A COMPARATIVE INVESTIGATION OF STREPTOCOCCUS AGALACTIAE ISOLATES FROM FISH AND CATTLE Except where reference is made to the work of others, the work described in this dissertation is my own or was done in collaboration with my advisory committee. This dissertation does not include proprietary or classified information. _____________________ Julio Carib Garc?a Certificate of Approval: _______________________ ____________________ Joyce J. Evans Phillip H. Klesius, Chair Affiliate Assistant Professor Affiliate Professor Fisheries and Allied Fisheries and Allied Aquacultures Aquacultures _______________________ ____________________ Craig A. Shoemaker Covadonga R. Arias Affiliate Assistant Professor Associate Professor Fisheries and Allied Fisheries and Allied Aquacultures Aquacultures _______________________ Joe F. Pittman Interim Dean Graduate School A COMPARATIVE INVESTIGATION OF STREPTOCOCCUS AGALACTIAE ISOLATES FROM FISH AND CATTLE Julio Carib Garc?a A Dissertation Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy Auburn, Alabama May 10, 2007 iii A COMPARATIVE INVESTIGATION OF STREPTOCOCCUS AGALACTIAE ISOLATES FROM FISH AND CATTLE Julio Carib Garcia Permission is granted to Auburn University to makes copies of this dissertation at its discretion, upon request of individuals or institutions and at their expense. The author reserves all publication rights __________________________ Signature of Author __________________________ Date of Graduation iv VITA Julio Carib Garc?a Febres, son of Julio Ram?n Garc?a Loubriel and Aida Teresa Febres Hern?ndez was born June 23, 1976, in San Juan, Puerto Rico. He graduated with a Bachelor of Arts degree (Environmental Science) from Washington College at Chestertown, MD in 2000. Two years later he entered Graduate School, in the Department of Fisheries and Allied Aquaculture, College of Agriculture, Auburn University and graduated in December 2004 with a Master of Science degree (Fisheries and Aquaculture). Upon completion he continued his education in the Department of Fisheries and Allied Aquaculture, College of Agriculture, Auburn University. v DISSERTATION ABSTRACT A COMPARATIVE INVESTIGATION OF STREPTOCOCCUS AGALACTIAE ISOLATES FROM FISH AND CATTLE Julio Carib Garc?a Doctor of Philosophy, May 10, 2007 (M. S., Auburn University, 2004) (B.A., Washington College, 2000) 87 Typed Pages Directed by Phillip H. Klesius Streptococcus agalactiae is the causative bacterium of streptococcosis and causes severe economic losses in wild and cultured fish and cattle, worldwide. In fish, infection can result in septicemia with hemorrhages on the body surface and in the external and internal organs. Streptococcus agalactiae may be isolated from brain, nares, head kidney and eye of infected fish. Streptococcus agalactiae also causes bovine mastitis and the organism can be isolated from milk samples. Streptococcus agalactiae is classified as Lancefield?s group B Streptococcus (GBS). There are also nine GBS capsular vi polysaccharide serotypes. Despite the significance of the disease, only limited information is available on the identification and characterization of the S. agalactiae isolates from fish and cattle. In the present study, GBS isolates from fish (n = 36), bottlenose dolphin (n=1) and cattle GBS isolates (n=10) were found to have a number of common phenotypic characteristics of the S. agalactiae reference strains (n=4). However, the phenotypic characteristics of these GBS isolates were different from the reference strains of Streptococcus dysgalactiae (n=2), Streptococcus phocae (n=1), Streptococcus iniae (n=1) and Lactococcus garvieae (n=1). The results showed that GBS isolates of fish and cattle origins could be distinguished from each other by capsular serotype, CAMP factor, D-lactose and D-trehalose fermentation, growth pattern in fluid medium and whole cell fatty acid profiles. Studies were performed to determine the pathogenicity of cattle isolates for Nile tilapia (Oreochromis niloticus) and channel catfish (Ictalurus punctatus). The cattle isolates were found not to be infectious for either fish species. No diseases signs or isolation of GBS cattle isolates from the brain or head kidney of fish at 24 and 48 hour post-injection were noted. Fish isolates were evaluated to determine whether a single clone or multi-clones of S. agalactiae were responsible for streptococcal disease in mullet and seabream during the Kuwait Bay, Kuwait epidemic by amplified fragment length polymorphism (AFLP). The results indicated that the infection was not caused by a single clone of S. agalactiae. The results suggest that five GBS S. agalactiae clones were involved in the epidemic that affected primarily mullet and seabream. vii ACKNOWLEDGEMENT I wish to express my appreciation to Paige Mumma and Ryan Wood for their laboratory assistance. In addition, I extend my appreciation to Drs. Phil Klesius, Joyce J. Evans, Covadonga Arias, Craig Shoemaker, Oscar Olivares-Fuster, and Victor Panangala for their research assistance, guidance and support. Further, I wish to thank Drs. John Hawke and Dina Zilberg for providing some of the bacteria used in this study. I also wish to thank everyone from the USDA-ARS Aquatic Animal Health Research Laboratory and Auburn University who helped me with the research. I wish to thank my family for their unending support during all of my endeavors. Finally and specifically, thank you, Nancee, for all of your endless support and encouragement, without you, this endeavor could not have been completed. viii Style manual used - Guideline for authors Journal of the World Aquaculture Society. Computer software used - Microsoft Word XP Standard ix TABLE OF CONTENTS LIST OF TABLES..............................................................................................................X LIST OF FIGURES .......................................................................................................... XI I. INTRODUCTION AND LITERATURE REVIEW ........................................................1 II. MATERIALS AND METHODS..................................................................................10 III. RESULTS ....................................................................................................................16 IV. DISCUSSION..............................................................................................................35 V. SUMMARY..................................................................................................................41 VI. LITERATURE CITED................................................................................................45 VII. APPENDIX Table A. Streptococcus agalactiae isolates, sources and origins from fish, bottlenose dolphin, cattle and reference strains of S. agalactiae, S. difficile, S. dysgalactiae and S. phocae..........................................................................57 VIII. APPENDIX Table B. Target gene, oligonucleotide primers and target size for polymerase chain reaction (PCR) ..................................................................................... 59 IX. APPENDIX Table C. Phenotypic properties of GBS Streptococcus agalactiae isolates from fish, bottlenose dolphin, cattle and reference type strains of S. agalactiae, S. difficile, S. dysgalactiae and S. phocae..............................................60 X. APPENDIX Table D. API-20 Strep results of ?-galactosidase (? -Gal), ?-glucosidase (?-Gluc), ?-galactosidase (?-Gal), alkaline phosphatase (PAL), aminopepidase (LAP) properties for GBS Streptococcus agalactiae isolates from fish, bottlenose dolphin, cattle and reference strains of S. agalactiae, S. difficile, S. dysgalactiae and S. phocae................................................................................................62 x XI. APPENDIX Table E. Capsule properties, hyaluronidase activity and pigment production for GBS Streptococcus agalactiae isolates from fish, bottlenose dolphin, cattle and reference strains of S. agalactiae, S. difficile, S. dysgalactiae and S. phocae...64 XII. APPENDIX Table F. Growth properties in fluid medium and soft agar for GBS Streptococcus agalactiae isolates from fish, bottlenose dolphin, cattle and reference strains of S. agalactiae, S. difficile, S. dysgalactiae and S. phocae ...................66 XIII. APPENDIX Table G. Fatty acid methyl esters (FAME), Gram-positive identification system (GPI), API-20 Strep, polymerase chain reaction (PCR), for Streptococcus agalactiae isolates from fish, cattle, bottlenose dolphin and reference strains of S. agalactiae, S. difficile, S. dysgalactiae and S. phocae ...................................68 XIV. APPENDIX Table H. Percent of whole-cell fatty acid composition of GBS Streptococcus agalactiae isolates from fish, cattle and reference strains determined by RCLIN 50 rapid method ...............................................................................................72 XV. APPENDIX Table I. Streptococcus agalactiae isolates from marine fish used for amplified fragment length polymorphism (AFLP) fingerprinting ...............................73 xi LIST OF TABLES III.1. Phenotypic characteristics of GBS Streptococcus agalactiae isolates from fish and cattle .....................................................................................................................30 III. 2. Phenotypic and genotypic properties of GBS Streptococcus agalactiae isolates from mullet, seabream, Nile tilapia, Gulf killifish, hybrid striped bass, cattle and S. agalactiae reference strains...........................................................................32 III. 3. Summary of cost, discrimination level, disadvantages and advantages of methods utilized for the comparison of Streptococcus agalactiae isolates from cattle and fish .....................................................................................................................43 xii LIST OF FIGURES III. 1. Dendogram of fatty acid profiles of GBS Streptococcus agalactiae isolates from fish, cattle and references strains. Analysis of profiles based on Pearson?s product-moment. The cutoff value of 94 % similarity .....................................................23 III. 2. The PCR product of GBS streptococcal species based on16S-23S rDNA intergenic sequence.................................................................................................25 III. 3. The PCR product of GBS streptococcal species based on 23S rDNA sequence..................................................................................................................26 III. 4. The PCR product of GBS streptococcal species based on 16S-23S intergenic spacer region rDNA sequence .........................................................................27 III. 5. Dendogram of amplified fragment length polymorphic (AFLP) profiles of GBS S. agalactiae isolates from mullet and seabream collected in Kuwait Bay, Kuwait...........................................................................................................29 1 I. INTRODUCTION AND LITERATURE REVIEW Streptococcus species are considered a diverse group, ranging from commensal organisms which occupy various niches of the human body to pathogens that have the capacity to infect a wide range of hosts (Fishcetti 2000). Further, Streptococcus spp. are considered among the most important pathogens affecting humans and mammals and have been reported to affect cold blooded animals including fish (Eldar et al. 1994). In fish, Streptococcus spp. have been reported to cause considerable fish morbidity and mortality worldwide, estimated to be $150 million annually (Klesius et al. 2000). Streptococcus agalactiae is a group B Streptococcus (GBS) (Lancefield 1933). In addition, nine GBS capsular polysaccharide serotypes have been identified (Hickman et al. 1999; Kong et al. 2002). Streptococcus agalactiae is generally characterized as a Gram-positive coccus, catalase (-), oxidase (-), CAMP (+) and hippurate hydrolysis (-). The identification of S. agalactiae is partly based on its hemolytic reaction on blood agar and Lancefield grouping. The types of hemolysis are alpha (?