanford University Comparative Medicine Department. Bacteria were successfully cultured by microbiologist Dr. Barry Lifland at RAF from the brain tissue of four freshly beached specimens. Standard microbiological tests were performed to determine the type of bacteria that appeared to be infecting these sharks, and the field was narrowed down to the generus of Carnobacteria. Species of Carnobacteria are found frequently in the intestine and gills of both fresh and salt water fishes, in aquaculture and in wild populations (eg Ringo et al., 2000; Ringo et al., 2001; Joborn, 1999). The bacteria of this genus are generally considered to be part of the normal microfloral community in the gut and on the gill surfaces, and pathenogenesis of the bacteria of this genus is rarely considered. In fact, species of Carnobacteria are frequently found to produce bacteriocins, proteins that kill or inhibit the growth of other bacteria, and this propensity has led to much interest in the use of Carnobacteria species as a probiotic in aquaculture (Robertson, 2000). When fish are physiologically stressed, however, they become more prone to infection (Herman et al., 1985), and Carnobacteria can become septicemic and damage the kidneys, liver, and brain (Baya et al., 1991). Hui et al. (1984) cultured Carnobacteria sp. (then still in the Lactobacillus genus) from diseased rainbow trout, cutthroat trout, and Chinook salmon, all from fish that had been stressed by handling or by spawning activity. Carnobacteria has been shown to cause mortalities in stressed cultured fish challenged with the bacteria (Baya et al., 1991). Carnobacteria have not been reported as normal flora in sharks, and this is the first report of Carnobacteria pathogenesis in any shark species. Identifying the bacteria at the species level would aid greatly in understanding the epidemiology of this infection. Once the bacteria is identified, the route of infection may be pinpointed as environmental, dietary, or congenital in future research. Methods Bacteria were isolated from four infected salmon shark brains and were cultured by Dr. Barry Lifland of the Stanford University Comparative Medicine Department. Liquid cultures were prepared for PCR analysis. Primers were ordered from Integrated DNA Technologies (IDT). Carnol6sF (5’- ATACATGCAAGTCGAACGC-3’) and Carnol6sR (5’¬ ATAAATCCGGACAACGCTTGCCA-3’) were custom designed to amplify a -500 bp region of the Carnobacterium 16s ribosomal DNA gene for analysis. CarnolTS-F (5’- TGGTTCGAGTCCATTTAGGCCCA-3’) and CarnolTS-R (5’¬ CTTACAGCTCCCCAAAGCATAT-3’) were custom designed to amplify a region of the large intergenic spacer region determined by Kabadjova (2002) to differentiate between C. piscicola, mobile, divergens, and gallinarum. PCR reactions were carried out in 25 uL volumes containing 18 uL ddH2O, 2.5 uL PCR buffer, 2.5 uL 0.8 umol dNTP, 0.5 uL of each primer, 0.06 uL of AmpliTag (WHO), and 1 uL of bacterial liquid culture. Denaturation was at 94 °C for 1.5 minutes. Successful annealing temperatures for Carno 16sF and Carno l6sR ranged from 53.8 °C to 59.7 °C, maintained for 30s. Extension was performed at 72 °C for 1 minute, and the cycle was carried out 33x. For ITS amplification, the cycle was 94 °C for 30 seconds, 59.5 °C for 30 seconds, and extension at 72 °C for 1 minute, carried out 33x. Completed PCR reactions were screened on 1.5% agarose gels for successful amplification. To remove ssDNA and primers from the PCR reactions and make the amplified DNA inert, SAP/EXO reactions were performed in 7 uL volumes containing 0.5 uL of Shrimp Alkaline Phosphatase (SAP) enzyme, 0.5 uL of Exonuclease, 0.25 uL SAP buffer, and 5 uL of PCR template buffer per reaction. The reaction was carried out at 37 °C for 30 minutes and then the enzymes were denatured at 80 °C for 15 minutes. Sequences of 16s and ISR were determined by the dideoxynucleotide chain termination method. Both forward and reverse sequencing reactions were carried for l6s and ITS sequencing in 10 uL volumes with 6 uL ddH2O, 1.5 uL 5x buffer, 1 uL Big Dye, 0.5 uL of either forward or reverse primer, and 1 uL of SAP/EXÖ template. The product DNA was precipitated with 40 uL of 75% isopropanol per reaction. The reaction tubes were centrifuged for 1 hour at - 3600 rpm, and were then spun upside down for 1 minute at 700 rpm to remove the isopropanol. The pelleted DNA of was then resuspended in 20 uL of Hi Dye, denatured for 2 minutes at 96 °C, and inserted into an ABI 3100 Genetic Analyzer. Forward and reverse sequences were edited by eye and the consensus sequences were sent to GenBank to garner initial similarities. The consensus sequences were aligned with known Carnobacterium species 16s and ISR sequences. Using ClustalW, PAUP was used to create neighbor-joining phylogenetic trees comparing the amplified sequences to sequences from known Carnobacterium spp. Results PCR with primers Carnol6sF and Carno l6sR amplified a region of-541 base pairs that was identical to all samples, and nearly identical to the 16s rDNA of C. piscicola and to a recently examined species from the Arctic, Carnobacterium sp Arctic P-2 (Lee et al., 2004 unpublished data) (Fig. 1). The sole difference from C. piscicola is a single base pair insertion in the C. piscicola sequence that is absent in the sequence from our bacteria. The sole difference from the Arctic species is a single base change. The 16s isolate sequence clusters with C. piscicola and the Arctic species (Fig. 2). PCR with CarnolTS-F and CarnolTS-R