Flea market finds and global exports: Four multistate outbreaks of human Salmonella infections linked to small turtles,United States—2015

Zoonotic transmission of Salmonella infections causes an estimated 11% of salmonellosis annually in the United States. This report describes the epidemiologic, traceback and laboratory investigations conducted in the United States as part of four multistate outbreaks of Salmonella infections linked to small turtles. Salmonella isolates indistinguishable from the outbreak strains were isolated from a total of 143 ill people in the United States, pet turtles, and pond water samples collected from turtle farm A, as well as ill people from Chile and Luxembourg. Almost half (45%) of infections occurred in children aged <5 years, underscoring the importance of the Centers for Disease Control and Prevention recommendation to keep pet turtles and other reptiles out of homes and childcare settings with young children. Although only 43% of the ill people who reported turtle exposure provided purchase information, most small turtles were purchased from flea markets or street vendors, which made it difficult to locate the vendor, trace the turtles to a farm of origin, provide education and enforce the United States federal ban on the sale and distribution of small turtles. These outbreaks highlight the importance of improving public awareness and education about the risk of Salmonella from small turtles not only in the United States but also worldwide.     

Detection of Salmonella enterica Serovar Montevideo and Newport in Free‐ranging Sea Turtles and Beach Sand in the Caribbean and Persistence in Sand and Seawater Microcosms

Salmonellae are Gram‐negative zoonotic bacteria that are frequently part of the normal reptilian gastrointestinal flora. The main objective of this project was to estimate the prevalence of non‐typhoidal Salmonella enterica in the nesting and foraging populations of sea turtles on St. Kitts and in sand from known nesting beaches. Results suggest a higher prevalence of Salmonella in nesting leatherback sea turtles compared with foraging green and hawksbill sea turtles. Salmonella was cultured from 2/9 and identified by molecular diagnostic methods in 3/9 leatherback sea turtle samples. Salmonella DNA was detected in one hawksbill turtle, but viable isolates were not recovered from any hawksbill sea turtles. No Salmonella was detected in green sea turtles. In samples collected from nesting beaches, Salmonella was only recovered from a single dry sand sample. All recovered isolates were positive for the wzx gene, consistent with the O:7 serogroup. Further serotyping characterized serovars Montevideo and Newport present in cloacal and sand samples. Repetitive‐element palindromic PCR (rep‐PCR) fingerprint analysis and pulsed‐field gel electrophoresis of the 2014 isolates from turtles and sand as well as archived Salmonella isolates recovered from leatherback sea turtles in 2012 and 2013, identified two distinct genotypes and four different pulsotypes, respectively. The genotyping and serotyping were directly correlated. To determine the persistence of representative strains of each serotype/genotype in these environments, laboratory‐controlled microcosm studies were performed in water and sand (dry and wet) incubated at 25 or 35°C. Isolates persisted for at least 32 days in most microcosms, although there were significant decreases in culturable bacteria in several microcosms, with the greatest reduction in dry sand incubated at 35°C. This information provides a better understanding of the epizootiology of Salmonella in free‐ranging marine reptiles and the potential public health risks associated with human interactions with these animals in the Caribbean.     

Hepatic CT attenuation differs in three species of freshwater turtles and hepatic Hounsfield units increase with folliculogenesis in wild Blanding's turtles (Emydoidea blandingii)

Freshwater turtle species are suffering from anthropocentric‐caused population declines, making preservation of professionally managed populations increasingly important. Turtles under professional care have an increased risk to develop hepatic lipidosis, potentially resulting in early death. Computed tomography can provide an antemortem screening for increased fatty liver composition. A goal of this prospective analytical cross‐sectional study was to assess the hepatic attenuation measured as Hounsfield units (HU) in a wild population of a freshwater turtle species, and then compare hepatic HU to freshwater turtles under professional care. Ninety‐five wild Blanding's turtles (BT; Emydoidea blandingii) as well as 10 Vietnamese Pond turtles (VPT; Mauremys annamensis) and six Northern Snake‐Necked turtles (NSNT; Chelodina oblonga) under professional care underwent CT with quantification of hepatic HU. Hepatic HU were correlated to serum chemistry findings and the presence of follicles was recorded in BT. The mean (±SD) hepatic attenuation for 95 wild BT was 97.5 HU ±9.6. There were significant differences in hepatic attenuation among VPT, NSNT, and BT, with median HU values (range) of 5.39 HU (–6.45 to 61.50), 71.74 HU (59.44‐94.49), and 95.43 HU (74.41‐124.56), respectively. Aspartate aminotransferase (AST) values had a weak negative correlation to HU with a coefficient of –0.85 (P < .001). The hepatic attenuation was significantly higher for individuals undergoing folliculogenesis (P = .007). The decreased HU values were significantly negatively correlated with AST values. Findings supported the use of CT as an aid for guiding future management practices in freshwater turtles.     