-), beta (?-) and non- hemolytic. Beta hemolysis is characterized as a clear zone around the colony on blood agar. Alpha hemolysis is a partial lysis of blood cells recognized by greenish zone around the colony. Lancefield?s grouping is based on the serological types of polysaccharide antigen that are extracted by acid from the bacterium. The identification of S. agalactiae is also based on phenotypic characterization using conventional and 2 rapid identification tests (Jayarao et al. 1991; MacFaddin 2000; Facklam 2002). Genotypic characterization has also been used to identify S. agalactiae (Berridge et al. 2001; Yildirim et al. 2002b; Duarte et al. 2004). Highly discriminative typing techniques, whole cell fatty acid composition and amplified fragment polymorphism fingerprinting, have been performed in Gram negative bacteria (Stead 1992; Arias et al. 2003; Arias et al. 2004; Shoemaker et al. 2005), but these tests have not been applied for characterization of S. agalactiae from fish and cattle. These highly discriminative typing techniques can reveal the possible polyclonal nature of S. agalactiae. Polymerase chain reactions has been used in identification schemes for S. agalactiae (Berridge et al. 2001; Mata et al. 2004; Kawamura et al. 2005). Vandamme et al. (1996) and Arias et al. (2003) emphasized the importance of a consensus approach to bacterial identification or a polyphasic approach using conventional and rapid phenotypic tests, molecular and genotypic identification schemes for the identification and discrimination of environmentally and medically important bacteria. Thus, a polyphasic approach was employed to identify and discriminate S. agalactiae from fish and cattle. Streptococcus agalactiae is considered a pathogen responsible for causing infections in chicken, cattle, camels, dogs, bottlenose dolphin, horses, emerald monitors, cats, fishes, frogs, hamsters, humans, mice, monkeys and nutria (Wilkinson et al. 1973; Elliott et al. 1990; Wagner et al. 1997; Evans et al. 2002; Yildirim et al. 2002a; Yildirim et al. 2002b; Hetzel et al. 2003; Zappulli et al. 2005; Evans et al. 2006a; Evans et al. 2006b). 3 Wild and cultured saltwater and freshwater fish species that have been reported to be infected with GBS are golden shiner (Notemigonus crysoleucas) (Robinson and Meyer 1966), sea catfish (Arius felis), striped mullet (Mugil cephalus), pinfish (Lagodon rhomboids), Atlantic croaker (Micropogon undulatus), spot (Leiostomus xanthrus), string ray (Dasyatis sabina), Gulf menhaden (Brevoortia patronus) (Plumb et al. 1974), bullminnows (Fundulus grandis) (Rasheed and Plumb 1984), striped bass (Morone saxatilis), bluefish (Pomatomus saltatrix), sea trout (Cynoscion regalis) (Baya et al. 1990), hybrid tilapia (Oreochromis niloticus x Oreochromis aureus) (Eldar et al. 1994; Eldar et al. 1995), mullet (Liza klunzingeri), seabream (Sparus auratus) (Evans et al. 2002), Nile tilapia (O. niloticus) (Evans et al. 2002; Suanyuk et al. 2005; Salvador et al. 2005) and silver pomfret (Pampus argenteus) (Duremdez et al. 2004). These infections have been documented in the United States, Japan, Kuwait, Colombia, Brazil, Thailand and Israel (Evans et al. 2006). Evans et al. (2006b; 2006c) have reviewed the geographical distribution of S. agalactiae in tilapia and other host. Hoshina (1958) reported the first case of Streptococcus sp. in fish. Robinson and Meyer (1966) reported what is believed to be the first case of GBS S. agalactiae in fish. Plumb et al. (1974) isolated a nonhemolytic, group B, Type Ib Streptococcus sp. from 90 % of fish examined involving multiple fish species from a fish kill in Soldier Creek located in the gulf coast of Alabama and Florida. The phenotypic properties of the group B isolates were previously described by Wilkinson et al. (1973). The fish species affected reported by Plumb et al. (1974) were menhaden, sea catfish, mullet, pinfish, Atlantic croaker and stingray. Cook et al. (1975) conducted experimental fish infectivity 4 trials using a group B isolate from a moribund menhaden from Soldier Creek that had been isolated by Plumb et al. (1974). Five of the eight fish species involved in the Alabama-Florida fish kill were challenged and mortality recorded. Mortality was noted in Atlantic croaker, sea catfish, Gulf menhaden, striped mullet and spot. A streptococcal epidemic episode involving bluefish, striped bass and sea trout was reported in the Chesapeake Bay and some of the tributaries during the summer of 1988 (Baya et al. 1990). Similar signs to those reported by Rasheed et al. (1985) were observed in these fish. Yellow or reddish-yellow ascites fluid was found in the abdominal cavity of the majority of the diseased fish. Baya et al. (1990) compared their isolates with the isolate of Plumb et. al. (1974) and found them to be similar biochemically and serologically. Elliot et al. (1990) examined a number of nonhemolytic GBS Streptococcus type Ib isolated from humans, mice, cattle, frogs and fish to determine whether these isolates were related. Using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), they compared the whole-cell protein profiles of these isolates from different animals. Elliot et al. (1990) concluded the PAGE profiles were very similar, suggesting that the isolates may have had a common ancestor. In August and September 2001, Kuwait Bay, Kuwait experienced a massive fish kill involving wild mullet due to the bacterium S. agalactiae (Evans et al. 2002). This pathogen was isolated from cultured gilthead seabream (Evans et al. 2002) and bottlenose dolphin (Tursiops truncatus) (Evans et al. 2006a). Evans et al. (2002) reported the Streptococcus isolates were group B serogroup. Phenotypic characteristics of the isolates 5 compared favorably with those of American Type Culture Collection (ATCC) reference strains of S. agalactiae. The GBS S. agalactiae isolates were pathogenic for Nile tilapia by intraperitoneal (IP) injection (Evans et al. 2002). Capsular polysaccharide serotypes and GBS surface protein antigens are believed to be important to the pathogenesis of GBS disease (Kong et al. 2002). Rasheed and Plumb (1984) isolated a non-hemolytic Streptococcus sp. from diseased Gulf killifish. Rasheed and Plumb (1984) used the isolate to determine the pathogenicity of the non- hemolytic Streptococcus sp. through different modes of infection, including IP injection, immersion and oral administration, to Gulf killifish. Mortality was produced by experimental IP injection, but not by immersion or oral administration. Furthermore, Rasheed et al. (1985) attributed the pathogenicity of streptococci to an exotoxin, but no evidence was provided to demonstrate the presence of an exotoxin. They noted that streptococcal infections in bullminnows were systemic infections localized in the eye, liver and spleen. Signs caused by Streptococcus sp. in fish species reported by Rasheed et al. (1985) included unilateral or bilateral exophthalmia, hemorrhage in the eye, dark coloration, erratic swimming and splenomegaly. Evans et al. (2002) reported that wild mullet had abnormal behavior including erratic swimming, swimming and whirling on the surface and ?C? shaped body curvature while at the surface. Ocular abnormalities that included opacity, peri-orbital and intraocular hemorrhages, purulence and exophthalmia were reported. Reddening and hemorrhage were observed in the integumental and muscoskeletal system (Evans et al. 2002). Cranial and body surface 6 hemorrhages were observed. An atypical sign of a clear opercula ?window to the gills? was also noted (Evans et al. 2002). Gram positive cocci were isolated from tilapia and trout by Eldar et al. (1994). Two distinct phenotypic groups were found, including a nonhemolytic and ?-hemolytic group. These isolates were tested with DNA-DNA hybridization and by DNA base composition. These two isolates belonged to two separate DNA groups. Eldar et al. (1994) proposed the nonhemolytic isolate be named S. difficile and the ?-haemolytic be named S. shiloi. Several studies have been conducted to correctly identify and name the nonhemolytic Streptococcus spp. Vandamme et al. (1997) using whole-cell protein SDS- PAGE analysis reported that the S. difficile strain (LMG 15799) was indistinguishable from a S. agalactiae strain. Moreover, Vandamme et al. (1997) was able to serologically type S. difficile. The capsular antigen was identified as Ib serotype (Vandamme et al. 1997). Streptococcal isolates are often unspeciated (Robinson and Meyer, 1966, Plumb et al. 1974) or are improperly identified as other Streptococcus spp. (Wilkinson et al. 1973, Kusuda and Komatsu 1978, Elliott et al. 1990, Eldar et. al 1994). However, S. agalactiae is the only group B species of the genus. Thus, it is very important to determine both Lancefield group and serotype of the isolate to determine its identity. Molecular biological techniques such as ribotyping, oligonucleotide probes and polymerase chain reaction (PCR) have been proposed for the more accurate identification of the various species of Streptococcus (Kawata et al. 2004). Recently, studies performed by Berridge 7 et al. (2001), Kawata et al. (2004) and Mata et al. (2004) focused on developing species- specific primers that target the 16S-23S rDNA intergenic spacer region and the 23S rDNA PCR product specifically for S. agalactiae. Berridge et al. (2001) developed species-specific PCR primers for the detection and identification of the 16S-23S rDNA intergenic spacer region of S. agalactiae and S. difficile. Berridge et al. (2001) demonstrated that the S. difficile fish isolates possess a common sequence found in the S. agalactiae type strain (ATCC-13813) and S. difficile type strain (ATCC-51487). However, the species-specific primers were unable to produce amplicons from related Streptococcus spp., further demonstrating the homology of S. agalactiae and S. difficile. Kawata et al. (2004) targeted the 23S rDNA PCR sequence for the identification of eight streptococcal species, including S. agalactiae. Kawata et al. (2004) primers produced an amplicon of 875 bp. Their results showed that 100 % of the S. agalactiae isolates (N=35) used in their investigation were correctly identified. They also reported that the PCR method was able to identify