yielded a piece of-540 base pairs. All of the sequences obtained from bacterial culture were 100% homologous to each other. The consensus sequence was not, however, identical to any Carnobacterium spp. in GenBank (Fig. 3). The isolate sequence clustered with C. piscicola, but is only 92% homologous to C. piscicola, and is no more closely related to any other Carnobacterium species in GenBank (Fig. 4). Discussion The 16s and ITS sequence data indicate that the bacteria isolated from the four individual sharks are identical, though the sharks were from separate beaching events geographically and temporally. Of these four, the southernmost beaching was at Sunset Beach in Orange County, and the northernmost was Bodega Bay in Sonoma County, CA. One shark had beached in August 2002, while the other three were from 2003. Since 16s sequences from species within the Carnobacteria genus generally share 97-98% or more homology (Genbank comparisons), the 16s sequence from our isolate, which is more than 99% homologous to C. piscicola and averages about 98% homology to other Carnobacteria species, confirms the biochemical and serological findings that place this isolate within the Carnobacteria. The ITS data indicate that the bacterial isolate is significantly different, however, from any currently known species of Carnobacteria. Interspecific ITS variation within the Carnobacteria is approximately 4- 7% (GenBank comparisons). Our isolate is 8% different from C. piscicola, its closest matching relative. Our isolate may prove to be the recently described arctic species C. sp. Arctic P-2, the large intergenetic spacer region of which has not yet been sequenced, or it may be an entirely new species within the Carnobacteria. It could be that the young sharks, like many wild and cultured fish, carry Carnobacteria naturally in their gut or on their gill surfaces. Though this particular isolate has not yet been described from other fish species, colonization of the gut may occur via the salmon shark diet, which in the adults consists primarily of salmonid fish known to carry Carnobacteria species. If the sharks harbor this Carnobacteria isolate naturally and then encounter stressful conditions along northern California, the bacteria may proliferate and infect the brain, causing disorientation and beaching. The infections may be peculiar to salmon shark juveniles due to some unique behavior or aspect of the juvenile life history, such as coastal migration, not shared by the adults or other species. Alternatively, since salmon sharks are ovoviviparous, the young sharks could contract the bacteria at birth, if their mothers are infected. Adult salmon sharks may be affected with the bacterial meningitis, and may simply beach in areas that aren’t heavily accessed by the public, since these sharks inhabit the northern Pacific where coastal communities are less frequent. Or perhaps the pelagic adult life prevents sick individuals from beaching on shore where they would be found. Why other sharks are unaffected by this bacterial meningoencephalitis is not clear. If exposure to the bacteria comes through the diet, then other shark species that prey on salmonid fishes, herring, or any other piscine species known to harbor Carnobacteria, should be affected. Yet no species other than salmon sharks have been observed to beach as regularly or as predictably as salmon sharks do. Conclusion An unusual Carnobacterium spp. has been isolated from the brains of beached salmon sharks suffering from acute meningoencephalitis with intralesional bacteria. The isolates share 100% homology with each other at both the 16s and large ITS regions. The isolates do not match with any known Carnobacteria species known to Genbank, but cluster most closely to C. piscicola, and an isolate from the Arctic, C. spp P-2. The epidemiology of this disease will be understood with further investigation into the identity and natural habitat of the isolate, and the lifestyle of juvenile and adult salmon sharks. Acknowledgements I greatly appreciate the support, expertise, and guidance of Dr. Corrine Davis, Dr. Barry Lifland, Dr. Stephen Palumbi, and the graduate students of the Palumbi Lab. Fig. 1. Alignment of the isolate consensus 16s rDNA sequence with other Carnobacteria sequences from GenBank. Fig. 2. Neighbor joining tree of the 16s rDNA sequences. Each bacteria is labeled with its GenBank accession number. Fig. 3. Alignment of isolate consensus large ISR sequence with other Carnobacteria sequences from GenBank. Fig. 4. Neighbor joining tree of ISR sequences. Each bacteria is labeled with its Gen Bank accession number. —8 —8 S .. .. .. .. . .. .. .. . 885 t- o aa a- - - 62 0 - oöd 88 55 5 - . . - * . odöoo 8 :LOVe SEE 8 aaaa- . -9 ::: aaa- 3ö o . 88888888 8. 2 0 hööoo 88 saaaa- 55 88 aaa- 28 8880 888 . 5 2 5 . aaaaa- oöo — 88 88 39 O oööo 88 . aaaaaa- 8888 8888 3888 88 ooooo . 3 . aBogsu ööo 9 38 S ACGG saaaaaa- 38 a- a- - oöo - a. 3 3. - 90 59. 3 - - a oööoo a- Fig 2. Consensus C.piscicola AF184247 C. spp arctic AY573049 — C. gallinarum AJ387905 C.divergens X54270 — C.mobile AJ317963 — C.funditum A1535202 - 0.01 substitutions/site — C.viridans AF 425608 8 —8 888 . 8888 kaa- HOOOO 88 88 88 88 5888 a 5 Hööoo 888 Höööö 8 388 2. - a- Höööö 8 — 5 —8 8 8 - Höööé 2.. EE 8 . a- Höööo .. .. 8 a- Höööé o - 0 aaa- * . . oo 56 5 O - a. hooo C — C. divergens AF374294 005 substionsfste — C. gallinarum AF374287 - Consensus - C. mobile AF374291 Fig. 4. 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