Hematology and serum biochemistry comparison in wild and captive Central American river turtles (Dermatemys mawii) in Tabasco, Mexico

Hematological and serum biochemistry analyses were determined on 51 Central American river turtles (Dermatemys mawii) during the dry and rainy seasons of 2006. Turtles came from two sites: Pantanos de Centla Biosphere Reserve and a turtle breeding farm, both located in Tabasco State, Mexico. Physical examination and body measures of animals were performed. Incidence and prevalence of hemoparasites were explored.Captive organisms were in poor physical condition while wild turtles were apparently healthy. There were differences in several hematological parameters related with the condition and the season. During the dry season captive turtles exhibited higher levels of uric acid and urea, as well as lower levels of glucose. Haemogregarina sp. was detected in 100% of the wild individuals, but not in captive individuals. Its incidence was greater during the rainy season. This is the first health assessment and hematology study of this critically endangered species.     

Salmonella Prevalence Among Reptiles in a Zoo Education Setting

Clinically healthy reptiles may shed Salmonella and therefore act as a potential zoonotic threat. Most people in Northern European countries are rarely exposed to reptiles, but many zoos have education departments where children have direct contact with this group of animals. The objectives of this study were to determine the prevalence and serotype distribution of Salmonella among reptiles in the Education Department (n = 55) at Copenhagen Zoo and compare it to the Zoo’s main reptile collection (n = 145) to evaluate the zoonotic risk. Salmonella was isolated from cloacal swabs by selective enrichment, and a single isolate from each positive sample was further identified by biochemical tests and serotyped. The overall prevalence was 35% (69/200) with significant difference between the Education Department (64%, 35/55) and the main reptile collection (23%, 34/145). A total of 28 serotypes were detected. Ten serotypes were isolated from more than one specimen and four from more than one species. Salmonella enterica subsp. enterica serovar Eastbourne was the predominant serotype (32%, 22/69) and was also the serotype isolated from most reptile species (n = 7). Transmission of serotypes from one department to another was very limited indicated by the serotype distribution. Despite the relative high prevalence observed among the reptiles in the Zoo’s Education Department compared to the reptiles in the Zoo’s main reptile collection, no Salmonella cases have been linked to the Zoo, and Salmonella ser. Eastbourne is very rarely isolated from humans in Denmark. Simple hygienic procedures such as hand washing which is consistently carried out following handling of reptiles at the Education Department may reduce the risk and therefore contribute to this low prevalence.     

Wild‐Caught and Farm‐Reared Amphibians are Important Reservoirs of Salmonella,A Study in North‐East Thailand

The role of amphibians as Salmonella reservoirs has not been as well studied as in reptiles, where the literature is abundant. Recent outbreaks of salmonellosis associated with exotic pet frogs have occurred in United States. Frog farming and wild frog harvesting have increased the international trade in these species. This necessitates a better understanding of the risk of salmonellosis transmission from amphibians to humans. We explored the presence of Salmonella in amphibians (frogs and toads) in Thailand, where farmed and wild frogs as well as toads are present. These live animals are easily found in the local markets and are used as food. Exportation of frog meat from Thailand is common. During March–June 2014, ninety‐seven frogs were collected from several habitats, including frog farms, urban areas and protected natural areas. The collected amphibians were tested for the presence of Salmonella. The overall prevalence of Salmonella was 69.07% (90.00% in farm animals, 0% in urban area animals and 44.83% in protected area animals). Eight serovars of Salmonella were isolated: subsp. diarizonae ser. 50:k:z, Hvittingfoss, Muenchen, Newport, Stanley, Thompson, Panama and Wandsworth. Six of the identified serovars, Hvittingfoss, Newport, Panama, Stanley, Thompson and Wandsworth, have been detected in humans in Thailand. According to our results, amphibians are reservoirs of Salmonella and can be a public health concern when used as a source of protein for humans.     