biochemically typical GBS isolates as well as atypical GBS isolates. Mata et al. (2004) developed a multiplex PCR designed to simultaneously detect Streptococcus spp. associated with warmwater fish. These results showed that the species specific PCR primers successfully identified S. agalactiae, S. difficile isolates and were similar to the S. agalactiae reference strains by PCR. Zlotkin et al. (1998) utilized restriction fragment length polymorphism (RFLP) to ribotype S. iniae strains associated with outbreaks in wild and cultured fish species in the Mediterranean Sea. The RFLP results confirmed that outbreaks of S. iniae were 8 produced by a single S. iniae clone. This allowed Zlotkin et al. (1998) to establish that, epidemiologically, all fish were infected by descendants of a single clone of S. iniae. Further, results suggest that cross-infection that occurred between wild and cultured fish was caused by a single clone. Whole cell fatty acids have been used for identification of bacterial fish pathogens (Shoemaker et al. 2005). Further, growth medium, time and temperature of incubation differ among bacterial genus species and groups. Thus, the selection and the use of a standardized medium that produces good growth of the organisms is vital for optimal results (Shoemaker et al. 2005). The pathogenesis of fish GBS isolates is poorly understood. Eldar et al. (1994) reported that Streptococcus spp. caused meningoencephalitis in fish. Pathogenicity studies conducted with GBS isolates from fish indicated that it is a highly virulent fish pathogen capable of infecting a wide variety of freshwater and saltwater fish (Evans et al. 2002). The infectivity of cattle GBS isolates for fish is unknown. No other information is available on cross-transmission of infection between the same fish species or among unrelated fish species. The hypothesis of this investigation was that S. agalactiae isolates from fish and cattle are phenotypically and genetically distinct. To test this hypothesis, three objectives were proposed. The first objective was to compare GBS isolates from fish (n=36) and GBS from cattle (n=10) for phenotypic and genotypic characteristics. The second objective was to determine the infectivity of GBS isolates from cattle for Nile tilapia and channel catfish. The third objective was included in order to determine whether single or 9 multiple clones of GBS isolates were responsible for streptococcal disease in saltwater mullet and seabream during the epidemic event in Kuwait Bay, Kuwait by amplified fragment length polymorphism (AFLP) analysis. 10 II. MATERIALS AND METHODS Experimental Design Thirty six S. agalactiae isolates collected from infected mullet, seabream, tilapia, Gulf killifish and hybrid striped bass in Kuwait, United States of America, Israel, Honduras and Brazil were compared to 10 cattle isolates collected from Wisconsin, USA, for phenotypic and genotypic characteristics (Appendix VII. Table A). In addition, an isolate collected from a bottlenose dolphin was also investigated. American Type Culture Collection (ATCC) S. agalactiae, S. difficile, S. dysgalactiae, and S. phocae reference strains were included for comparison. All cultures were grown on tryptic soy broth (TSB) or sheep blood agar (SBA) for 24 hours at 32 ?C. Phenotypic characteristics were determined for hemolysin production (Evans et al. 2002), CAMP reaction (Darling 1975), Lancefield?s grouping (Lancefield 1933), capsular serotyping (group B typing antisera, Denka Seiken, Tokyo, Japan) (Wilkinson et al. 1973), pyrrolidonylarylamidase reaction (PYR), Voges-Proskauer reaction (VP) by API Strep-20 (bioM?rieux, Hazelwood, MO, USA); hippurate hydrolysis (HIP) (Jokipii and Jokipii 1976); urea hydrolysis (URE) (MacFaddin 2000); starch hydrolysis (Evans et al. 2004); arginine decarboxylase (ARG), and esculine hydrolysis (ESC) (Jokipii and Jokipii 1976); fermentation of D-sorbitol (SOR), D- trehalose (TRE), D-ribose (RIB), inulin (INU), D-mannitol (MAN), D-xylulose (XYL) and D-lactose (LAC) (Evans et al. 2002). 11 Hyaluronidase production (Smith and Willett 1968), pigment production (Merritt and Jacobs 1976), growth characteristics in soft agar and fluid medium (Kane et al. 1975) and encapsulation (Atlas et al. 1995) of GBS isolates were also evaluated. Fatty Acid Methyl Ester (FAME) Fatty acid methyl esters (FAME) were extracted according to the procedure described in the Microbial Identification System Operating Manual MIDI (2000) to generate a GBS library for the analysis of FAME profiles. GBS isolates were grown in SBA for 24 hours at 32 ?C. Briefly, 20 + 0.5 mg (wet wt.) of bacterial cells were harvested and placed in 13-mm X 100-mm glass screw cap test tubes (Stead 1992). The bacterial cells were treated with 1 ml of a strong methanolic base (45g sodium hydroxide, 250 ml methanol, 150 ml deionized water) and boiled in a water bath for 30 min to saponify the cells. The fatty acids were methylated (2 ml of 325 ml 6.0N hydrochloric acid, 275 ml of methanol) in a 100 ?C water bath for 5 min. After methylation, the FAME were extracted in hexane and methyl tert-butyl ether. Residual fatty acids were removed from the organic phase by washing with dilute base for 5 min. After extraction, the FAME were injected into an Agilent 6850 gas chromatograph for analysis by the rapid RCLN50 method (MIDI, 2000). A library report was generated by MIDI Procedure (MIDI, 2000). The validation report uses comparative matrices to determine the number of correct matches to the genus and species levels in relation to other profiles available in the MIDI software (MIDI, 2000). The validation data were imported into the BioNumerics version 4 software package (Applied Maths, Sint-Martens-Latem, Belgium). A similarity matrix and the corresponding dendrogram using the Pearson?s 12 product-moment correlation coefficient were generated. The reproducibility of the results was determined by results generated from three cultures grown and extracted on three different days. These results established a 94 % cutoff level. API-20 Strep GBS isolates grown on SBA plates for 24 hr at 32 ?C were used in the API-20 Strep strip test. The cells were harvested in API GP medium (0.5 g L-cystine, 20 g tryptone, 5 g sodium chloride, 0.5 g sodium sulfite, 0.17 g phenol red and 1000 ml demineralized water) and the bacterial suspensions were adjusted to a turbidity ? 4 McFarland (bioM?rieux, Durham, NC, USA). Each well of the API-20 Strep plate was inoculated with 100 ?l of the bacterial suspension and incubated for 4 hr or 16 hr at 35 ?C. The color changes in each individual well of the API-20 Strep plate were read and analyzed using the MINI API (bioM?rieux) system. Results generated by the MINI API system were automatically compared against the system database for genus and species identification and the degree of identification. Gram positive identification (GPI) Streptococcus agalactiae isolates grown on SBA for 24 hr at 32 ?C were utilized to inoculate the GPI 32 cards according to manufacture?s directions (Vitek, bioM?rieux). After inoculation, the GPI cards were incubated in the Vitek GPI system for 24 hours. The GPI validation reports were used to generate a GBS library. The validation reports provided the genus and species and probability for a correct identification. 13 Genotypic characterization. Genomic DNA templates were prepared using the High Pure PCR preparation Kit (Roche Diagnostic, Indianapolis, USA). The GBS isolates were incubated in TSB at 32 ?C for 24 hr. Samples were then added to a sterile 2 ml microcentrifuge tube and centrifuged for 10 min at 5,000 x g. Following centrifugation, the sample supernatant was discarded. The bacterial samples were prepared according to the manufacturer?s instructions. After centrifugation, the eluted DNA was stored at 20 ?C until PCR analysis. The methods described to amplify S. agalactiae 16S -23S rDNA (Mata et al. 2004), 23S rDNA (Kawata et al. 2004) and 16S-23S rDNA intergenic spacer region (Berridge et al. 2001) were used to amplify species-specific targets by PCR to identify the forty seven S. agalactiae isolates (VIII. Appendix Table B). The PCR reagent mixture contained 1 X reaction buffer, 2.5 mM MgCl, 0.8 mM each of dATP, dCTP, dGTP and dTTP, 0.2 ?M each oligonucleotide primer, 0.5 ?L of bovine serum albumin, 1.25 units of Taq DNA polymerase, 50-100 ng template DNA, and sterile water (Berridge et al. 2001). Briefly, PCR mixture were cycled 35 times at 94 ?C for 1 min, 50 ?C for 1 min and 72 ?C for 1 min with a final extension for 5 min at 72 ?C. The annealing temperature was adjusted to 60 ?C for the specific S. agalactiae primers. The PCR products were analyzed by 1.0 % agarose gel electrophoresis, stained with ethidium bromide and visualized under UV light. Samples were considered positive when a clear band of the expected molecular weight was observed under UV light. 14 Amplified Fragment Length Polymorphism (AFLP) Twelve isolates of S. agalactiae collected from 6 wild mullet and 6 cultured seabream were tested for interspecific diversity by AFLP analysis. These fish were collected from a Streptococcus epidemic in Kuwait Bay, Kuwait. The GBS isolates were incubated in TSB at 32 ?C for 24 hr and the cells harvested by centrifugation. Briefly, genomic DNA templates were prepared using the DNeasy Tissue Kit (Qiagen Science, Maryland, USA). The kit was used according to manufacturer?s instructions and Arias et al. (2004). One hundred nanograms of total DNA was digested with HindIII and TaqI (Promega, Madison, WI, USA). Next, the adapters were ligated to the restricted fragments using T4 DNA lipase (Promega). The HindIII primer was labeled with IR700 Fluorochrome (LICOR, Lincoln, NE, USA). The presence of PCR products was determined by electrophoresis in a NEN Global Edition IR2 DNA analyzer (LICOR) according the manufacture?s directions. The AFLP data were processed with BioNumerics and levels of similarity between GBS isolates were calculated with the Pearson?s product moment correlation coefficient and a dendrogram of the GBS isolates was generated. Experimental infectivity trials ? Nile tilapia and channel catfish exposed to GBS isolates from cattle Ten S. agalactiae isolates from cattle were grown in TSB for 24 hr at 32 ?C. The cattle isolates were taken from original frozen stock cultures stored at -80 ?C. The freshly prepared broth cultures of each isolate were adjusted to an optical density (OD) of 1.0 at 540 nm using a SmartSpec?3000 (Bio Rad, Foster City, CA) and then adjusted to give final concentrations of 1 x 10 9 and 1 x 10 10 colony forming unit (CFU)/mL. Two 15 hundred noninfected Nile tilapia (15.23 + 1.03 g mean weight) and two hundred channel catfish (15.0 + 1.20 g mean weight) were IP injected with 0.1 mL at 1 x 10 9 CFU/mL and 1 x 10 10 CFU/mL of each of the GBS cattle isolates. Two separate tanks containing ten control fish each were inoculated with 0.1 mL of TSB. Two separate tanks with ten fish each served as positive controls and were inoculated with 0.1 mL of a mullet isolate (KU- MU-11-Br) at 1 x 10 9 CFU/mL and 1 x 10 10 CFU/mL, respectively. This mullet isolate (KU-MU-11-Br) had previously been shown to be virulent for Nile tilapia (Evans et al. 2002), even after repeated passages in TSB. Mean water temperature was 32 + 0.6 ?C. Fish were exposed to a light and dark period of 12:12 hours and flow- through water (0.5 L/hour). Aeration was supplied through air stones. The fish were fed daily to satiation with Aquamax Grower 400 (Brentwood, MO). The inoculated fish were then held and observed in individual 57 L aquaria per group for 14 days. Moribund fish were removed and head kidneys and brains were cultured for S. agalactiae. 