Meningoencephalitis and Arthritis Associated with Brucella ceti in a Short-beaked Common Dolphin (Delphinus delphis)

Transcription

1 DOI: / Journal of Wildlife Diseases, 49(3), 2013, pp # Wildlife Disease Association 2013 Meningoencephalitis and Arthritis Associated with Brucella ceti in a Short-beaked Common Dolphin (Delphinus delphis) Nicholas J. Davison, 1,2,5 James E. F. Barnett, 1 Lorraine L. Perrett, 3 Claire E. Dawson, 2,3 Matthew W. Perkins, 4 Robert C. Deaville, 4 and Paul D. Jepson 4 1 Animal Health and Veterinary Laboratories Agency, Polwhele, Truro, Cornwall TR4 9AD, UK; 2 Scottish Marine Animal Stranding Scheme, SAC Veterinary Services, Drummondhill, Inverness, Scotland IV2 4JZ, UK; 3 Department of Bacteriology Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, UK; 4 Cetacean Strandings Investigation Programme (CSIP), Institute of Zoology, Zoological Society of London, Regents Park, London NW14RY, UK; 5 Corresponding author ( nick.davison@sac.co.uk) ABSTRACT: Brucella species infection in marine mammal species has been reported to have a global distribution. In 2007, the description of Brucella ceti was published and formally adopted for those isolates originating from cetaceans and pathologic lesions similar to those seen in terrestrial mammals infected with Brucella spp. have been associated with its isolation. Brucella ceti infection specific to the central nervous system has been described in two species of cetacean: striped dolphins (Stenella coeruleoalba) in Europe and Costa Rica and an Atlantic white-sided dolphin (Lagenorhynchus acutus) in the UK. We describe the first report, to our knowledge, of B. ceti associated meningitis and arthritis in a third species, the shortbeaked common dolphin (Delphinus delphis), in an animal that stranded in the UK. Key words: Animal Health and Veterinary Laboratory Agency, arthritis, Brucella ceti, Delphinus delphis, Institute of Zoology, isolation, meningitis, United Kingdom. In September 2009, a juvenile shortbeaked common dolphin (Delphinus delphis; reference: 22/M74/10/09) was found dead at Carbis Bay beach, Cornwall, UK (50u129N, 5u289W). The animal was taken to the Animal Health and Veterinary Laboratories Agency, Polwhele, Truro, for standardized, cetacean necropsy (Kuiken and Hartman, 1991). The mean of three standard blubber-thickness measurements was 10.6 mm, and the nutritive condition of the animal was moderate (Kuiken and Hartman, 1991). The carcass was 168 cm from the tip of the rostrum to the tail notch and weighed 50.5 kg. Numerous fresh rake (teeth) marks/scars with mm spacing were present on the rostrum, head (Fig. 1a), dorsal fin, thorax, ventral abdomen, tail stock, and tail fluke, and there were fresh abrasions over the tips of the rostrum. There were up to 2-cm-diameter discrete areas of hemorrhage/bruising in the blubber and subcutaneous fascia over the head and thorax. Subcutaneous hemorrhages were deep to the rostral abrasions. A 134-cm area of purulent, necrotic, subcutaneous tissue, rimmed by hemorrhage, was present on the right flank. The oropharynx and blowhole contained sand and teeth were loose in both arcades. Comminuted fractures of the rostral, mandible, and maxilla were present, and there was separation of the periosteum from the bone caudal to both fractures, with associated hemorrhage between the periosteum and maxillary bone. The left lung was markedly congested, the right lung less so. Occasional, 3 4-mm-diameter lesions were visible on the lung surfaces, consistent with Halocercus delphini infection. All compartments of the stomach were empty; four coalescing, 3 20-mm-diameter, superficial ulcers were present in the cardiac stomach mucosa, and a Pholeter gastrophilus associated lesion was found in the fundic stomach wall. The liver was congested. The atlanto-occipital joint contained approximately30mlofinspissated,caseous material (Fig. 1b). No obvious gross abnormalities were noted in either the brain or the cranial spinal cord, and no other abnormalities were seen in the remainder of the carcass. Routine bacterial cultures of the inspissated, caseous material of the atlantooccipital joint and the brain were inoculated directly onto sheep blood agar (Oxoid, Basingstoke, UK) and MacConkey agar 632

