Eradication of Arizona hinshawii from artificially infected turtle eggs

Turtle eggs, 24 h old, were infected with Arizona hinshawii and treated 48 h later with gentamicin sulfate (Garasol; Shering Corp., Allantown, N.J.) by pressure differential egg dip treatment to ascertain the concentration of this reagent required to eradicate this pathogen from eggs. Infected eggs treated with 1,000 or 1,500 micrograms of gentamicin per ml of dip solution eliminated detectable A. hinshawii from eggs as determined by testing shells and embryo-yolk homogenates of 12-day-old eggs and the gastrointestinal tracts, kidneys, livers and gall bladders, and yolks of 50-day-old embryos. Treated eggs produced hatchlings which did not excrete detectable A. hinshawii at 72 h or 30 days after hatching, nor was this organism recovered from the visceral organs of these hatchlings when necropsied 30 days after hatching. Bacteriological assays on infected nontreated eggs showed that greater than 70% of the eggs harbored A. hinshawii, and eggs in this group produced hatchlings which actively excreted and harbored A. hinshawii. Eggs not infected or treated also produced turtles which excreted and systemically carried A. hinshawii and Salmonella spp. though not at the same level as did the turtles produced from infected, nontreated eggs.     

Eradication of Arizona hinshawii from artificially infected turtle eggs.

Turtle eggs, 24 h old, were infected with Arizona hinshawii and treated 48 h later with gentamicin sulfate (Garasol; Shering Corp., Allantown, N.J.) by pressure differential egg dip treatment to ascertain the concentration of this reagent required to eradicate this pathogen from eggs. Infected eggs treated with 1,000 or 1,500 micrograms of gentamicin per ml of dip solution eliminated detectable A. hinshawii from eggs as determined by testing shells and embryo-yolk homogenates of 12-day-old eggs and the gastrointestinal tracts, kidneys, livers and gall bladders, and yolks of 50-day-old embryos. Treated eggs produced hatchlings which did not excrete detectable A. hinshawii at 72 h or 30 days after hatching, nor was this organism recovered from the visceral organs of these hatchlings when necropsied 30 days after hatching. Bacteriological assays on infected nontreated eggs showed that greater than 70% of the eggs harbored A. hinshawii, and eggs in this group produced hatchlings which actively excreted and harbored A. hinshawii. Eggs not infected or treated also produced turtles which excreted and systemically carried A. hinshawii and Salmonella spp. though not at the same level as did the turtles produced from infected, nontreated eggs.     

Treatment of Salmonella-Arizona-Infected Turtle Eggs with Terramycin and Chloromycetin by the Temperature Differential Egg Dip Method

Attempts to eliminate Salmonella and Arizona infection from newly hatched turtles were made by dipping fresh eggs in cold solutions of Terramycin and Chloromycetin at 1,000, 1,200, 1,500 and 2,000 mug per ml for either 10, 20, or 30 min. Control groups consisted of hatchings produced from nondipped eggs or eggs dipped in chilled water. In two of the four experiments 5 to 10 eggs were blended on days 15, 30, and 45 post antibiotic dip treatment. Twenty-five to 60 hatchlings from each control or experimental dip groups were held in containers and the water was tested (excretion method) for Salmonella and Arizona every 15 or 30 days for 180 to 210 days after hatching. Representative turtles were homogenized (blending method) to determine if systemic infections were present. All specimens tested were enriched in tetrathionate and selenite cystine broth. Nondipped eggs and water-dipped eggs routinely showed Salmonella and Arizona present in egg homogenate and hatchlings emerging from these eggs excreted these pathogens. Terramycin- and Chloromycetin-dipped eggs were uniformly negative for these pathogens, only if fresh eggs were dipped. Bacteriological assay of container water and whole turtle homogenate from hatchlings were negative for Salmonella and Arizona if eggs were dipped in 1,000 mug of Terramycin early in the egg laying season or if eggs were dipped in 1,500 or 2,000 mug of Terramycin per ml late in the egg laying season. The results of temperature-differential egg dip studies suggest that this is a feasible and promising method by which to eradicate Salmonella and Arizona from the turtle.     