16 III. RESULTS Serotypes Differences in capsular serotypes between fish and cattle isolates were found (Tables 1 and 2, IX. Appendix Table C). Mullet (n=11), seabream (n=6) and bottlenose dolphin (n=1) isolates collected from the Kuwait Bay epidemic and Nile tilapia from Brazil and Mississippi were serotypes Ia. One hybrid striped bass GBS isolate (Israel) was Ia serotype. In contrast, the cattle isolates (n=10) were non-typable (NT). The other Nile tilapia GBS isolates from Brazil and Honduras were NT. The GBS isolates from Gulf killifish (n=3) were also NT. Further, S. agalactiae references strains ATCC-27956 (n=1) and CECT-183 (n=1) were NT. The S. agalactiae reference strain ATCC-13813 (n=1) was serotype II. The S. difficile reference strain ATCC-51487 (n=1) was serotype Ib. Hemolytic characteristics The results of hemolytic activity showed variations between fish and cattle GBS isolates (Tables 1 and 2, IX. Appendix Table C). All of the mullet, seabream and bottlenose dolphin GBS isolates from the Kuwait Bay epizootic were ?-hemolytic, while five of the cattle isolates were ?-hemolytic and the other five were ?-hemolytic. The GBS Nile tilapia isolates from Larvas, Brazil were non-hemolytic while the GBS Nile tilapia isolates from Parana State, Brazil were ?-hemolytic. The GBS hybrid striped bass isolate (Israel) was also non-hemolytic and the GBS isolates (Louisiana Veterinary 17 Diagnostic Laboratory, LVDL) from tilapia, Gulf killifish and hybrid striped bass were non-hemolytic. CAMP characteristics The results of CAMP factor production revealed that all of the mullet, bottlenose dolphin, and cattle isolates were positive. Three seabream isolates also yielded a positive CAMP test (Tables 1 and 2, IX. Appendix Table C). Nile tilapia GBS isolates from both regions of Brazil and LVDL were found to be CAMP factor negative. The killifish and hybrid striped bass isolates were CAMP negative. Seventy five percent of GBS reference strains were shown to be CAMP factor positive while one of the GBS reference strain was CAMP factor negative. Biochemical characteristics Phenotypic similarities and differences between the GBS fish and cattle isolates were observed (Tables 1 and 2). The mullet, seabream, bottlenose dolphin and cattle isolates were positive for Voges-Proskauer (VP) activity by API-20 Strep and negative for pyrrolidonylarylamidase (PYR) activity by conventional tests. Sixty four percent of the GBS isolates hydrolyzed hippurate (HIP). Of these, 27 % of the mullet isolates were positive. All of the seabream isolates hydrolyzed hippurate. The bottlenose dolphin GBS isolate was negative for hippurate hydrolysis. Hippurate was hydrolyzed by 86 % of the Nile tilapia isolates. Thirty three percent of killifish isolates hydrolyzed hippurate, whereas one hundred percent of the hybrid striped bass were hydrolytic. Hippurate hydrolysis was observed for 50 % of the cattle GBS isolates. The GBS reference strains 18 hydrolyzed hippurate. None of the fish, cattle GBS and reference strains hydrolyzed starch (results not shown). Arginine decarboxylation (ARG) varied between mullet, seabream, bottlenose dolphin and cattle GBS isolates. Mullet and bottlenose dolphin isolates hydrolyzed arginine. Fifty percent of seabream GBS isolates were arginine positive. Sixty seven percent of the Nile tilapia GBS isolates was found not to decarboxylate arginine. Sixty seven percent of the killifish and fifty percent of the hybrid striped bass isolates were arginine positive. Only 50 % of cattle isolates were positive. GBS reference strains decarboxylated arginine. None of the GBS isolates hydrolyzed esculin (ESC) and urea (results not shown). Two GBS reference strains were positive for ESC. Fermentation of D-lactose (LAC) also varied between mullet, seabream and cattle GBS isolates. Mullet, seabream (93 %) and bottlenose dolphin isolates were negative. Only one GBS isolate from Larvas, Brazil were positive for D-lactose. None of the killifish and hybrid striped bass isolates were positive. Ninety percent of cattle GBS isolates fermented D-lactose. Seventy five percent of the GBS reference strains fermented D-lactose. The mullet, seabream and bottlenose dolphin isolates fermented D- trehalose (TRE). One of 14 Nile tilapia was positive. None of the killifish and hybrid striped bass isolates was D-trehalose positive. The S. agalactiae reference strains were positive. All GBS isolates and reference strains fermented ribose. Negative results were noted for sorbitol, inulin and xylulose (results not presented). Mannitol (MAN) was also negative. 19 Rapid enzyme characteristics The results of API-20 Strep determination for ?-galactosidase (?-Gal), ?- glucouronidase (?-Glu), ?-galactosidase (?-Gal) enzyme activities, (PAL) and (LAP) reactions are presented in Tables 1 and 2 and X. Appendix Table D. The seabream and bottlenose dolphin isolates were positive for ?-Gal while about half of the mullet isolates were positive. Only two GBS Nile tilapia isolates from Brazil were ?-Gal positive. None of Nile tilapia GBS isolates from LVDL were positive. The killifish and hybrid striped bass isolates were ?-Gal negative. All of the GBS isolates from cattle were negative. One GBS reference strain was ?-Gal positive. Seabream GBS isolates were negative for ?-Gluc while four mullet GBS isolates were negative (n=4) and six mullet were positive (n=6) for ?-Gluc activity. The Nile tilapia GBS isolates from Larvas, Brazil were ?-Gluc positive while the GBS isolates from Parana State, Brazil were ?- Gluc negative except for one positive isolate. The LVDL tilapia, killifish and hybrid striped bass and Israel hybrid striped bass GBS isolates were observed to be ?-Gluc negative. The cattle isolates were positive. Fifty percent of the GBS reference isolates were ?-Gluc negative. The assays for ?-Gal activity revealed that none of GBS isolates from fish were positive while 2 cattle isolates were positive. The GBS dolphin isolate and reference strains were ?-Gal negative. All GBS fish, cattle isolates and reference strains were PAL and LAP positive. 20 Capsule and hyaluronidase characteristics All of the GBS isolates were encapsulated. The mullet, seabream, bottlenose dolphin and cattle produced hyaluronidase (Tables 1 and 2, XI. Appendix Table E). GBS isolates of Nile tilapia from Brazil did not produced hyaluronidase. Two killifish isolates produced hyaluronidase in contrast to the hybrid striped bass isolates that were negative. One half of the GBS reference strains did not produce this enzyme. Pigment production Pigment production was variable among fish and cattle isolates (Tables 1 and 2, XI. Appendix Table E). The mullet, seabream and bottlenose dolphin isolates produced pigment. None of the Nile tilapia, Gulf killifish, hybrid striped bass or cattle isolates produced pigment. All of the GBS reference strains produced pigment. The growth patterns of GBS isolates in fluid medium and soft agar are presented in Tables 1 and 2 and XII. Appendix Table F. The growth patterns of the GBS isolates from mullet and cattle in fluid medium were sedimentary while the growth pattern of seabream, bottlenose dolphin and the majority Nile tilapia isolates were turbid. In soft agar, GBS displayed generally diffused colony growth (XII. Appendix Table F). FAME characteristics The results for FAME profiles of GBS S. agalactiae from fish and cattle produced by the Microbial Identification System (MIS) and RCLIN50 method are presented in XIII. Appendix Table G. The FAME similarity indexes showed that fish and cattle isolates were a correct match at genus and species levels for S. agalactiae. The FAME similarity indexes (SI) for the GBS S. agalactiae isolates from fish, bottlenose dolphin 21 and reference strain were very good to excellent (0.3 ? 0.9 SI). Lower discrimination at the genus level was noted for one mullet isolate (0.27 SI) and for two cattle isolates (0.18 and 0.12 SI). The SI for ATCC -51487, S. difficile was 0.61 and was correctly matched at the genus and species levels for S. agalactiae. Cattle and fish GBS isolates differed with regard to percent whole cell fatty acid composition (XIV. Appendix Table H). Cattle isolates had 17:iso w5c, 18:1 w5c and 19:1 iso1 whole cell fatty acids while fish isolates and reference strains lacked these whole cell fatty acids. The four GBS S. agalactiae reference strains lacked the 17:0 fatty acid. GBS S. agalactiae isolates from fish were higher in 12:0 and 14:0 whole cell fatty acid percent than found in cattle (16:0, followed by 18:1 w9c, 18:1 w7c and 18:0). FAME data were exported from the GBS library to BioNumerics version 3.0 software package (Applied Maths,) using Pearson?s product-moment correlation coefficients to generate a dendrogram for grouping of the GBS isolates and reference strains with a cut off level of 94 % similarity (Figure 1). The GBS S. agalactiae fish, cattle and reference strains belonged to one of nine groups based on their similarities in whole cell fatty acids. The majority of mullet isolates were grouped together with the S. difficile ATCC-51973 reference strain. The majority of cattle isolates also grouped together in the next cluster. The ATCC-12388 and -12394, S. dysgalactiae reference strains were noted to be similar to the whole cell fatty acid profiles of a Brazil tilapia isolate (04ARS-BZ-TN-005) and a cattle (MOS-59998-17) isolate. Another group of GBS S. agalactiae was represented by the Brazil and Israel striped bass isolates. One Brazil tilapia isolate (03ARS-BZ-TN-01) clustered separately. The largest group 22 consisted of the majority of the tilapia, seabream, killifish, hybrid striped bass, bottlenose dolphin isolates and S. agalactiae reference strains. At 85 % similarity, two major clusters could be observed that consisted of mullet and cattle isolates and isolates from tilapia and other fish species. The FAME analysis appeared to discriminate between the GBS S. agalactiae isolates at the host species and/or origin levels. Gram Positive Identification (GPI) characteristics The results of the GPI analyses showed the genus and species identification was acceptable to very good ranging from 77-99 % probability for the mullet and seabream GBS S. agalactiae isolates (XIII. Appendix Table G). However, the identification of GBS S. agalactiae isolates from cattle was generally unacceptable. Four of the ten cattle isolates were not identified as S. agalactiae. The Israeli hybrid striped bass isolate was not identified as S. agalactiae and had the lowest identification probability at 21 %. GPI analyses correctly identified the reference strains. 