Pharmacokinetics of marbofloxacin in Green sea turtles (Chelonia mydas) following intravenous and intramuscular administration at two dosage rates

Limited pharmacokinetic information to establish suitable therapeutic plans is available for green sea turtles. Therefore, the present study was conducted to evaluate the pharmacokinetic characteristics of marbofloxacin (MBF) in the green sea turtle, Chelonia mydas, following single intravenous (i.v.) or intramuscular (i.m.) administration at two dosages of 2 and 4 mg/kg body weight (b.w.). Blood samples were collected at assigned times up to 168 hr. MBF in plasma was extracted using liquid–liquid extraction and analyzed by a validated high-performance liquid chromatography (HPLC). MBF was quantifiable from 15 min to 96 hr after i.v. and i.m. administrations at two dose rates. A noncompartmental model was used to fit the plasma concentration of MBF versus time curve for each green sea turtle. The t_1/2λz value, similar for both the dosages (22–28 hr), indicated that the overall rate of elimination of MBF in green sea turtles is relatively slow. The average i.m. F% ranged 88%–103%. MBF is a concentration-dependent drug and the AUC/MIC ratio is the best PK/PD predictor for its efficacy. The MBF dosage of 4 mg/kg appeared to produce an appropriate value of the PK-PD surrogate that predicts antibacterial success for disease caused by susceptible bacteria. In contrast, i.m. administration of MBF at a dosage of 2 mg/kg b.w. was not found to produce a suitable PK-PD surrogate index. However, further studies of multiple doses and plasma binding proteins are warranted to confirm an appropriate dosage regimen.     

Serovars of Salmonella from captive reptiles

The distribution on serovars of 60 Salmonella isolates from reptiles kept in captivity in Denmark during the period 1995–2006 was investigated. The isolates were all recovered from clinical specimens submitted to the National Veterinary Institute. A majority of the samples were from reptiles in zoological gardens or similar, while a minor number was from reptiles kept in private homes. A total of 43 serovars were detected, most of them being what is usually called exotic serotypes, and many not having a trivial name, while a few isolates belonged to well‐known human pathogenic serovars, such as S. Enteritidis, S. Typhimurium, S. Bovismorbificans. One isolate was rough and two were non‐typeable. Isolates from turtles belonged to the subspecies enterica, while many isolates from both sauria and snakes belonged to other subspecies. The findings underline the potential zoonotic risk by handling reptiles in zoological garden or other public settings, or keeping pet reptiles in private homes.     

Prevalence and Spatial Distribution of Salmonella Infections in the Pennsylvania Raccoon (Procyon lotor)

A study was conducted to determine the prevalence and spatial distribution of Salmonella infection in Pennsylvania raccoons (Procyon lotor), common wildlife mammals known to occupy overlapping habitats with humans and domestic food animals. The Pennsylvania Game Commission provided a total of 371 raccoon intestinal samples from trapped and road‐killed raccoons collected between May and November 2011. Salmonella was isolated from the faeces of 56 (15.1%) of 371 raccoons in 35 (54%) of 65 counties across Pennsylvania. The five most frequently isolated serotypes were Newport (28.6%), Enteritidis (19.6%), Typhimurium (10.7%), Braenderup (8.9%) and Bareilly (7.1%). Pulsed‐field gel electrophoresis (PFGE) analysis of the Salmonella isolates and subsequent comparison to the Pennsylvania Department of Health human Salmonella PFGE database revealed 16 different pulsetypes in Salmonella isolates recovered from raccoons that were indistinguishable from pulsetypes of Salmonella collected from clinically ill humans during the study period. The pulsetypes of seven raccoon Salmonella isolates matched those of 56 human Salmonella isolates by month and geographical region of sample collection. Results from Clustered Regularly Interspaced Short Palindromic Repeats and Multi‐Virulence Locus Sequence Typing (CRISPR‐MVLST) analysis corroborated the PFGE and serotyping data. The findings of this study show that several PFGE pulsetypes of Salmonella were shared between humans and raccoons in Pennsylvania, indicating that raccoons and humans might share the same source of Salmonella.     