Acquisition of Salmonella flora by turtle hatchlings on commercial turtle farms

A commercial turtle pond in South Louisiana was studied to identify the mechanism by which turtle hatchlings acquire Salmonella flora. The visceral organs and mature eggs removed from 31 adult gravid female turtles over the course of two egg-laying seasons and from 37 adult females during one winter dormant period were examined bacteriologically for Salmonella. Pond water, egg nest soil, and hatchlings produced by eggs removed from the oviducts and nest soil were also tested. Eighty-eight turtles hatched from eggs removed from the oviducts of 15 turtles at necropsy did not excrete or harbor systemically Salmonella, nor were these pathogens isolated from ovarian tissue or immature eggs. The findings suggest transovarian transmission of these pathogens does not occur frequently. Turtles hatched from eggs retrieved from soil nests 1 to 2 h after deposition harbor and excrete these organisms. This result coupled with the isolation of these pathogens from the cloaca, colon contents, and bursal fluid from 18 females captured in the act of egg laying supports the cloaca to egg and nest soil to egg mode for salmonellae infection in the resultant hatchling. Salmonella arizonae and Salmonella serogroups B, C2, and E1 were isolated from the cloaca, colon contents, pond water, and nest soil, and were excreted by hatchlings produced from eggs removed from the soil nests. These same serogroups were isolated from the colon contents of 19 of 37 females tested during the dormant period, suggesting the salmonellae persist in the pond environment in the adult throughout the year.     

Plasmid-mediated high-level gentamicin resistance among enteric bacteria isolated from pet turtles in Louisiana

The sale of small turtles is banned by the Food and Drug Administration from the U.S. market due to concerns about their excretion of Salmonella spp. To produce a safe pet for the export market, the Louisiana pet turtle industry uses gentamicin sulfate baths (1,000 microg/ml) to eradicate Salmonella spp. from turtle eggs. In 1999, we analyzed bacterial samples recovered from turtle farms and found that strains of Salmonella enterica subsp. arizonae and other bacteria, such as Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia, were resistant to high concentrations of gentamicin (>2,000 microg/ml) and to other aminoglycosides. The goal of this study was to identify the gene(s) which contributes to the high-level gentamicin resistance phenotype observed in bacteria from environmental samples with turtle farming activity, particularly the salmonellae, and to estimate the incidence of such genes in these bacteria. R plasmids from gentamicin-resistant strains were transferred by conjugation and transformation to naive Escherichia coli cells. Cloning and sequencing of the gentamicin resistance determinants on these plasmids revealed the presence of the aminoglycoside acetyltransferase genes aac(3)-IIa and aac(3)-VIa; the latter was present as a gene cassette of a class 1 integron. Multiplex PCR assays showed that every gentamicin-resistant isolate carried one of these acetyltransferase genes. Pulsed-field gel electrophoresis and restriction enzyme digestion analysis of R plasmids carrying these genes revealed different restriction profiles and sizes, indicating a dissemination of the gentamicin resistance genes through mobile molecular elements. The data presented highlight the need to develop an alternate method for the eradication of Salmonella spp. from turtle eggs.     

Invertebrate infestation on eggs and hatchlings of the loggerhead turtle, Caretta caretta, in Dalaman, Turkey

The damage caused by some invertebrates to the eggs and hatchlings of loggerhead turtles, Caretta caretta, was investigated during the summer of 2002 on Dalaman beach, Turkey. The specimens, identified to family or genus levels, from nine families representing seven orders were recorded as infesting nests of loggerhead turtles. The heaviest impacts on loggerhead turtle nests was made by Pimelia sp. (Tenebrionidae, Coleoptera). Twenty-four (36.3%) out of 66 intact loggerhead hatched nests were affected by these larvae. Larval damage by Pimelia sp. was recorded in 188 (10.6%) out of 1773 eggs, but only in two (0.28%) hatchlings. The results show that fewer insects were in the nest the further from vegetation and therefore the relocation of nests from the water's edge to further inland close to vegetation may increase the infestation rate of the eggs.     