23 10098969492908886848280787674 KU-MU-26-INT KU-MU-35-EYE MOS-61185-10 KU-MU-18-HK KU-MU-36-BR KU-MU-25-HK KU-MU-7-BR KU-SB-37-HK KU-SB-38-BR KU-MU-21-BR KU-MU-5-BR ATCC-51973 MOS-61321-2 MOS-61337-3 MOS-59918-14 MOS-61321-11 MOS-61321-3 M0S-55100-2 KU-MU-27-BR ATCC-12388 04ARS-BZ-TN-005 ATCC-12394 MOS-59998-17 ATCC-12386 ATCC-13813 CECT-183 KU-SB-1-HK KU-SB-1-BR KU-SB-1-EYE KU-SB-37-BR ATCC-31475 KU-DL-MUSCLE KU-MU-12-BR LADLA-97-151 03ARS-BZ-TN-05 LADL-00-192 LADL-00-351A LADL-05-108A 03ARS-BZ-TN-04 03ARS-BZ-TN-09 ATCC-27956 KU-MU-20-HK MOS-61185-5 03ARS-BZ-TN-06 LADL-90-503 LADL-02-097 03ARS-BZ-TN-01 04ARS-BZ-TN-007 MOS-59998-7 IS-ET-03-09 04ARS-BZ-TN-003 04ARS-BZ-TN-004 04ARS-BZ-TN-006 04ARS-BZ-TN-002 ATCC-51487 ATCC-43921 % similarity Figure 1. Dendogram of fatty acid profiles of GBS Streptococcus agalactiae from fish, cattle and references strains. Analysis of profile based on Pearson?s product-moment. Cutoff value is 94 % similarity. 24 API-20 Characteristics The API-20 Strep system provided accurate identification of GBS S. agalactiae fish and cattle isolates that ranged from excellent to good identification (X. Appendix Table D). The bottlenose dolphin GBS isolate and the hybrid striped bass isolate from Israel gave a good identification similar to S. agalactiae. The majority of Nile tilapia GBS isolates and cattle isolates varied from good to very good identification. Genotypic characterization Molecular characterization of the GBS isolates was conducted by PCR with specific primers designed against the 23S rRNA gene and 16S-23S intergenic spacer region (ISR). Two different sets of primers were used against the ISR yielding a 190 bp and 130 bp product respectively (XIII. Appendix Table I). The PCR results with 16S- 23S (190 bp) specific primer did not yield the specific amplicon for one seabream and nine Nile tilapia GBS isolates. Positive PCR tests were observed for all GBS cattle and dolphin isolates. The 16S-23S (190 bp) primers did not amplify one seabream isolate, hybrid striped bass isolates, one Gulf killifish isolate, and 11 of the Nile tilapia isolates. A representative PCR pattern for four S. agalactiae, S. dysgalactiae, S. phocae and S. iniae isolate is presented in Figure 2. The 190 bp reactions were observed for all S. agalactiae isolates, but not for the other streptococcal isolates. The 23S specific primer (866 bp) did not amplify 3 seabream, 12 Nile tilapia and 9 cattle isolates. The 23S PCR failed to amplify three reference strains. Figure 3 shows that PCR pattern for the 23S PCR primer. The 866 bp reactions were observed in all S. agalactiae isolates, but not for the other streptococcal isolates. The specific intergenic region 16S-23S (130 bp) primers 25 amplified specific DNA fragments from all fish, cattle, dolphin GBS isolates and reference strains. Figure 4 shows an acceptable PCR pattern for 130 bp reactions. M 1 2 3 4 5 6 7 8 190 bp? Figure 2. The PCR products of streptococcal species based on 16S-23S intergenic rDNA 1 sequence. Lanes: 1, S. agalactiae KU-MU-5-BR; 2, S. agalactiae KU-DL-MUSCLE; 3, S. agalactiae Cattle-55100-2; 4, S. agalactiae type strain ATCC-12386; 5, S. dysgalactiae type strain ATCC-12388; 6, S. phocae type strain ATCC-51973; 7, S. agalactiae type strain ATCC-51487; 8, S. iniae; M, molecular DNA size markers (Bio-Rad, Hercules, CA, USA). 1 Targeted sequence used for analysis of 16S-23S intergenic rDNA obtained from Mata et al. 2004. 26 875 bp? M 1 2 3 4 5 6 7 8 Figure 3. The PCR products of streptococcal species based on 23S rDNA 1 sequence. Lanes: 1, S. agalactiae KU-MU-5-BR; 2, S. agalactiae KU-DL-MUSCLE; 3, S. agalactiae Cattle-55100-2; 4, S. agalactiae type strain ATCC-12386; 5, S. dysgalactiae type strain ATCC-12388; 6, S. phocae type strain ATCC-51973; 7, S. agalactiae type strain ATCC-51487; 8, S. iniae; M, molecular DNA size markers(Bio-Rad, Hercules, CA, USA). 1 Targeted sequence used for analysis of 16S-23S rDNA obtained from Kawata et al. 2004. 27 M 1 2 3 4 5 6 7 8 130 bp? Figure 4. The PCR products of streptococcal species based on 16S-23S intergenic rDNA 1 sequence. Lanes: 1, S. agalactiae KU-MU-5-BR; 2, S. agalactiae KU-DL-MUSCLE; 3, S. agalactiae Cattle-55100-2; 4, S. agalactiae type strain ATCC-12386; 5, S. dysgalactiae type strain ATCC-12388; 6, S. phocae type strain ATCC-51973; 7, S. agalactiae type strain ATCC-51487; 8 S. iniae; M, molecular DNA size markers (Bio-Rad, Hercules, CA, USA). 1 Targeted sequence used for analysis of 16S-23S rDNA obtained from Berridge et al. 2001. 28 Experimental virulence trials The cattle isolates were not infectious (0 % mortality) for Nile tilapia and channel catfish. GBS cattle isolates were also not recovered from S. agalactiae inoculated fish at 24 and 48 hr post-injection. Clinical signs and/or mortality did not occur in the challenged or control fish. Tilapia IP injected with mullet isolate (KU-MU-11-Br) had 60 and 90 % mortality at 1 x 10 9 CFU/mL and 1 x 10 10 CFU/mL, respectively (positive control). AFLP characteristics DNA samples from mullet and seabream isolates from Kuwait Bay epizootic were analyzed by AFLP for genetic similarity between the mullet and seabream isolates (XV. Appendix Table I). Seabream isolates had AFLP profiles similar to the mullet isolates (Figure 5). At 90 % similarity, five distinct clonal groups were identified among these isolates. Clones I contained isolates from mullet and clone II contained isolates from seabream. Clone III contained four mullet and one seabream isolate. A single seabream isolate was observed in clone IV. Clone V contained two seabream isolates. No particular clone could be correlated with a particular fish species. Clone IV contained one seabream isolates that presented the most dissimilar AFLP profile. Interestingly, two seabream isolates recovered from the same fish (SB-37-HK and SB-37- BR) belonged to different AFLP clones. The results suggested that infection was not caused by a single clone of S. agalactiae that originated from either seabream or mullet. The results seem to suggest that at least five different S. agalactiae clones were responsible for the GBS S. agalactiae epidemic. 29 Polyphasic characteristics The results of the comparative investigation of GBS S. agalactiae isolates indicate that phenotypic characteristic were different between fish and cattle origins (Table 10). The differences between fish and cattle isolates were noted in capsule serotype, growth pattern in fluid medium, pigment production, production of hyaluronidase, CAMP factor production, fermentation of D-trehalose and D-lactose and FAME. The results indicated that fish and cattle isolates can be differentiated from each other by the use of a polyphasic approach. Figure 5. Dendogram of amplified fragment length polymorphic (AFLP) profiles of twelve Streptococcus agalactiae isolates from marine fish. The tracks show the processed band patterns after conversion, normalization, and background subtractions. Pearson product-moment similarity coefficients were used to determine linkage levels. Pearson correlation [25.8%-89.2%] AFLP-Hind-A-700 10080 . . . . . . . . . . . . MU-31-EYE MU-5-BR SB-1-BR SB-1-EYE MU-1-BR MU-21-BR MU-12-BR MU-27-BR SB-37-HK SB-1-HK SB-37-BR SB-38-BR I II III IV V 10080 10080 30 Table 1. Phenotypic characteristics of GBS S. agalactiae isolates of fish and cattle and percent of isolates that expressed that characteristic. (NT=non-typable) Percent of isolates (%) Characteristics Fish Cattle n=36 n=10 Catalase production - - Oxidase production - - Lancefield group B + + (100) (100) Serotype Ia NT (56) (100) Capsule + + (100) (100) Turbidity pattern in Sedimentary Sedimentary fluid medium (41) (100) Colony morphology in Diffuse colonies Diffuse colonies soft agar (97) (100) Pigment + - (50) (100) Production of + + hyaluronidase (53) (100) VP test + + (100) (100) Hemolysis ? ? ( 67) (50) CAMP factor - + (61) (100) Hippurate hydrolysis + + (61) (50) Esculine hydrolysis - - (100) (100) Arginine Decarboxylase + + (47) (50) Starch hydrolysis - - (100) (100) Urea hydrolysis - - (100) (100) Pyrolidonylarylamidase - + (100) (10) 31 Table 1. Continued % isolates ?-Galactosidase + - (38) (100) ?-Glucoronidase + + (22) (100) ?-Galactosidase - + (100) (20) Acid production from: D-Sorbitol - - (100) (100) D-Trehalose + - (50) (100) D-Ribose + + (100) (100) Inulin - - (100) (100) D-Mannitol - - (100) (100) D-Xyulose - - (100) (100) D-Lactose + + (6) (90) 1 The symbols denote: n= number of isolates; (-) = negative; (+) = positive; percent of isolates = (%). Phenotypic variations were observed among fish GBS isolates from different species and origins (Table 2). The mullet, seabream and a minority of the Nile tilapia isolates were Ia serotypes, while the Gulf killifish, hybrid striped bass and cattle were NT. A similar pattern of differences between these species was observed for the growth pattern in fluid medium. Nile tilapia, Gulf killifish and hybrid striped bass isolates did not produce pigment. ?-hemolysis was observed in the mullet, seabream, cattle and in a minority of Nile tilapia isolates. CAMP factor production was absent for the Nile tilapia, Gulf killifish and hybrid striped bass isolates. 32 Table 2. Phenotypic and genotypic properties of GBS Streptococcus agalactiae isolates from mullet, seabream, Nile tilapia, Gulf killifish, hybrid striped bass, and S. agalactiae reference strains 1 (Percent of isolates) Phenotypic and genotypic properties Mullet n=11 Seabream n=6 Tilapia n=14 Gulf killifish n=3 Hybrid striped bass n=2 S. agalactiae reference strains n=4 Serotype Ia Ia NT NT NT NT (100) (100) (97) (100) (100) (50) Capsule + + + + + + (100) (100) (100) (100) (100) (100) Growth Sed 2 Tur Tur Sed Sed Sed in fluid medium (100) (100) (85) (100) (50) (50) Colony Diffuse Diffuse Diffuse Diffuse Diffuse Diffuse morphology colonies colonies colonies colonies colonies colonies in soft agar (100) (100) (93) (100) (50) (100) Pigment + + - - - + production (100) (100) (100) (100) (50) (50) Production of + + - + - + hyaluronidase (100) (100) (100) (66) (100) (14) VP test + + + + + + (100) (100) (100) (100) (100) (100) ?-Hemolysis + + + - - + (100) (100) (43) (100) (100) (50) CAMP + + - - - + reaction (100) (50) (100) (100) (100) (75) Hippurate + + + + + + (27) (100) (86) (33) (100) (50) Esculine - - - - - + (100) (100) (100) (100) (100) (50) Arginine + + - + + + (100) (50) (31) (67) (50) (100) Starch - - - - - - (100) (100) (100) (100) (100) (100) Urea - - - - - - (100) (100) (100) (100) (100) (100) ?-Gal + + + - - + (45) (100) (14) (100) (100) (50) 33 Table 2. Continued Phenotypic and genotypic properties Mullet n=11 Seabream n=6 Tilapia n=14 Gulf killifish n=3 Hybrid striped bass n=2 S. agalactiae reference Strains n=4 ?-Gluc + - - - - + (64) (100) (92) (100) (100) (75) ?-Gal - - - - - - (100) (100) (93) (100) (100) (100) D-Sorbitol - - - - - - (100) (100) (100) (100) (100) (100) D-Trehalose + + + - - + (100) (100) (7) (100) (100) (100) D-Ribose + + + + + + (100) (100) (100) (100) (100) (100) Inulin - - - - - - (100) (100) (100) (100) (100) (100) D-Mannitol - - - - - - (100) (100) (100) (100) (100) (100) D-Xylulose - - - - - - (100) (100) (100) (100) (100) (100) D-Lactose - + + - - + (100) (17) (7) (100) (100) (100) Specific PCR 16S- 23S + + + + + + rDNA 130bp (100) (100) (100) (100) (100) (100) Specific PCR 16S- 23S + + + - + + rDNA 190bp (100) (83) (31) (67) (50) (100) Specific PCR -23S rDNA + + + - - + 866 bp (100) (67) (15) (100) (100) (75) 1 The symbols denote:n = number of isolates; (-) = negative; (+) = positive; (% isolates). 