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SALMONELLA ENTERICA PREVALENCE IN LEATHERBACK SEA ...

Journal of Zoo and Wildlife Medicine44(3): 765 768, 2013 Copyright 2013 by American Association of Zoo VeterinariansSALMONELLA ENTERICAPREVALENCE IN LEATHERBACK SEATURTLES (DERMOCHELYS CORIACEA) IN ST. KITTS, WESTINDIESC layton S. Dutton, Floyd Revan, Chengming Wang, , , Chuanling Xu, Terry M. Norton, , Dipl. , Kimberly M. Stewart, , , Bernhard Kaltenboeck, , ,and Esteban Soto, , : Salmonellaspp. are gram-negative bacteria capable of causing diseases in a wide range of aquatic andterrestrial animals, including humans. Sea and terrestrial turtles have been recognized as carriers of this zoonoticpathogen. In this project, conventional and molecular diagnostic methods were combined to investigate theprevalence ofSalmonella entericain LEATHERBACK sea turtles (Dermochelys coriacea) that used the island of St.

SALMONELLA ENTERICA PREVALENCE IN LEATHERBACK SEA TURTLES (DERMOCHELYS CORIACEA) IN ST. KITTS, WEST ... in the eastern Caribbean used by leatherback sea turtles as nesting ground from March through ... Salmonella strains and 87 non-Salmonella strains,12 All oligonucleotides were designed by use of the

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Transcription of SALMONELLA ENTERICA PREVALENCE IN LEATHERBACK SEA ...

1 Journal of Zoo and Wildlife Medicine44(3): 765 768, 2013 Copyright 2013 by American Association of Zoo VeterinariansSALMONELLA ENTERICAPREVALENCE IN LEATHERBACK SEATURTLES (DERMOCHELYS CORIACEA) IN ST. KITTS, WESTINDIESC layton S. Dutton, Floyd Revan, Chengming Wang, , , Chuanling Xu, Terry M. Norton, , Dipl. , Kimberly M. Stewart, , , Bernhard Kaltenboeck, , ,and Esteban Soto, , : Salmonellaspp. are gram-negative bacteria capable of causing diseases in a wide range of aquatic andterrestrial animals, including humans. Sea and terrestrial turtles have been recognized as carriers of this zoonoticpathogen. In this project, conventional and molecular diagnostic methods were combined to investigate theprevalence ofSalmonella entericain LEATHERBACK sea turtles (Dermochelys coriacea) that used the island of St.

2 Kitts,West Indies as a nesting ground during 2011 (n 21). Isolates obtained from selective media were screened andcolonies suspected of beingSalmonellaspp. were confirmed by fluorescence resonance energy transfer polymerasechain reaction. The PREVALENCE ofS. entericawithin this sample population during this period was found to Moreover, due to the increasing risk of antibiotic resistance in enteric bacteria, antimicrobial susceptibilitywas investigated in all recoveredSalmonellaspp. isolates utilizing the broth microdilution method. All isolateswere susceptible to the lowest concentration of kanamycin, gentamicin, ciprofloxacin, enrofloxacin, nalidixic acid,and trimethoprim/sulfamethoxazole tested. Further research should be pursued to understand the interaction ofthis bacterial pathogen with the environment, host, and other microbial communities, and to further developfaster, more sensitive, and more specific diagnostic words: LEATHERBACK sea turtles, PCR, SALMONELLA , St.

3 Kitts, COMMUNICATIONL eatherback sea turtles (Dermochelys coriacea)are listed as critically endangered species by theInternational Union for Conservation of a worldwide distribution, they prefer tonest on isolated beaches in tropic areas adjacentto deep water, laying an average of six clutches perreproductive island of St. Kitts(178159N, 628409W) is a 168-km2island locatedin the eastern caribbean used by LEATHERBACK seaturtles as nesting ground from March are gram-negative facultativeintracellular bacteria that belong to the familyEnterobactericeae. Members of this genus maypose health risks to both aquatic and terrestrialanimals, including ,14 The genusSalmo-nellais composed of two species,SalmonellabongoriandSalmonella ENTERICA , but there aremore than 2,000 serovars come from ingestion ofcontaminated water and/or food (fecal-oral trans-mission);10,13,14however, domesticated animals areconsidered to be the primary reservoirs ofSalmo-nellathat may infect ,14 Several studieshave reported the PREVALENCE ofSalmonellaincaptive and wild ,2,6,9,16 19,21 Nevertheless,the PREVALENCE ofSalmonellaspp.

4 In wild seaturtles and risk factors associated withSalmonellatransmission from wild sea turtles to human islargely unknown, and only in a few cases has thisbeen ,15,17,18,21 This study was con-ducted to investigate the PREVALENCE and antimi-crobial susceptibility of these potentialpathogenic bacteria in nesting LEATHERBACK seaturtles on the island of St. Kitts during the 2011nesting swabs were taken from 21 leatherbacksea turtles that nested on St. Kitts during the 2011nesting season. Immediately following egg laying,a sterile BactiSwabt(Remel, Lenexa, Kansas66215, USA) was introduced 5 to 6 cm into thecloaca and rotated five times. The swabs werestored on wet ice immediately following sampleacquisition and then submitted to the RossUniversity School of Veterinary Medicine MarineLaboratory.

5 These swabs were then used toFrom the Department of Pathobiology, Ross UniversitySchool of Veterinary Medicine, Island Main Rd., WestFarm, St. Kitts, West Indies (Dutton, Revan, Soto); Sea Turtle Monitoring Network, Basseterre, St. Kitts,West Indies (Stewart, Norton); Yangzhou UniversityCollege of Veterinary Medicine, Yangzhou, Jiangsu225009, China (Wang, Xu); Georgia Sea Turtle Center,214 Stable Road, Jekyll Island, Georgia 31527, USA(Norton); and Auburn University School of VeterinaryMedicine, Auburn University, Alabama 36849, USA(Kaltenboeck). Correspondence should be directed to MacConkey agar plates (Remel). Theinoculated plates were incubated for 48 hr at from primary isolation agar plates werereplated for purity of culture onSalmonella Shigellaagar (Remel), used to select forSalmonellaspp.