2 SHORT COMMUNICATIONS FIGURE 1. Short-beaked common dolphin (Delphinus delphis) found dead at Carbis Bay beach, Cornwall, UK. Postmortem examination confirmed Brucella ceti associated meningitis and arthritis. (a) Fresh rake (teeth) marks/scars with mm spacing on the rostrum and head. White card is 6 cm wide. (b) Atlanto-occipital joint containing large quantity of caseous pus. (c) Brain showing nonsuppurative meningoencephalitis associated with perivascular mononuclear cell infiltration within the meninges. Bar5100 mm. 633 (Oxoid), were incubated at 37 C in a capnophilic (10% CO2) atmosphere, and were examined daily for 7 days. Cultures on sheep blood agar produced a profuse, pure growth resembling Brucella after 3 days. No bacterial growth was recorded on Mac Conkey agar. The isolates were subcultured onto Farrell medium, and subsequent growths were Oxidase-positive, gram-negative, and modified Ziehl-Neelsen positive coccobacilli. Further subcultures were made on sheep blood agar and incubated aerobically at 37 C, revealing that the isolates were not CO2 dependent; they were tentatively identified as B. ceti. Isolates were confirmed initially as B. ceti by classic biotyping. The isolates produced lysis of phages BK2 (Berkeley), Wb (Weybridge), and Fi (Firenze) (Corbel et al., 1979; Table 1). Amplification of an IS711 element downstream of the bp26 gene by PCR (Cloeckaert et al., 2000) confirmed that the isolates possessed this unique feature specific to marine mammal strains of Brucella. Molecular characterization of the outer membrane protein (omp2) of the strain with a selection of restriction enzymes revealed the type to be N(K), found in short-beaked common dolphins and striped dolphins (Stenella coeruleoalba; Cloeckaert et al., 2001). Serum from the animal was analyzed by a competitive and by two indirect enzymelinked immunosorbent assays (celisa and ielisa) to detect Brucella antibodies. Positive/negative thresholds for these assays were set with some uncertainty but were based on those used for testing a wide range of terrestrial mammals from the UK for brucellosis (McGiven et al., 2003). The serum sample had detectable Brucella antibodies by all three ELISA tests (Table 1). Brain and atlanto-occipital joint were fixed in 10% neutral-buffered formalin and routinely processed and stained with hematoxylin and eosin before microscopic examination. The cerebrum showed chronic, nonsuppurative meningoencephalitis, characterized by patchy congestion of the

3 634 JOURNAL OF WILDLIFE DISEASES, VOL. 49, NO. 3, JULY 2013 TABLE 1. Phenotypic characteristics of Brucella ceti isolated from a short-beaked common dolphin (Delphinus delphis) compared with other Brucella species and immunologic results for the presence of Brucella antibodies in serum. a BF Th Monospecific sera Lysis by Phage at RTD Species Hydrolyzation of urea H2S produced CO2 Required 1/50,000 w/v A M Wb Tb BK2 Fi R/C Short-beaked common dolphin (Delphinus delphis) 22/M74/10/ CL NL CL Plq NL Brucella melitensis NL NL CL NL NL Brucella abortus CL CL CL CL NL Brucella suis CL NL CL PL NL Brucella ceti b + + (2) (+) (+) + (2) L b NLe L b NL/PL NL Brucella pinnipedialis b + + (+) + + (+) (2) L b NLe L b NL/PL NL ielisa (AS7) ielisa (16M) celisa (16M) Interpretation Serum Positive a (+) 5 most strains positive; (2) 5 most strains negative; 16M 5 Brucella melitensis antigen; AS7 5 Brucella abortus antigen; b 5 lysis occurs in most strains; BF 5 basic fuchsin at 20 ml/ml (1/50,000 w/v); BK2 5 Berkeley; celisa 5 competitive enzyme-linked immunosorbent assays,,60% positive; CL 5 confluent lysis; e 5 lysis occurs in a few strains; Fi 5 Firenze; ielisa 5 indirect enzyme-linked immunosorbent assay,.10% positive; L 5 lysis; NL 5 no lysis; PL 5 partial lysis; Plq 5 plaques; R/C5 phage for identifying rough strains of Brucella; Tb5 Tibilisi; Th 5 thionin at 20 ml/ml (1/50,000 w/v); Wb 5 Weybridge. b Characteristics as noted in Foster et al. (2007) and Whatmore (2009).