2 The following symbols denote: sed = sediment; tur = turbid. 34 Hydrolysis of hippurate was more pronounced for cattle isolates. Fifty percent of the cattle isolates deaminate arginine while fish isolates did not. The majority of Nile tilapia did not ferment D-trehalose while cattle, mullet and seabream did ferment this sugar to acid. The majority of the cattle isolates fermented D-lactose and other fish isolates were non-fermentative. In addition, 16S-23S (190 bp) and 23S (866 bp) primers did not identify all the fish isolates by PCR. The phenotypic characteristics and 16S-23S intergenic spacer region primer (130 bp) for S. agalactiae by PCR clearly revealed that the S. difficile was S. agalactiae (IX. and XIII. Appendix C, Appendix G). S. agalactiae isolates and reference strains were differentiated from the S. dysgalactiae, S. phocae, S. iniae and Lactococcus garviae by bacteriological properties and specific primers for 16S-23S intergenic region (130 bp) by PCR. 35 IV. DISCUSSION GBS S. agalactiae is the cause of severe disease affecting fish and cattle. However, the possibility of transmission of GBS fish isolates infecting cattle or vice versa has not been established. Based on phenotypic, biological, molecular characteristics and mortality results from infectivity studies using cattle GBS isolates to infect tilapia and catfish, the potential likelihood for cattle GBS isolates to infect fish appears to be limited or nonexistent. The hypothesis that phenotypic and genotypic characteristics of GBS fish isolates were different from GBS cattle isolate was verified. The phenotypic characteristics of GBS S. agalactiae isolates from fish were similar to the properties of the ATCC S. agalactiae reference strains and different from ATCC S. dysgalactiae, S. iniae, S. phocae and L. garviae reference strains. The ATCC S. difficile reference strain was found to be identical to S. agalactiae from fish and cattle and to the ATCC S. agalactiae reference strains. Finch and Martin (1984) demonstrated that cattle and human isolates were separate populations that shared the common group B antigen. In a comparative molecular study between GBS S. agalactiae of bovine mastitis and human origins, Bohnsack et al. (2004) reported that bovine and human GBS isolates were largely unrelated. Yildirim et al. (2002a) reported that cattle GBS isolates were nonpigmented, utilized lactose and hemagglutinated rabbit erythrocytes. The results also showed that GBS isolates from dogs, cats and humans were generally lactose negative, pigmented and 36 did not hemagglutinate rabbit erythrocytes. In the present study, we observed that GBS isolates from cattle were not pigmented and generally fermented lactose. Yildirim et al. (2002a), also reported that the capsular serotype of GBS cattle isolates was generally NT with a few IV and Ia serotypes. In the present study, GBS isolates from cattle were NT serotype. The results of this study also agree with those of Yidirim et al. (2002a) for the growth patterns of GBS cattle isolates in fluid medium and soft agar, sedimentary and diffuse colony morphology, respectively. Yildirim et al. (2002a) reported that serotype, growth properties in fluid medium, soft agar and fermentation of lactose may be generally used to distinguish between GBS isolates from cattle, humans, dogs and cats. A similar conclusion that fish and cattle isolates may be generally distinguished based on these bacteriological characteristics was derived here. Merl et al. (2003) observed that GBS cattle isolates were generally nontypable, pigmented, expressed sedimentary growth in fluid medium and diffuse colony morphology on soft agar. The only bacteriological characteristic difference between their study and those of the present study was pigmentation. McDonald et al. (2005) reported that the bacteriological characteristics of GBS S. agalactiae isolates from cattle were group B, CAMP factor positive, Voges-Proskauer and esculin negative, hippurate positive, lactose, ribose and trehalose positive. A small number of isolates were mannitol and sorbitol positive. The principal differences in bacteriological characteristics between results of McDonald et al. (2005) and the present study using cattle isolates from Wisconsin were hippurate hydrolysis, sorbitol and trehalose fermentation. 37 Phenotypic characteristics of GBS S. agalactiae from mullet and seabream were reported using conventional and API Rapid ID 32 tests by Evans et al. (2002). The results of Evans et al. (2002) and those of the present study were generally identical for mullet and seabream GBS isolates with regard to hemolysis, oxidase, catalase, leucine aminopeptidase, Voges-Proskauer, hydrolysis of hippurate, arginine deamination , ?- galactosidase, ?-galactosidase, ?-glucuronidase, alkaline phosphatase, fermentation of ribose, trehalose and lactose. The phenotypical results of GBS S. agalactiae isolates from silver pomfret (Pampus argenteus) using the API -20 Strep system reported by Duremdez et al. (2004) were generally similar to those of the present study and Evans et al. (2002) for mullet and seabream GBS isolates. A major difference was that Evans et al. (2002) reported that GBS isolates were ?-hemolytic and Duremdez et al. (2004) reported that the GBS isolates were ?-hemolytic. Hetzel et al. (2003) reported that emerald monitors (Varanus prasinus) became septicemic when infected by S. agalactiae that was acquired from wild mice. Genotypic analysis of DNA from the S. agalactiae isolates from monitors and mice revealed that they were identical. However, identification of the zoonotic species of cattle streptococci and their relevance to fish is likely to be unimportant. Cross-species transmission of GBS cattle isolates is unlikely to occur. The result of the present study indicated that cattle GBS S. agalactiae isolates were not virulent for experimentally challenged Nile tilapia and channel catfish (Hypothesis II). Sukhnanand et al. (2005) concluded from the results of molecular and biochemical characterization of GBS S. agalactiae isolates from cattle and humans that they appeared different with regard to 38 host specificity and pathogenesis. Sukhnanand et al. (2005) suggested that the potential for transmission of S. agalactiae from a bovine host to a human host or vice versa was low. Transmission of S. agalactiae within a fish species and between fish species is probable. Evans et al. (2002) demonstrated that tilapia were susceptible to mullet and seabream S. agalactiae, even after culture There is a limited possibility that the single passage of the cattle isolate in culture immediately before the experimental challenge trials affected virulence. In the present study, the GBS disease in mullet and seabream was caused by five clones of S. agalactiae, but it was not possible to determine whether the disease was transmitted from seabream to mullet or vice versa (objective III). The virulence and evolutionary adaptation of GBS S. agalactiae for fish makes S. agalactiae a source of infection for susceptible fish from dead diseased fish. The duration of S. agalactiae survival and pathogenicity was investigated by Garcia (2004). He suggested that S. agalactiae can survive for long periods of time in fresh, brackish and marine waters and remains pathogenic for Nile tilapia. The S. difficile reference strain was identified as a S. agalactiae isolate from tilapia as previously reported by Vandamme et al. (1997), Berridge et al. (2001) and Kawamura et al. (2005). In our studies, bacteriological properties and 16S-23S intergenic region (130 bp) specific primer for S. agalactiae by PCR clearly revealed that the S. difficile was undistinguishable from S. agalactiae. Streptococcus agalactiae isolates and reference strains were differentiated from the S. dysgalactiae and S. phocae by bacteriological properties and specific primers for 16S-23S intergenic region by PCR. 39 The phenotypic characteristics used in this study were able to identify and differentiate GBS S. agalactiae from fish and cattle origins. The phenotypic characteristics of S. agalactiae reference strains were similar to those of the fish and cattle isolates. Each fish isolate possessed the group B antigen, turbidity growth pattern in fluid medium and produced hyaluronidase and fermented D-trehalose. In contrast, the cattle isolates possessed the group B antigen, produced sediment growth pattern in fluid medium and pigment, were CAMP positive, hydrolyzed hippurate, decarboxylated ?- glucoronidase, fermented D-lactose and had a generally defined FAME profile. The results suggest that these phenotypic characteristics can be used to differentiate between fish and cattle GBS S. agalactiae. However, CAMP test and hydrolysis of hippurate were noted not to be presumptive identification tests for GBS S. agalactiae isolates as suggested by Facklam (2002). Fuchs et al. (1978) demonstrated that presumptive tests of CAMP, hippurate hydrolysis and pigment production correlated with human clinical GBS cases. Our results do not support the use of CAMP, hippurate hydrolysis and pigment production for the presumptive identification of GBS S. agalactiae isolates from fish. The PCR tests with specific primer, API-32 Strep and Gram positive identification system (GPI) did not differentiate GBS S. agalactiae isolated from fish from those of cattle origin. On the other hand, FAME did produce a good degree of correlation with the isolate cluster and the host species. Jayarao et al. (1991) reported that GPI and API Rapid Strep systems gave comparable results for the identification of GBS S. agalactiae from cattle. The majority 40 of cattle isolates were identified at the excellent level. In the present study, we found the GPI system to be less accurate than API-20 Strep system in the identification of GBS S. agalactiae from cattle. The API-20 Strep system gave very good to excellent identification of fish and cattle isolates. Molecular genetic methods such as AFLP may be useful to distinguish S. agalactiae isolates between host species at the clonal level. Rapid and accurate PCR tests using specific 16S-23S intergenic region primers can identify S. agalactiae isolates from fish and cattle. Future identification and strain differentiation tests may be based on specific monoclonal antibody and/or molecular genetic technologies. 