Outbreak of human infections with uncommon Salmonella serotypes linked to pet bearded dragons, 2012–2014

Reptiles are one of the fastest growing sectors in the United States pet industry. Reptile‐associated salmonellosis (RAS) continues to be an important public health problem, especially among children. We investigated an outbreak of human Salmonella infections resulting from serotypes Cotham and Kisarawe, predominately occurring among children. An outbreak of illnesses was identified in persons with exposure to pet bearded dragon lizards. Human and animal health officials, in cooperation with the pet industry, conducted epidemiologic, traceback and laboratory investigations. Onsite sampling was conducted at two US breeding facilities, one foreign breeding facility, and a large pet retail chain. A total of 166 patients in 36 states were identified with illness onset dates from 02/2012–06/2014. The median patient age was 3 years (range, <1–79 years), 57% were aged ≤5 years, and 37% were aged ≤1 year. Forty‐four patients (37%) were hospitalized, predominantly children. Sampling at breeding facilities and a national pet store chain resulted in isolation of outbreak serotypes at each facility; isolation proportions ranged from 2%–24% of samples collected at each facility.Epidemiologic, microbiologic and traceback evidence linked an outbreak of uncommon Salmonella serotypes to contact with pet bearded dragons. The high proportion of infants involved in this outbreak highlights the need to educate owners about the risk of RAS in children and the potential for household contamination by pet reptiles or their habitats. Strategies should be developed to improve breeding practices, biosecurity and monitoring protocols to reduce Salmonella in the pet reptile trade.     

Preliminary findings of Salmonella spp. in captive green iguanas (Iguana iguana) and their environment

Captive reptiles are routinely identified as reservoirs of Salmonella spp. and reports of reptile-associated salmonellosis are increasing. Unfortunately, little is known about the epidemiology of Salmonella spp. and green iguanas. We did a limited survey of a green-iguana farm in El Salvador to identify sources of Salmonella spp. in green iguanas and their environment. A limited number of samples for microbiological culture were collected from iguanas (adult, hatchling, and embryos) and their environment (food, water, soil, shelter, insects, and wild-caught lizards). Salmonella spp. was isolated from the intestine of both adult (3/20) and hatchling iguanas (8/20). There was no evidence of Salmonella spp. in the reproductive tracts of female iguanas (0/10). Salmonella spp. was isolated from the surface of 40% (7/16) of the egg surfaces tested. Salmonella spp. was not identified from the externalized yolk-sac of the iguana embryos tested. Soil samples from a breeding pen and a nest were both positive for Salmonella spp. Eight different Salmonella spp. serotypes were identified in this survey. These results suggest that horizontal transmission of Salmonella spp. is a potential source of exposure to hatchling iguanas at this facility.     

Phenotypic and Genotypic Characterization of Salmonella enterica in Captive Wildlife and Exotic Animal Species in Ohio,USA

The purpose of this study was to investigate the occurrence, antimicrobial resistance patterns, phenotypic and genotypic relatedness of Salmonella enterica recovered from captive wildlife host species and in the environment in Ohio, USA. A total of 319 samples including faecal (n = 225), feed (n = 38) and environmental (n = 56) were collected from 32 different wild and exotic animal species in captivity and their environment in Ohio. Salmonellae were isolated using conventional culture methods and tested for antimicrobial susceptibility with the Kirby–Bauer disc diffusion method. Salmonella isolates were serotyped, and genotyping was performed using the pulsed‐field gel electrophoresis (PFGE). Salmonella was detected in 56 of 225 (24.9%) faecal samples; six of 56 (10.7%) environmental samples and six of 38 (15.8%) feed samples. Salmonella was more commonly isolated in faecal samples from giraffes (78.2%; 36/46), cranes (75%; 3/4) and raccoons (75%; 3/4). Salmonella enterica serotypes of known public health significance including S. Typhimurium (64.3%), S. Newport (32.1%) and S. Heidelberg (5.3%) were identified. While the majority of the Salmonella isolates were pan‐susceptible (88.2%; 60 of 68), multidrug‐resistant strains including penta‐resistant type, AmStTeKmGm (8.8%; six of 68) were detected. Genotypic diversity was found among S. Typhimurium isolates. The identification of clonally related Salmonella isolates from environment and faeces suggests that indirect transmission of Salmonella among hosts via environmental contamination is an important concern to workers, visitors and other wildlife. Results of this study show the diversity of Salmonella serovars and public health implications of human exposure from wildlife reservoirs.     