4 SHORT COMMUNICATIONS 635 meningeal blood vessels and patchy mild to marked infiltration with mononuclear cells (lymphocytes, plasma cells, and macrophages) cuffing blood vessels in brain tissue and meninges. In some foci, the meninges were thickened because of cellular (macrophage) infiltration or fibrosis (Fig 1c). Microscopic examination of the atlantooccipital joint showed chronic, nonsuppurative and suppurative arthritis, characterized by dense infiltrates of predominantly mononuclear cells (lymphocytes, plasma cells, and macrophages) in the articular surface (cartilage) with marked pyogranulomatous reaction (predominantly mononuclear cells, mainly macrophages) cuffing the articular lesion. Brucella sp. has been isolated from the central nervous system of striped dolphins (Stenella coeruleoalba) from Europe, notably Spain, the UK, and Costa Rica (González et al., 2002; Jepson, 2006; Muňoz et al., 2006; Hernández-Mora et al., 2008; Davison et al., 2009). Striped dolphins appear more susceptible to severe B. ceti associated, central nervous system (CNS) lesions (González-Barrientos et al., 2010; Guzmán- Verri et al., 2012). Brucella spp. have also been associated with meningoencephalitis and an abnormal, chronically arthritic atlanto-occipital joint in an Atlantic whitesided dolphins (Lagenorhynchus acutus ) from Scotland (Jepson, 2006; Dagleish et al., 2007). However, none of these conditions has been confirmed in the short-beaked common dolphins, despite greater numbers of this species being examined in the UK. Brucella ceti has been isolated from short-beaked common dolphins off the coasts of the UK (Patterson et al., 2000; Foster et al., 2002; Barnett et al., 2009) but without associated pathology, including grossly and microscopically normal reproductive organs (Jepson and Deaville, 2009; Deaville, unpubl.). Three cases of hydrocephalus in UK-stranded common dolphins were associated with microscopic evidence of chronic, nonsuppurative meningoencephalitis and choroiditis, but microbiologic culture of the brain was only attempted in one animal, and B. ceti was not recovered (Davison et al., 2009). More recently, B. ceti was recovered from the brains of three short-beaked common dolphins from Cornwall, UK, but histopathologic examination was not conducted (Barnett et al., 2009; Deaville, unpubl.). The chronic, nonsuppurative meningitis and chronic, nonsuppurative arthritis (atlanto-occipital joint) in association with the isolation of B. ceti are consistent with descriptions of striped dolphins and Atlantic white-sided dolphins with Brucella spp. (González et al., 2002; Jepson, 2006; Muňoz et al., 2006; Dagleish et al., 2007; Hernández-Mora et al., 2008; Davison et al., 2009; Guzmán-Verri et al., 2012). To our knowledge, this is the first confirmed report of B. ceti associated CNS and musculoskeletal pathology in a shortbeaked common dolphin. We acknowledge Cornwall Wildlife Trust Marine Strandings Network and British Divers Marine Life Rescue volunteers for carcass retrieval. Postmortem investigation was carried out under the aegis of the UK Cetacean Strandings Investigation Programme, which is jointly funded by the Department for Environment Food and Rural Affairs (Defra) and the UK Devolved Administrations. LITERATURE CITED Barnett J, Davison N, Deaville R, Monies R, Loveridge J, Tregenza N, Jepson P Post mortem evidence of interactions of bottlenose dolphins (Tursiops truncatus) with other dolphin species in south-west England. Vet Rec. 165: Cloeckaert A, Grayon M, Grepinet O An IS711 element downstream of the bp26 gene is a specific marker of Brucella spp. isolated from marine mammals. Clin Diagn Lab Immunol. 7: Cloeckaert A, Verger J-M, Grayon M, Paquet J-Y, Garin-Bastuji B, Foster G, Godfroid J Classification of Brucella spp. isolated from marine mammals by DNA polymorphism at the omp2 locus. Microbes Infect. 3: Corbel MJ, Bracewell CD, Thomeas EL, Gill KPW Techniques in the identification and classification of Brucella. Identification methods for microbiologists. In: Identification methods