41 V. SUMMARY The collection of GBS S. agalactiae cattle isolates used in the present study was heterogeneous in regards to phenotypic characteristics, and GBS fish isolates could be distinguished from GBS cattle isolates by selected phenotypic properties. The hypothesis that the GBS fish and cattle populations were distinct was validated. FAME profiles were able to distinguish between the major clusters of fish and cattle isolates that had similar profiles. Mullet and seabream S. agalactiae isolates were distinguishable from Nile tilapia isolates, both phenotypically and with regard to whole cell fatty acid profiles. The cattle S. agalactiae isolates were found not to be infectious for experimentally inoculated Nile tilapia. The twelve S. agalactiae isolates from mullet and seabream isolated during the Kuwait Bay epidemic were revealed to belong to five distinct clonal groups by AFLP. Two seabream isolates recovered from the brain and head kidney from the same fish belonged to separate AFLP clones. Therefore, the GBS epidemic involved the dissemination of multi clones within the fish population rather than the dissemination of a single clone of S. agalactiae. This finding validated the final objective that infection between the two fish populations was not due to a single clone of S. agalactiae. To our knowledge, this is the first report to describe phenotypic, genotypic characteristics, API-20 Strep analysis, GPI and FAME characterization of GBS S. agalactiae isolates from fish and cattle. A polyphasic approach should be used to identify and distinguish isolates of GBS S. agalactiae similar to that proposed by Arias et 42 al. (2003, 2004). Genotypic, bacteriological and specific monoclonal antibody characterization may contribute to a more complete understanding of the epidemiological aspects of GBS isolates from fish from a regional and global standpoint. 43 Table 3: Summary of cost, discrimination level, disadvantages and advantages of methods utilized for the comparison of Streptococcus agalactiae isolates from cattle and fish. Method Advantage Disadvantages Discrimination Level 1 Estimated Cost 2 Phenotypic Methods Important for delineation of taxa. Easy to perform test. Proven methods. Laborious, time consuming, often difficult to standardized and interpret. Effective for delineation of taxa. $ - $$$$$* API 20 Strep strip Multi biochemical test, time efficient, semi automated systems. Results dependent on color reaction. Variable results dependent on reader. Effective for identification of bacterial species already identified by the API software. However, it can be misleading when used for the identification of a new species. $$$$ Gram positive identification (GPI) Time efficient, fully automated and computerized system. High equipment cost. Will not identify or will misidentify organisms not present in the software database. Effective for species level identification. $$$$ 44 Table 3. Continued Method Advantage Disadvantages Discrimination Level 1 Estimated Cost 2 Gram positive identification (GPI) Can be affected by growth phase, temperature and growth medium. Polymerase chain reaction (PCR) Time efficient, reproducible, semi automated system. Can be very sensitive. Good for identification and detection of bacterial samples. High equipment cost, expensive reagents, technologically complex. Prone to contamination. Depending on application, species or strains can be identified. $$$$ Amplified fragment length polymorphism Excellent discrimination level. Semi- automated. (AFLP) High equipment cost, time consuming, specific analyzing software required, success of technique relies in expert operator. Effective for discrimination between species of a genus or different strains of a single species. $$$$$ 1 Discrimination power of each of the methods utilized for the phenotypic and genotypic identification of Streptococcus agalactiae isolates from cattle and fish. Family, Genus, Single clone. 2 Estimated cost of methods used for identification of Streptococcus agalactiae isolates from cattle and fish. Estimated cost of individual sample; $ = $1-2, $$ = $3-4, $$$ = $5-6, $$$$ = $7-8, $$$$$ = $9-10. 45 Table 3. 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Isolate Fish species, organ of isolation, state and country of origin KU-MU-5-BR 1 Mullet, brain, Kuwait Bay, Kuwait KU-MU-7-BR Mullet, brain, Kuwait Bay, Kuwait KU-MU-12-BR Mullet, brain, Kuwait Bay, Kuwait KU-MU-18-HK Mullet, head kidney, Kuwait Bay, Kuwait KU-MU-20-HK Mullet, head kidney, Kuwait Bay, Kuwait KU-MU-21-BR Mullet, brain, Kuwait Bay, Kuwait KU-MU-25-HK Mullet, head kidney, Kuwait Bay, Kuwait KU-MU-26-INT Mullet, intestine, Kuwait Bay, Kuwait KU-MU-27-BR Mullet, brain, Kuwait Bay, Kuwait KU-MU-35-EYE Mullet, eye, Kuwait Bay, Kuwait KU-MU-36-BR Mullet, brain, Kuwait Bay, Kuwait KU-SB-1-BR Seabream, brain, Kuwait Bay, Kuwait KU-SB-1-HK Seabream, head kidney KU-SB-1-EYE Seabream, eye, Kuwait Bay, Kuwait KU-SB-37-BR Seabream, brain, Kuwait Bay, Kuwait KU-SB-37-HK Seabream, head kidney, Kuwait Bay, Kuwait KU-SB-38-BR Seabream, brain, Kuwait Bay, Kuwait KU-DL-MUSCLE Bottlenose dolphin, muscle, Kuwait bay, Kuwait 03ARS-BZ-TN-01 2 Nile tilapia, brain, Larvas, Brazil 03ARS-BZ-TN-03 Nile tilapia, brain, Larvas, Brazil 03ARS-BZ-TN-04 Nile tilapia, brain, Larvas, Brazil 03ARS-BZ-TN-05 Nile tilapia, brain, Larvas, Brazil 03ARS-BZ-TN-06 Nile tilapia, brain, Larvas, Brazil 03ARS-BZ-TN-09 Nile tilapia, brain, Larvas, Brazil LADL-90-50 3 Hybrid striped bass, brain, Louisiana, USA LADL-97-151 Gulf killifish, brain, Louisiana, USA LADL-00-192 Gulf killifish, brain, Louisiana, USA LADL-00-351a Nile tilapia, brain, Mississippi, USA LADL-02-097 Gulf killifish, brain, Louisiana, USA LADL-05-108a Nile tilapia, brain, Honduras IS-ET-09-03 4 Hybrid striped bass, brain, Israel 04ARS-BZ-TN-002 5 Nile tilapia, brain, Parana State, Brazil 04ARS-BZ-TN-003 Nile tilapia, brain, Parana State, Brazil 04ARS-BZ-TN-004 Nile tilapia, brain, Parana State, Brazil 04ARS-BZ-TN-005 Nile tilapia, brain, Parana State, Brazil 04ARS-BZ-TN-006 Nile tilapia, brain, Parana State, Brazil 04ARS-BZ-TN-007 Nile tilapia, brain, Parana State, Brazil Cattle-55100-2 6 Bovine mastitis, milk sample, Wisconsin, USA Cattle-59918-14 Bovine mastitis, milk sample, Wisconsin, USA Cattle-59998-7 Bovine mastitis, milk sample, Wisconsin, USA 59 VII. Appendix Table A. Continued Isolate Fish species, organ of isolation, state and country of origin Cattle-59998-1 Bovine mastitis, milk sample, Wisconsin, USA Cattle-61185-5 Bovine mastitis, milk sample, Wisconsin, USA Cattle-61185-10 Bovine mastitis, milk sample, Wisconsin, USA Cattle-61321-2 Bovine mastitis, milk sample, Wisconsin, USA Cattle-61321-3 Bovine mastitis, milk sample, Wisconsin, USA Cattle-61321-11 Bovine mastitis, milk sample, Wisconsin, USA Cattle-61337-3 Bovine mastitis, milk sample, Wisconsin, USA ATCC-12386 7 Streptococcus agalactiae, grouping strain, American Type Culture Collection (ATCC) ATCC-13813 Streptococcus agalactiae, bovine mastitis, milk samples United Kingdom, ATCC ATCC-27956 Streptococcus agalactiae, bovine mastitis, milk samples Wisconsin, USA, ATCC ATCC-31475 Streptococcus agalactiae, human meningitis, Texas, USA, ATCC ATCC-51487 Streptococcus difficile (S. agalactiae), tilapia sp., brain, Israel, ATCC ATCC-51973 Streptococcus phocae, group strain, USA, seal ATCC-12388 Streptococcus dysagalactiae, grouping strain, ATCC ATCC-12394 Streptococcus dysagalactiae, grouping strain, ATCC ATCC-43921 Lactococcus garvieae, bovine mastitis, milk, USA, ATCC CECT-138 8 Streptococcus agalactiae, bovine mastitis, Spain, Colecci?n, Espa?ola De Cultivos 1 Streptococcus agalactiae isolates from Evans et al. 2002. 2 Streptococcus agalactiae isolates from Nile tilapia (Oreochromis niloticus), reared in cage culture in lakes and ponds in the region of Larvas, Brazil. 3 Streptococcus agalactiae isolates from Dr. John Hawke, Louisiana Veterinary Diagnostic Laboratory, Louisiana State University, Baton Rouge, Louisiana. 4 Dr. Dina Zilberg, The Albert Katz Department of Dryland Biotechnologies, Ben- Gurion, University of the Negev, Israel. 5 Streptococcus agalactiae isolates from Nile tilapia, (Oreochromis niloticus) reared in cage culture in lakes and ponds in the region of Parana State, Brazil. 6 Streptococcus agalactiae cattle mastitis milk isolates from Department of Pathobiology, Diagnostic Laboratory, College of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 7 References strains from American Type Culture Collection, Washington, DC, USA. 8 References strains from Colecci?n Espa?ola De Cultivos, Universidad de Valencia y Unidad Asociada al Consejo Superior de Investigaciones Cient?ficas, Spain 60 VIII. Appendix Table B. Target gene, oligonucleotide sequence and target size for polymerase chain reaction (PCR) used in the present study. Target Gene Sequence PCR amplicon base pair (bp) 16S-23S rDNA Forward CCACGATCTAGAAATAGATTG 130 intergenic spacer Reverse TGCCAAGGCATCCACC 16S-23S rDNA Forward AGGAAACCTGCCATTTGCG 190 intergenic spacer 2 Reverse CAATCTATTTCTAGATCGTGG 23s rDNA 3 Forward AACAGCCTCGTATTTAAAATGATAGATTAAC 875 Reverse TCCTACCATGACACTAATGTGTC 1 Berridge et al. 2001, 2 Mata et al. 2004, and 3 Kawata et al. 2004. The amplicons for each set of primers were sequenced by these authors. 61 IX Appendix Table C. Phenotypic properties of GBS Streptococcus agalactiae isolates from fish, bottlenose dolphin, cattle isolates and reference strains of S. agalactiae, S. dysagalactiae, S. difficile and S. phocae Isolate Ser 1 He Ca Py Hip Arg Es Tre Man Lac KU-MU-5-BR Ia ? + - - + - + - - KU-MU-7-BR Ia ? + - - + - + - - KU-MU-12-BR Ia ? + - + + - + - - KU-MU-18-HK Ia ? + - + + - + - - KU-MU-20-HK Ia ? + - + + - + - - KU-MU-21-BR Ia ? + - - + - + - - KU-MU-25-HK Ia ? + - - + - + - - KU-MU-26- INT Ia ? + - - + - + - - KU-MU-27-BR Ia ? + - - + - + - - KU-MU-35- EYE Ia ? + - - + - + - - KU-MU-36-BR Ia ? + - - + - + - - KU-SB-1HK Ia ? + - + + - + - + KU-SB-37HK Ia ? + - + + - + - - KU-SB-38BR Ia ? + - + + - + - - KU-SB-1-EYE Ia ? - - + - - + - - KU-SB-37-Br Ia ? - - + - - + - - KU-SB-1-Br Ia ? - - + - - + - - KU-DL- MUSCLE Ia ? + - - + - + - - 03ARS-BZ- TN-01 NT - - - + - - - - - 03ARS-BZ- TN-04 NT - - - - - - - - - 03ARS-BZ- TN-05 Ia - - - + - - + - + 03ARS-BZ- TN-06 NT - - - + + - - - - 03ARS-BZ- TN-09 NT - - - + - - - - - LADL-90-503 NT - - - + + - - - - LADL-97-151 NT - - - + + - - - - LADL-00-192 NT - - - + + - - - - LADL-00- 351A Ia - - - + + - - - - LADL-02- 097 NT - - - - - - - - - LADL-05- 108A NT - - - + + - - - - IS-TN-09-03 NT - - - + - - - - - 62 IX. Appendix Table C. Continued Isolate Ser 1 He Ca Py Hip Arg Es Tre Man Lac 04ARS-BZ- TN-003 NT ? - - + - - - - - 04ARS-BZ- TN-004 NT ? - - + - - - - - 04ARS-BZ- TN-006 NT ? - - + - - - - - 04ARS-BZ- TN-005 NT ? - - + - - - - - 04ARS-BZ- TN-007 NT ? - - + - - - - - Cattle-55100-2 NT ? + - + - - - - - Cattle -59918- 14 NT ? + - + - - - - + Cattle -59998-7 NT ? + - - - - - - + Cattle -59998- 17 NT ? + - + - - - - + Cattle -61185-5 NT ? + - - + - - - + Cattle -61185- 10 NT ? + + + + - - - + Cattle -61321-2 NT ? + - - + - - - + Cattle -61321-3 NT ? + - + + - - - + Cattle -61321- 11 NT ? + - - + - - - + Cattle -61337-3 NT ? + - - - - - - + ATCC-12386 NT ? + + + + + + - + ATCC-13813 II ? + - + + - + - + ATCC-27956 NT ? - - + + + + - + ATCC-31475 III ? + + + + - + - - ATCC-51487 lb - - - + - - - - CECT-183 NT - + - + - - + - + ATCC-12388 NT ? - - - - + + - - ATCC-12394 NT ? - - - + + + - + ATCC-51973 NT ? + - - - - + + - 1 Capsule serotype determined by typing antisera (Ia, Ib, II and III), 270023, Denka Seiken, Tokyo, Japan. The following symbols denote: He = type of hemolysis on 5% sheep blood agar; Ca = CAMP reaction; Py = Pyrrolidonylarylamidase reaction; 63 IX. Appendix Table C. Continued Vp = Voges-Prokauers reaction, all isolates negative, not shown; Hip = Hippurate hydrolysis; Arg= Arginine hydrolysis; Es = Esculin hydrolysis; Fermentation of Tre, D-trehalose; Man, D-mannitol; Lac, D-lactose; and (+) = positive reaction and (-) = negative reaction. 