Sea turtle species vary in their susceptibility to tropical cyclones

Severe climatic events affect all species, but there is little quantitative knowledge of how sympatric species react to such situations. We compared the reproductive seasonality of sea turtles that nest sympatrically with their vulnerability to tropical cyclones (in this study, “tropical cyclone” refers to tropical storms and hurricanes), which are increasing in severity due to changes in global climate. Storm surges significantly decreased reproductive output by lowering the number of nests that hatched and the number of hatchlings that emerged from nests, but the severity of this effect varied by species. Leatherback turtles (Dermochelys coriacea) began nesting earliest and most offspring hatched before the tropical cyclone season arrived, resulting in little negative effect. Loggerhead turtles (Caretta caretta) nested intermediately, and only nests laid late in the season were inundated with seawater during storm surges. Green turtles (Chelonia mydas) nested last, and their entire nesting season occurred during the tropical cyclone season; this resulted in a majority (79%) of green turtle nests incubating in September, when tropical cyclones are most likely to occur. Since this timing overlaps considerably with the tropical cyclone season, the developing eggs and nests are extremely vulnerable to storm surges. Increases in the severity of tropical cyclones may cause green turtle nesting success to worsen in the future. However, published literature suggests that loggerhead turtles are nesting earlier in the season and shortening their nesting seasons in response to increasing sea surface temperatures caused by global climate change. This may cause loggerhead reproductive success to improve in the future because more nests will hatch before the onset of tropical cyclones. Our data clearly indicate that sympatric species using the same resources are affected differently by tropical cyclones due to slight variations in the seasonal timing of nesting, a key life history process.     

Monitoring and conservation of critically reduced marine turtle nesting populations: lessons from the Cayman Islands

Historically, nesting marine turtles were abundant in the Cayman Islands and were an integral part of the economy and culture. Today, nesting of loggerhead turtle Caretta caretta and green turtles Chelonia mydas takes place at very low levels. Hawksbill Eretmochelys imbricata nesting has not been recorded since 1999. We overview highly detailed monitoring data gathered over a 6-year period allowing insight into the magnitude and spatial and temporal patterns of marine turtle nesting, cost-effectiveness of monitoring such reduced populations, impacts of development on reproductive success and current threats to the recovery of the population. Nesting is diffuse and widely distributed for both nesting species on Grand and Little Cayman. Modelled nesting detection profiles for Grand Cayman show that in order to maintain data resolution, most sandy coastline must be surveyed throughout each season. However, in Little Cayman it may be possible to reduce effort. Legal take of adults and illegal take of eggs may be significantly impacting the remaining population. Surprisingly, we observed no significant correlation between density of coastal development and clutch density, adult emergence success or hatching success for either species. A significant relationship exists however, between density of coastal development and incidence of misorientation events in loggerhead hatchlings but not in green turtle hatchlings. Effective protection of known nesting habitat and the elimination of exploitation of remaining adults and eggs within the population are critical to its recovery.     

Orientation and behaviour of hatchling green turtles (Chelonia mydas) in the sea

To investigate the initial stage of the ‘lost-year puzzle’ of sea turtle ecology, both hatchlings from the natural nesting groung at Tortuguero, Costa Rica and hatchlings from Tortuguero eggs that had hatched in a beach on Bermuda were tracked individually after their departure from these beaches. Of the Bermuda beach most were tracked by swimmers equipped with face mask and flippers and followed by a boat. Tracking off Tortuguero was done by an observer in a following boat. The data showed that non-random departure courses were maintained even when swimming hatchlings and moved over the horizon from all fixed objects on the shore. Observations were made on swimming and diving behaviour and on predator relationships of travelling turtles. The procedures described are useful research techniques and will be used for more extensive tracking in future seasons.     

Evaluation of Methods for Isolating Salmonella and Arizona Organisms from Pet Turtles

Two methods for isolating Salmonella and Arizona organisms from turtles, blending and excretion, were evaluated, and the percentage of isolates obtained by each method was compared with the percentage of isolates obtained by culture of turtle organs. The blending and excretion methods were equally effective in detecting the overall incidence of Salmonella and Arizona infections in turtles. The percentage of isolates obtained by specific organ culture, however, was less than the percentage obtained by the other two methods. The blending method detected a greater number of turtles with Arizona infections than did the excretion method, but there was no difference in the number of Salmonella infections detected by the two methods. The frequency of isolation of Arizona organisms from organs other than the small intestine and colon was higher than that of Salmonella.     