6 , and incubated overnight at 378C. Once singlecolonies were observed and purity of the isolatewas determined, the isolates were frozen at 808 Cin 13phosphate-buffered saline (PBS) containing20%glycerol (Thermo Fisher Scientific, Waltham,Massachusetts 02451, USA) for later use. Allrecovered bacteria isolates were initially Gram-stained and tested for cytochrome oxidase andcatalase activity (Thermo Fisher Scientific). Mo-lecular identification of gram-negative, rod-shaped bacteria that did not produce cytochromeoxidase was achieved using fluorescence reso-nance energy transfer polymerase chain reaction(FRET PCR).20 Briefly, a loop of the bacteriumwas suspended in 500ll of sterile 13 PBS andsubjected to DNA extraction using the DNeasyblood and tissue kKit (Qiagen Inc.)

7 , Valencia,California 91355, USA) following the manufac-turer s suggested protocol for gram-negative bac-teria. Extracted DNA was stored at 208C untilfurther part of thettrRSBCA locus encoding proteinsfor tetrathionate respiration was chosen as thetarget to design a FRET PCR inclusivity and exclusivity of this techniquewas found to be 100%while applying 110 Salmonellastrains and 87 non-Salmonellastrains,12 All oligonucleotides were designed by use of theVector NTI software (InvitrogenTMCorporation,Carlsbad, California 92008, USA). The copynumber ofSalmonellaspp. genomes was deter-mined by FRET-PCR performed in a LightCy-clertreal-time platform with software version (Roche Molecular Biochemicals, Indianapolis,Indiana 46250, USA) in a similar approach aspreviously primers (upstream:59-CAC TCATCATTGAAG AAG TGC TGG CTC-39; downstream:59-GGT AAT CGC ACA GGT AAT GGC AATCAG-39) and the probes (59-TTT GCC TGT TATCTT CAC TGG CGG AA-[6-FAM]-39;59-[Bod-ipy 630/650]-AAA GAC GCC GCA ACA GAAGAA AAT CG-[phosphate]-39) were designed toamplify and detect all fluorescein probe was 39-labeled with car-boxyfluorescein (6-FAM) and used unpurified as aFRET energy donor probe excited by 488-nmlight.

8 The Bodipy 630/650 probe was 59-labeled,39-phosphorylated, high performance liquid chro-matography purified, and used as acceptor probe(Integrated DNA Technologies, Coralville, Iowa52241 USA). Nucleotide fragments representingpartialttrRSBCA were synthesized and inserted inthe pIDTSMART cloning vector (IntegratedDNA Technologies). The plasmid was linearizedwithHindIII (Promega, Madison, Wisconsin53711, USA), followed by inactivation of therestriction enzyme at 658C for 20 min. DNA wasquantified by PicoGreentDNA fluorescenceassay (Molecular Probes, Inc., Eugene, Oregon97402, USA) for preparation of quantitativestandards. Specificity of theSalmonellaFRET-PCR was confirmed by amplification of fourisolates ofSalmonellaTyphimurium and the syn-thesized target gene ofSalmonella, and a lack ofamplification from extracted DNAs ofEscherichiacoli,Pasteurella multocidaandPseudomonas aerugi-nosa.

9 PCR product was verified in 4%MetaPhoragarose gel electrophoresis, and isolated andpurified for automated DNA sequencing with aQIAquick PCR purification kit (Qiagen). Nucleo-tide sequencing was performed using an ABIautomatic DNA sequencer (Model 377; Perkin-Elmer, Waltham, Massachusetts 02451, USA) atthe Genomic Sequencing Laboratory (AuburnUniversity, Auburn, Alabama, USA) using theforward and antisense primers described minimal inhibitory concentrations of anti-microbial agents toS. entericaisolates fromleatherback sea turtles and quality control (E. coliATCC 25922) were tested using the SensititreUrinary Plate Format, plate code CMV1 BURF;Sensititre Staphylococci Plate, plate code EUST;and the Sensititre Custom Plate Format, platecode CMV2 AGNF (Trek Diagnostic System,Inc.)

10 , Cleveland, Ohio 44131, USA), using themanufacturer s suggested protocol and the Clin-ical and Laboratory Standards Institute , 171 bacterial isolates were isolated onMacConkey agar plates. After use of selectivemedia screening ( SALMONELLA Shigellaagar) andFRET PCR, seven isolates were identified The seven isolates came from five of the21 turtles that came to nest in St. Kitts during2011, indicating a PREVALENCE ofSalmonellato the nesting LEATHERBACK females sampledin 2011. The recoveredSalmonellawere suscepti-ble to kanamycin, gentamicin, ciprofloxacin, tri-methoprim/sulfamethoxazole, enrofloxacin, andnalidixic contrast to the findings in this project,previous studies reporting the frequency ofSal-monellain wild turtles have found the prevalenceto be very 18 Previously, researchers utilized766 JOURNAL OF ZOO AND WILDLIFE MEDICINE selective media and biochemical analysis toanalyze cloacal swabs from 94 wild-caught turtlesin central North Carolina, ,Salmonellawas not recovered from any of thecloacal another report, researchersidentified 189 bacterial isolates from 70 nestingleatherback sea turtle cloacal swabs in PacuareNature Reserve, Costa selectivemedia and biochemical analysis.


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