64 X. Appendix Table D. API-20 Strep results of ?-galactosidase (?-gal), ?- glucosidase (?-Gluc), ?-galactosidase, (?-Gal), alkaline phosphase (PAL), aminopepidase (LAP). Properties for GBS Streptococcus agalactiae isolates from fish, bottlenose dolphin, cattle isolates and reference strains of S. agalactiae, S. difficile, S. dysagalactiae and S. phocae Isolates ?-Gal ?-Gluc ?-Gal PAL LAP KU-MU-5-BR - 1 + - + + KU-MU-7-BR - + - + + KU-MU-12-BR + - - + + KU-MU-18-HK + - - + + KU-MU-20-HK + - - + + KU-MU-21-BR - + - + + KU-MU-25-BR - + - + + KU-MU-26-INT - + - + + KU-MU-27-BR - + - + + KU-MU-35-EYE + - - + + KU-MU-36-BR + - - + + KU-SB-1-HK + - - + + KU-SB-37-HK + - - + + KU-SB-38-BR + - - + + KU-SB-1-EYE + - - + + KU-SB-37-BR + - - + + KU-SB-1-BR + - - + + KU-DL-MUS + + - + + 03ARS-BZ-TN- 01 - + - + + 03ARS-BZ-TN- 04 - + - + + 03ARS-BZ-TN- 05 - + - + + 03ARS-BZ-TN- 06 - + - + + 03ARS-BZ-TN- 09 - + - + + LADL-90-503 - - - + + LADL-97-151 - - - + + LADL-00-192 - - - + + LADL-00-351A - - - + + LADL-02-097 - - - + + LADL-05-108A - - - + + IS-TN-09-03 - - - + + 04ARS-BZ-TN- 002 + - - + + 65 X. Appendix Table D. Continued Isolates ?-Gal ?-Gluc ?-Gal PAL LAP 04ARS-BZ-TN- 003 - - - + + 04ARS-BZ-TN- 004 - - - + + 04ARS-BZ-TN- 005 + + - + + 04ARS-BZ-TN- 006 - - - + + 04ARS-BZ-TN- 007 - - - + + Cattle-55100-2 - + - + + Cattle-59918-14 - + - + + Cattle-59998-7 - + + + + Cattle-59998-17 - + - + + Cattle-61185-5 - + - + + Cattle-61185-10 - + - + + Cattle-61321-2 - + - + + Cattle-61321-3 - + + + + Cattle-61321-11 - + - + + Cattle-61337-3 - + - + + ATCC-12386 - + - + + ATCC-27956 + - - + + ATCC-31475 - + - + + ATCC-12394 - + - + + ATCC-51487 - + - + + CECT-183 - + - + + ATCC-12388 - + - + + ATCC-13813 + - - + + ATCC-51973 - - - + + 1 The symbols denote: (+) = positive and (-) = negative reactions. 66 XI. Appendix Table E. Capsule properties, hyaluronidase activity and pigment production for GBS Streptococcus agalactiae isolates from fish, bottlenose dolphin, cattle isolates and reference strains of S. agalactiae, S. difficile, S. dysagalactiae and S. phocae. Isolate Capsule Property 1 Hyaluronidase activity 2 Pigment production 3 KU-MU-5-BR + + + KU-MU-7-BR + + + KU-MU-12-BR + + + KU-MU-18-HK + + + KU-MU-20-HK + + + KU-MU-21-BR + + + KU-MU-25-BR + + + KU-MU-26-INT + + + KU-MU-27-BR + + + KU-MU-35-EYE + + + KU-MU-36-BR + + + KU-SB-1-HK + + + KU-SB-37-HK + + + KU-SB-38-BR + + + KU-SB-1-EYE + + + KU-SB-37-BR + + + KU-SB-1-BR + KU-DL-MUSCLE + + + 03ARS-BZ-TN-01 + - - 03ARS-BZ-TN-04 + - - 03ARS-BZ-TN-05 + - - 03ARS-BZ-TN-06 + - - 03ARS-BZ-TN-09 + - - LADL-90-503 + - - LADL-97-151 + + - LADL-00-192 + - - LADL-00-351A + - - LADL-02-097 + + - LADL-05-108A + - - IS-TN-09-03 + + 04ARS-BZ-TN- 002 + - - 04ARS-BZ-TN- 003 + - - 04ARS-BZ-TN- 004 + - - 67 XI. Appendix Table E. Continued Isolate Capsule Property 1 Hyaluronidase activity 2 Pigment production 3 04ARS-BZ-TN- 005 + - - 04ARS-BZ-TN- 006 + - - 04ARS-BZ-TN- 007 + - - Cattle-55100-2 + + - Cattle-59918-14 + + - Cattle-59998-7 + + - Cattle-59998-17 + + - Cattle-61185-5 + + - CattleS-61185-10 + + - Cattle-61321-2 + + - Cattle-61321-3 + + - Cattle-61321-11 + + - Cattle-61337-3 + + - ATCC-12386 + + + ATCC-13813 + - + ATCC-27956 + - + ATCC-31475 + + + ATCC-51487 + - + CECT-183 + - ATCC-12388 + + - ATCC-12394 + + - ATCC-51973 + + - The symbols denote: (+) = positive and (-) = negative reactions. 1 Atlas et al. 1995. The negative capsule stain test were denoted by present of capsule (+) and absence of capsule (-). 2 Smith and Willett 1968. The hyaluronidase plate tests were denoted by present enzymes (+) and absence of enzyme (-). 3 Merritt and Jacobs 1976. 68 XII. Appendix Table F. Growth properties in broth and soft agar for GBS Streptococcus agalactiae isolates from fish, bottlenose dolphin, cattle isolates and reference strains of S. agalactiae, S. difficile, S. dysagalactiae, S. phocae 1 . Isolate Fluid medium 2 Soft agar medium 3 KU-MU-5-BR S D KU-MU-7-BR S KU-MU-12-BR S D KU-MU-18-HK S KU-MU-20-HK S D KU-MU-21-BR S KU-MU-25-BR S D KU-MU-26-INT S KU-MU-27-BR S D KU-MU-35-EYE S KU-MU-36-BR S D KU-SB-1-HK T KU-SB-37-HK T D KU-SB-38-BR T KU-SB-1-EYE T D KU-SB-37-BR T KU-SB-1-BR T D KU-DL-MUSCLE T 03ARS-BZ-TN-01 T D 03ARS-BZ-TN-04 T 03ARS-BZ-TN-05 T D 03ARS-BZ-TN-06 T C 03ARS-BZ-TN-09 T D LADL-90-503 S LADL-97-151 S D LADL-00-192 S LADL-00-351A S D LADL-02-097 S LADL-05-108A S D IS-TN-09-03 T 04ARS-BZ-TN-002 T D 04ARS-BZ-TN-003 T 04ARS-BZ-TN-004 T D 04ARS-BZ-TN-005 T 04ARS-BZ-TN-006 T D 04ARS-BZ-TN-007 T Cattle-55100-2 S D 69 XII. Appendix Table F. Continued Isolate Fluid medium Soft agar medium Cattle-59918-14 S D Cattle-59998-7 S Cattle-59998-17 S D Cattle-61185-5 S CattleS-61185-10 S D Cattle-61321-2 S Cattle-61321-3 S D Cattle-61321-11 S Cattle-61337-3 S D ATCC-12386 T ATCC-13813 S D ATCC-31475 S ATCC-27956 S C ATCC-51487 S D CECT-183 T ATCC-12388 T D ATCC-12394 S ATCC-51973 S D 1 Merl et al. 2003. The following symbols denote the results of growth characteristics in broth cultures; 2 S=sediment and clear supernatant and T = turbidity. 3 The results in soft agar were denoted by C = compact colonies and D = diffuse colonies. 70 XIII. Appendix Table G. Fatty acid methyl esters, Gram positive identification (GPI), API-20 Strep, polymerase chain reaction (PCR), Streptococcus agalactiae isolates from fish, Bottlenose dolphin, cattle and reference strains of S. agalactiae, S. dysagalactiae and S. phocae Isolates and species obtained from FAME 1 GPI Profile 2 API-20 Strep Profile 3 PCR 16S- 23S rDNA 4 190 bp PCR 23S rDNA 5 875 bp PCR 16S-23S intergenic rDNA 6 130 bp KU-MU- 5-BR 0.863 99.0% 3463014 Ex + + + KU-MU- 7-BR 0.798 89.0% 3463014 Ex + + + KU-MU- 12-BR 0.725 99.0% 3263010 VG + + + KU-MU- 18-HK 0.724 92.0% 3262010 G + + + KU-MU- 20-HK 0.833 99.0% 3463014 Ex + + + KU-MU- 21-BR 0.905 99.0% 3463014 Ex + + + KU-MU- 25-HK 0.827 99.0% 3463014 Ex + + + KU-MU- 26-INT 0.657 89.0% 3063014 Ex + + + KU-MU- 27-BR 0.270 79.0% 3063014 Ex + + + KU-MU- 35-EYE 0.539 99.0% 3463014 Ex + + + KU-MU- 36-BR 0.856 92.0% 3463014 Ex + + + KU-SB-1- HK 0.878 99.0% 3263014 VG - + + KU-SB- 37-HK 0.831 99.0% 3263014 VG + + + KU-SB- 38-Br 0.871 99.0% 3263014 VG + + + KU-SB-1- EYE 0.861 77.0% 3263014 VG + - + 71 XIII. Appendix Table G. Continued Isolates and species obtained from FAME 1 GPI Profile 2 API-20 Strep Profile 3 PCR 16S- 23S rDNA 4 190 bp PCR 23S rDNA 5 875 bp PCR 16S-23S intergenic rDNA 6 130 bp KU-SB- 37-Br 0.921 88.0% 3263014 VG + - + KU-SB-1- Br 0.885 77.0% 3263014 VG + - + KU-DL- MUS 0.704 71.0% 1663014 G + + + 03-ARS- BZ-TN-01 0.633 94.0% 3061000 G + + + 03-ARS- BZ-TN-04 0.815 94.0% 3061000 G + - + 03-ARS- BZ-TN-05 0.887 94.0% 3061000 G + + + 03-ARS- BZ-TN-06 0.825 94.0% 3061000 G - - + 03-ARS- BZ-TN-09 0.840 94.0% 3061000 G + - + LVDL-90- 503 0.784 94.0% 3023000 G - - + LVDL-97- 151 0.835 94.0% 3023000 G + - + LVDL-00- 192 0.695 94.0% 3023000 G + + LVDL-00- 351a 0.843 94.0% 3023000 G - - + LVDL-02- 097 0.587 94.0% 3023000 G - - + LVDL-05- 108a 0.658 94.0% 3023000 G - - + IS-TN-09- 03 0.860 21.0% 3463000 G - - + 04ARS- BZ-TN- 002 0.735 94.0% 3063000 VG - - + 04ARS- BZ-TN- 003 0.795 94.0% 3063000 VG - - + 72 XIII. Appendix Table G. Continued Isolates and species obtained from FAME 1 GPI Profile 2 API-20 Strep Profile 3 PCR 16S- 23S rDNA 4 190 bp PCR 23S rDNA 5 875 bp PCR 16S-23S intergenic rDNA 6 130 bp 04ARS- BZ-TN- 004 0.792 94.0% 3063000 VG - - + 04ARS- BZ-TN- 007 0.560 94.0% 3063000 VG - - + Cattle- 55100-26 0.510 UN 3463410 G + + + Cattle- 59918-14 0.645 93.0% 3463404 Ex + + + Cattle - 59998-7 0.584 UN 3472400 G + + + Cattle - 59998-17 0.353 82.0% 3463404 Ex + + + Cattle - 61185-5 0.752 99.0% 3563400 G + - + Cattle - 61185-10 0.746 94.0% 3563400 G + - + Cattle - 61321-2 0.269 88.0% 563400 G + + + Cattle - 61321-3 0.178 71.% 3563404 G + + + Cattle - 61321-11 0.134 UN 563410 Ex + + + Cattle - 61337-3 0.321 48.0% 3463404 VG + + + ATCC- 12386 0.529 73.0% 3563415 VG + - + ATCC- 13813 0.754 91.0% 3263414 VG + + + ATCC- 27956 0.557 53.0% 3261415 VG + + + ATCC- 31475 0.624 91.0% 3563014 VG + - + 73 XIII. Appendix Table G. Continued Isolates and species obtained from FAME 1 GPI Profile 2 API-20 Strep Profile 3 PCR 16S- 23S rDNA 4 190 bp PCR 23S rDNA 5 875 bp PCR 16S-23S intergenic rDNA 6 130 bp CECT- 183 0.759 91.0% 3463410 Ex + - + ATCC- 12394 0.380 99.0% 0463015 VG - - - ATCC- 12388 0.070 99.0% 5143110 D - - - The symbols denote: (+) = positive and (-) = negative reactions. 1 Shoemaker et al. 2005. Results were excellent identification of Streptococcus agalactiae at similarity indexes < 0.500, very good at 0.300- 0.499; acceptable at 0.200- 0.299; low discrimination at 0.100-0.199 and unidentified (UN) at 0.000-099. 2 Jayarao et al. 1991. Results were excellent identification of S. agalactiae at 98-99.9%; very good at 90-97%; acceptable at 70-89%; low discrimination at 50-69% and unidentified (UN) at 0-49%. 3 API-20 Strep system was evaluated according to manufacturer?s instructions. The results were read after 4-4.5 h and/or 24 h* incubation at 35?C. The generated profile number was recorded and then referenced to API-20 Rapid Strep Profile Index (SPI) for excellent (Ex), very good (VG), good (G), doubtful (D) and unidentified (UN) results. 4 Mata et al. 2004. The PCR reactions were denoted by present of specific PCR product (+) and absence of specific PCR product (-). 5 Kawata et al. 2004. The PCR reactions were denoted by present of specific PCR product (+) and absence of specific PCR product (-). 6 Berridge et al. 2001. The PCR reactions were denoted by present of specific PCR product (+) and absence of specific PCR product (-). 74 XIV. Appendix Table H. Percent of whole-cell fatty acid composition of GBS Streptococcus agalactiae isolates from fish, cattle and reference strains determined by RCLIN50 rapid method 1 . Percent in profile Fatty acid 2 Fish (n = 35) Cattle (n = 10) Reference (n = 4) 12:0 4.0 2.3 1.9 14:0 11.8 6.1 7.7 16:1 w9c 3.7 7.1 5.4 16:1 w5c 1.6 2.4 2.0 16:0 35.8 30.0 42.0 17:1 iso w5c - 1.9 - 17:0 1.5 1.7 - 18:1 w9c 16.3 16.6 13.3 18:1 w7c 10.4 15.7 15.6 18:1 w5c - 1.5 - 18:0 14.9 12.0 12.0 19:1 iso 1 - 2.8 - Total 77.29 77.34 77.13 1 The rapid method for the RCLIN 50 was used to obtain fatty acid methyl esters (FAME) for use in library generation (MIDI, 2002a) and analysis of fatty acid. 2 Percent of the total fatty acids for fish, cattle and reference strains. 75 XV. Appendix Table I. Streptococcus agalactiae isolates from marine fish used for amplified fragment length polymorphism (AFLP) fingerprinting. Isolate Fish species Organ of isolation Geographic origin KU-MU-5-BR 1 Mullet Brain Kuwait Bay, Kuwait KU-MU-7-BR Mullet Brain Kuwait Bay, Kuwait KU-MU-12-BR Mullet Brain Kuwait Bay, Kuwait KU-MU-21-BR Mullet Brain Kuwait Bay, Kuwait KU-MU-27-BR Mullet Brain Kuwait Bay, Kuwait KU-MU-35-EYE Mullet Eye Kuwait Bay, Kuwait KU-SB-1-BR Seabream Brain Kuwait Bay, Kuwait KU-SB-1-HK Seabream Head kidney Kuwait Bay, Kuwait KU-SB-1-EYE Seabream Eye Kuwait Bay, Kuwait KU-SB-37-BR Seabream Brain Kuwait Bay, Kuwait KU-SB-37-HK Seabream Head kidney Kuwait Bay, Kuwait KU-SB-38-BR Seabream Brain Kuwait Bay, Kuwait 1 Streptococcus agalactiae isolates from: Evans et al. 2002.