Effects of the egg incubation environment on turtle carapace development

Developing organisms are often exposed to fluctuating environments that destabilize tissue-scale processes and induce abnormal phenotypes. This might be common in species that lay eggs in the external environment and with little parental care, such as many reptiles. In turtles, morphological development has provided striking examples of abnormal phenotypic patterns, though the influence of the environment remains unclear. To this end, we compared fluctuating asymmetry, as a proxy for developmental instability, in turtle hatchlings incubated in controlled laboratory and unstable natural conditions. Wild and laboratory hatchlings featured similar proportions of supernumerary scales (scutes) on the dorsal shell (carapace). Such abnormal scutes likely elevated shape asymmetry, which was highest in natural nests. Moreover, we tested the hypothesis that hot and dry environments cause abnormal scute formation by subjecting eggs to a range of hydric and thermal laboratory incubation regimes. Shape asymmetry was similar in hatchlings incubated at five constant temperatures (26–30°C). A hot (30°C) and severely Dry substrate yielded smaller hatchlings but scutes were not overtly affected. Our study suggests that changing nest environments contribute to fluctuating asymmetry in egg-laying reptiles, while clarifying the conditions at which turtle shell development remains buffered from the external environment.     

A gravid fossil turtle from the Early Cretaceous reveals a different egg development strategy to that of extant marine turtles

Extant sea turtles develop and lay pliable (flexible) eggs; however, it is unknown whether they inherited this reproductive strategy from their closer fossil relatives or if it represents an evolutionary novelty. Here, we describe the first undisputable gravid marine fossil turtle ever found, from the early Cretaceous of Colombia, belonging to Desmatochelys padillai Cadena & Parham, which constitutes a representative of the Protostegidae. Using thin sectioning of one of the eggs, as well as scanning electron microscopy coupled with elemental characterization, cathodoluminescence, and computer tomography, we established that Desmatochelys padillai produced rigid eggs similar to those associated with some extant and fossil freshwater and terrestrial turtles. At least 48 spherical eggs were preserved inside this gravid turtle. We suggest that the development of rigid eggs in the extinct marine turtle Desmatochelys padillai resulted as an adaptation for egg‐embryo requirements dictated by the physical attributes of the nesting site.     

First fossil gravid turtle provides insight into the evolution of reproductive traits in turtles

Here we report on the first discovery of shelled eggs inside the body cavity of a fossil turtle and on an isolated egg clutch, both referable to the Cretaceous turtle Adocus. These discoveries provide a unique opportunity to gain insight into the reproductive traits of an extinct turtle and to understand the evolution of such traits among living turtles. The gravid adult and egg clutch indicate that Adocus laid large clutches of rigid-shelled spherical eggs and established their nests near rivers, traits that are shared by its closest living relatives, the soft-shelled turtles. Adocus eggshell, however, was probably more rigid than that of living turtles, based on its great thickness and structure, features that may represent unique adaptations to intense predation or to arid nest environments. In light of the reproductive traits observed in Adocus, the distribution of reproductive traits among turtles reveals that large clutches of rigid-shelled eggs are primitive for hidden-necked turtles (cryptodirans) and that spherical eggs may have evolved independently within this group.     

Isolation of Salmonella spp. from yacare caiman (Caiman yacare) and broad-snouted caiman (Caiman latirostris) from the Argentine Chaco