5 636 JOURNAL OF WILDLIFE DISEASES, VOL. 49, NO. 3, JULY 2013 for microbiologists, 2nd Ed., Skinner FA, Lovelock DW, editors. Academic Press, London, UK, pp Dagleish MP, Barley J, Howie FE, Reid RJ, Herman J, Foster G Isolation of Brucella species from a diseased atlanto-occipital joint of an Atlantic white-sided dolphin (Lagenorhynchus acutus). Vet Rec. 160: Davison NJ, Cranwell MP, Perrett LL, Dawson CE, Deaville R, Stubberfield EJ, Jarvis DS, Jepson PJ Meningoencephalitis associated with Brucella species in a live-stranded striped dolphin (Stenella coeruleoalba) in south-west England. Vet Rec. 165: Foster G, MacMillan AP, Godfroid J, Howie F, Ross HM, Cloekaert A, Reid RJ, Brew S, Patterson IAP A review of Brucella sp. infection of sea mammals with particular emphasis on isolates from Scotland. Vet Microbiol. 90: Foster G, Osterman BS, Godfroid J, Jacques I, Cloekaert A Brucella ceti sp. nov. and Brucella pinnipedialis sp. nov. for Brucella strains with cetaceans and seals as their preferred hosts. Int J Syst Evol Microbiol. 57: González L, Patterson IA, Reid RJ, Foster G, Barberán M, Blasco JM, Kennedy S, Howie FE, Godfroid J, MacMillan AP, et al Chronic meningoencephalitis associated with Brucella sp. infection in live-stranded striped dolphins (Stenella coeruleoalba). J Comp Pathol. 126: González-Barrientos R, Morales J-A, Hernández- Mora G, Baquero-Calvo E, Guzmán-Verri C, Chaves-Olarte E, Moreno E Pathology of striped dolphins (Stenella coeruleoalba) infected with Brucella ceti). J Comp Pathol 142: doi: /j.jcpa Guzmán-Verri C, González-Barrientos R, Hernández-Mora G, Morales J-A, Baquero-Calvo E, Chaves-Olarte E, Moreno E Brucella ceti and brucellosis in cetaceans. Front Cell Infect Microbiol. 2:3. doi: /ficmb Microbiology/ /fcimb /full. Accessed March Hernández-Mora G, González-Barrientos R, Morales J-A, Chaves-Olarte E, Guzmán-Verri C, Baquero-Calvo E, De-Miguel M-J, Marin C-M, Blasco JM, Moreno E Neurobrucellosis in stranded dolphins, Costa Rica. Emerging Infect Dis. 14: Jepson PD, editor Cetacean strandings investigation and co-ordination in the UK: Report to the Department for Food and Rural Affairs for the period 1 January December 2004, pp aspx?document5wp01011_8244_frp.pdf. Accessed March Jepson PD, Deaville R Investigation into the common dolphin mass stranding event in Cornwall, 9th of June Document.aspx?Document5WC0601_8031_TRP. pdf. Accessed March Kuiken T, Hartman MG, editors Cetacean pathology: Dissection techniques and tissue sampling. In: Proceedings of the first European cetacean society workshop on cetacean pathology. European Cetacean Society, Leiden, Netherlands, September; Eur Cetacean Soc Newsl 17, 39 pp. McGiven JA, Tucker JD, Perrett LL, Stack JA, Brew SD, MacMillan AP Validation of FPA and celisa for the detection of antibodies to Brucella abortus in cattle sera and comparison to SAT, CFT, and ielisa. J Immunol Methods 278: Muňoz PM, García-Castrillo G, López-García P, González-Cueli JC, Demiguel MJ, Marín CM, Barberán M, Blasco JM Isolation of Brucella species from a live-stranded striped dolphin (Stenella coeruleoalba) in Spain. Vet Rec. 158: Patterson IAP, Howie FE, Reid RJ, Ross HM, MacMillan AP, Foster G, Buxton D Brucella infections in marine mammals from Scottish waters. In: Proceedings of the Joint Conference of the American Association of Zoo Veterinarians and the International Association for Aquatic Animal Medicine, Baer CK, Patterson RA, editors. American Association of Zoo Veterinarians and American Association of Wildlife Veterinarians, New Orleans, Louisiana, September, pp Whatmore AM Current understanding of the genetic diversity of Brucella, an expanding genus of zoonotic pathogens. Infect Genet Evol. 9: Submitted for publication 11 June Accepted 22 December 2012.