Serotypes of Salmonella Isolated from Faeces of Patients with Acute Diarrhoea in Gwangju Area,Korea, During 2000–2009

The purpose of this study was to determine the changing pattern of Salmonella serotypes causing acute diarrhoea in humans in Gwangju area, Korea, during 2000–2009. A total of 596 Salmonella isolated from culture of 29 896 faecal samples of patients with acute diarrhoea were included in this study. Faecal samples were collected from local hospitals and clinics in Gwangju area during January 2000–December 2009. The mean annual frequency of isolates for the 10 years was 2.0% (range, 0.9–6.0). The isolates were serologically classified into 43 different serotypes. The 10 most common serotypes were Salmonella Enteritidis (47.9%), S. Typhimurium (20.4%), S. Braenderup (3.2%), S. Montevideo (2.9%), S. Paratyphi B (2.9%), S. London (2.3%), S. Bardo (1.7%), S. Virchow (1.7%), S. Infantis (1.5%) and S. Typhi (1.5%), accounting for 86% of all the isolates. Temporal variations were observed in the distribution of different Salmonella serotypes over the years, and only S. Enteritidis and S. Typhimurium were persistent throughout the study period. Although age specificity varied with serotypes, Salmonella was isolated most frequently from children below 5 years of age (179/596, 30.0%). A seasonal trend was apparent, and the highest rates were found in the summer months. This is the first report of the annual frequency of isolation of Salmonella serotypes, and seasonal and age‐specific patterns of salmonellosis in humans in Gwangju area, Korea, over a decade‐long period.     

Reptile‐Associated Salmonellosis in Minnesota, 1996–2011

Reptile‐associated salmonellosis (RAS) occurs when Salmonella is transmitted from a reptile to a human. This study describes the epidemiology of RAS in Minnesota during 1996–2011. All Minnesotans with confirmed Salmonella infections are reported to the Minnesota Department of Health (MDH). Case patients are interviewed about illness characteristics and risk factors, including foods eaten, drinking and recreational water exposures, contact with ill people, and animal contact. Willing RAS case patients can submit stool from the reptile for culture. Serotype and pulsed‐field gel electrophoresis (PFGE) subtype of Salmonella isolates from reptiles and case patients are compared. Of 8389 sporadic (not associated with an outbreak) non‐typhoidal salmonellosis case patients in Minnesotans during 1996–2011, 290 (3.5%) reported reptile exposure. The median age of case patients with reptile exposure was 11 years, 31% were under the age of 5 years and 67% were under the age of 20 years; 50% were female. The median illness duration was 8 days; 23% required hospitalization. The most commonly reported reptile exposures were lizard (47%), snake (20%), turtle (19%) and a combination of reptile types (14%). Eighty‐four per cent of isolates from case patients who reported reptile exposure were Salmonella enterica subspecies I. The three most common serotypes were Typhimurium (15%), Enteritidis (7%) and subspecies IV serotypes (7%). Of 60 reptiles testing positive for Salmonella, 36 (60%) yielded the same Salmonella serotype as the human isolate. Twenty‐six of 27 reptile isolates that were subtyped by PFGE were indistinguishable from the human isolate. Of these, 88% were subspecies I; the most common serotypes were Enteritidis (12%), Typhimurium (8%), and Bareilly (8%). RAS accounts for approximately 3.5% of salmonellosis cases in Minnesota, primarily affecting children. The majority of isolates from case patients and reptiles belonged to Salmonella subspecies I, suggesting that reptiles are a source of human infection with serotypes not traditionally considered to be reptile‐associated.     