Presence of Salmonella spp. was evaluated in yacare caiman (Caiman yacare) and broad-snouted caiman (Caiman latirostris) from a ranching facility in the Argentine Chaco. Crocodilian ranching programs are based on captive breeding of wild-harvested eggs and release of excess hatchlings into the wild. Samples for bacterial isolation were collected from 102 captive (35 C. yacare and 67 C. latirostris) and seven free-ranging caiman (four C. yacare and three C. latirositris) between 2001 and 2005 and from three artificially incubated C. yacare wild eggs. Two Salmonella spp. of known zoonotic potential, S. infantis and S. nottingham, were isolated from captive caiman in 2001 and 2002, respectively. This is the first report for S. nottingham in reptiles and of S. infantis in caiman. Salmonella spp. prevalence varied significantly between years, with a 77% prevalence peak in 2002. Although the cause of this increase was not confirmed, we found no correlation with the type of enclosure, caiman species, or body weight. Deteriorated physical condition of caiman hatchlings due to dietary changes in 2002 could have influenced Salmonella spp. shedding. However, external sources such as food, water, or enclosures could not be ruled out. Pathogenic Salmonella spp. present a risk for human infection. Inadvertent introduction of Salmonella spp. or other bacteria into the environment when caiman are released could pose a threat to wild caiman populations. Prophylactic measures to detect and decrease Salmonella spp. presence in caiman ranching facilities are recommended to reduce risk to humans and make caiman-ranching a sound conservation strategy for crocodilian species.     

Fungal infection of sea turtle eggs in the sea turtle hatcheries in Peninsular Malaysia

Fungal infection in sea turtle nests has become a potential threat to sea turtle embryos. We screened the hatchery nest sand, eggshells of failed eggs, and stillbirths of green turtles and hawksbills collected from hatcheries in Malacca, Pahang, Perak and Terengganu for the presence of fungi. The DNA sequence of the ITS region of the three highest occurring isolated fungi confirmed that these species were Pseudallescheria ellipsoidea (35.4%), Scedosporium aurantiacum (27.2%), and Fusarium solani (22.0%). Morphological characteristics of these fungi were recorded. Although the total fungi abundance had no significant effect on hatching success (p > 0.05), the abundance of P. ellipsoidea significantly increased mortality in the nests (r = 0.70, p < 0.05). Future research should focus on understanding the biological aspects of this species to establish a more effective mitigation technique for the prevention of fungal infection of sea turtle eggs and hatchery employees.     

A novel hypothesis for the adaptive maintenance of environmental sex determination in a turtle

Temperature-dependent sex determination (TSD) is widespread in reptiles, yet its adaptive significance and mechanisms for its maintenance remain obscure and controversial. Comparative analyses identify an ancient origin of TSD in turtles, crocodiles and tuatara, suggesting that this trait should be advantageous in order to persist. Based on this assumption, researchers primarily, and with minimal success, have employed a model to examine sex-specific variation in hatchling phenotypes and fitness generated by different incubation conditions. The unwavering focus on different incubation conditions may be misplaced at least in the many turtle species in which hatchlings overwinter in the natal nest. If overwintering temperatures differentially affect fitness of male and female hatchlings, TSD might be maintained adaptively by enabling embryos to develop as the sex best suited to those overwintering conditions. We test this novel hypothesis using the painted turtle (Chrysemys picta), a species with TSD in which eggs hatch in late summer and hatchlings remain within nests until the following spring. We used a split-clutch design to expose field-incubated hatchlings to warm and cool overwintering (autumn–winter–spring) regimes in the laboratory and measured metabolic rates, energy use, body size and mortality of male and female hatchlings. While overall mortality rates were low, males exposed to warmer overwintering regimes had significantly higher metabolic rates and used more residual yolk than females, whereas the reverse occurred in the cool temperature regime. Hatchlings from mixed-sex nests exhibited similar sex-specific trends and, crucially, they were less energy efficient and grew less than same-sex hatchlings that originated from single-sex clutches. Such sex- and incubation-specific physiological adaptation to winter temperatures may enhance fitness and even extend the northern range of many species that overwinter terrestrially.     

A new, nearly complete stem turtle from the Jurassic of South America with implications for turtle evolution

Turtles have been known since the Upper Triassic (210Myr old); however, fossils recording the first steps of turtle evolution are scarce and often fragmentary. As a consequence, one of the main questions is whether living turtles (Testudines) originated during the Late Triassic (210Myr old) or during the Middle to Late Jurassic (ca 160Myr old). The discovery of the new fossil turtle, Condorchelys antiqua gen. et sp. nov. from the Middle to Upper Jurassic (ca 160-146Myr old) of South America (Patagonia, Argentina), presented here sheds new light on early turtle evolution. An updated cladistic analysis of turtles shows that C. antiqua and other fossil turtles are not crown turtles, but stem turtles. This cladistic analysis also shows that stem turtles were more diverse than previously thought, and that until the Middle to Upper Jurassic there were turtles without the modern jaw closure mechanism.     