Pharmacokinetics of ceftriaxone in Green sea turtles (Chelonia mydas) following intravenous and intramuscular administration at two dosages

Green sea turtles are widely distributed in tropical and subtropical waters. Adult green sea turtles face many threats, primarily from humans, including injuries from boat propellers, being caught in fishing nets, pollution, poaching, and infectious diseases. To the best of our knowledge, limited pharmacokinetic information to establish suitable therapeutic plans is available for green sea turtles. Therefore, the present study aimed to describe the pharmacokinetic characteristics of ceftriaxone (CEF) in green sea turtles, Chelonia mydas, following single intravenous and intramuscular administrations at two dosages of 10 and 25 mg/kg body weight (b.w.). Blood samples were collected at assigned times up to 96 hr. The plasma concentrations of CEF were measured by liquid chromatography tandem mass spectrometry. The concentrations of CEF in the plasma were quantified up to 24 and 48 hr after i.v. and i.m. administrations at dosages of 10 and 25 mg/kg b.w., respectively. The C_max values of CEF were 15.43 ± 3.71 μg/ml and 43.48 ± 4.29 μg/ml at dosages of 10 and 25 mg/kg, respectively. The AUC_last values increased in a dose‐dependent fashion. The half‐life values were 2.89 ± 0.41 hr and 5.96 ± 0.26 hr at dosages of 10 and 25 mg/kg b.w, respectively. The absolute i.m. bioavailability was 67% and 108%, and the binding percentage of CEF to plasma protein was ranged from 20% to 29% with an average of 24.6%. Based on the pharmacokinetic data, susceptibility break‐point and PK‐PD index (T > MIC, 0.2 μg/ml), i.m. administration of CEF at a dosage of 10 mg/kg b.w. might be appropriate for initiating treatment of susceptible bacterial infections in green sea turtles.     

Salmonella in free living terrestrial and aquatic turtles

Detection of Salmonella in pet turtles has been the focus of extensive research, but its incidence in free living turtles is not well known. The aim of this study was to evaluate the incidence of Salmonella in terrestrial and aquatic species of chelonians inhabiting a National Park in southwestern Spain. Individuals of the terrestrial tortoise Testudo graeca (n = 16) and the aquatic turtles Emys orbicularis (n = 26) and Mauremys leprosa (n = 50) were investigated. Maximum incidence of Salmonella was recorded in the terrestrial species (100%). In contrast, the incidence of infected animals was low in the aquatic species, 12% in M. leprosa and 15.4% in E. orbicularis. Five serotypes of Salmonella belonging to subspecies enterica (I) and salamae (II) were identified. All serotypes were found in the terrestrial species, and three in the aquatic ones, suggesting that wild terrestrial chelonians are important reservoirs of Salmonella in our study area. Cloacal transmission during mating is the most probable mode of transmission among individuals.     

Evaluation of Pet‐Related Management Factors and the Risk of Salmonella spp. Carriage in Pet Dogs from Volunteer Households in Ontario (2005–2006)

The purpose of this study was to determine pet‐related management factors that may be associated with the presence of Salmonella spp. in feces of pet dogs from volunteer households. From October 2005 until May 2006, 138 dogs from 84 households in Ontario were recruited to participate in a cross‐sectional study. Five consecutive daily fecal samples were collected from each dog and enrichment culture for Salmonella spp. was performed. A higher than expected number of the dogs (23.2%; 32/138) had at least one fecal sample positive for Salmonella, and 25% (21/84) of the households had at least one dog shedding Salmonella. Twelve serotypes of Salmonella enterica subsp. enterica were identified, with the predominant serotypes being Typhimurium (33.3%; 13/39), Kentucky (15.4%; 6/39), Brandenburg (15.4%; 6/39) and Heidelberg (12.8%; 5/39). Univariable logistic regression models were created with a random effect for household to account for clustering. Statistically significant risk factors for a dog testing positive included having contact with livestock, receiving a probiotic in the previous 30 days, feeding a commercial or homemade raw food diet, feeding raw meat and eggs, feeding a homemade cooked diet, and having more than one dog in the household. In two‐variable models that controlled for feeding raw food, the non‐dietary variables were no longer statistically significant. These results highlight the potential public health risk of including raw animal products in canine diets.