Comparison of prevalence and antimicrobial susceptibilities of Campylobacter spp. isolates from organic and conventional dairy herds in Wisconsin

The prevalence and antimicrobial susceptibilities of Campylobacter spp. isolates from bovine feces were compared between organic and conventional dairy herds. Thirty organic dairy herds, where antimicrobials are rarely used for calves and never used for cows, were compared with 30 neighboring conventional dairy farms, where antimicrobials were routinely used for animals for all ages. Fecal specimens from 10 cows and 10 calves on 120 farm visits yielded 332 Campylobacter isolates. The prevalence of Campylobacter spp. in organic and conventional farms was 26.7 and 29.1%, and the prevalence was not statistically different between the two types of farms. Campylobacter prevalence was significantly higher in March than in September, higher in calves than in cows, and higher in smaller farms than in large farms. The rates of retained placenta, pneumonia, mastitis, and abortion were associated with the proportion of Campylobacter isolation from fecal samples. The gradient disk diffusion MIC method (Etest) was used for testing susceptibility to four antimicrobial agents: ciprofloxacin, gentamicin, erythromycin, and tetracycline. Two isolates were resistant to ciprofloxacin, and none of isolates was resistant to gentamicin or erythromycin. Resistance to tetracycline was 45% (148 of 332 isolates). Tetracycline resistance was found more frequently in calves than in cows (P = 0.042), but no difference was observed between organic and conventional farms. When we used Campylobacter spp. as indicator bacteria, we saw no evidence that restriction of antimicrobial use on dairy farms was associated with prevalence of resistance to ciprofloxacin, gentamicin, erythromycin, and tetracycline.     

Thermal plasticity of diving behavior, aquatic respiration, and locomotor performance in the Mary River turtle Elusor macrurus

Locomotion is a common measure of performance used in studies of thermal acclimation because of its correlation with predator escape and prey capture. However, for sedentary animals such as freshwater turtles, we propose that diving behavior may be a more ecologically relevant measure of performance. Increasing dive duration in hatchling turtles reduces predator exposure and therefore functions as an ecological benefit. Diving behavior is thermally dependent, and in some species of freshwater turtles, it is also reliant on aquatic respiration. This study examined the influence of thermal acclimation on diving behavior, aquatic respiration, and locomotor performance in the endangered, bimodally respiring Mary River turtle Elusor macrurus. Diving behavior was found to partially acclimate at 17 degrees C, with turtles acclimated to a cold temperature (17 degrees C) having a significantly longer dive duration than hatchlings acclimated to a warm temperature (28 degrees C). This increase in dive duration at 17 degrees C was not a result of physiological alterations in metabolic rate but was due instead to an increase in aquatic oxygen consumption. Increasing aquatic oxygen consumption permitted cold-acclimated hatchlings to remain submerged for significantly longer periods, with one turtle undertaking a dive of over 2.5 d. When burst-swimming speed was used as the measure of performance, thermal acclimation was not detected. Overall, E. macrurus demonstrated a partial ability to acclimate to changes in environmental temperature.     

Salinity of incubation media influences embryonic development of a freshwater turtle

Variations in water potential have marked effects on aspects of embryological development in reptiles. Therefore variation in the salinity of the incubation environment is likely to have significant consequences on the early life stage. The combination of an extended incubation period, coupled with the real threat of soil salinisation within their range makes Chelodina expansa an ideal model to assess the influence of salinity on turtle embryology. We quantified the influence of salt on the development of C. expansa hatchlings in four substrate treatments varying in salinity. Embryos incubated in higher salinities had 39 % less survival than those incubated in substrates with freshwater. Hatchlings that emerged from eggs in saline treatments were smaller with higher concentrations of plasma sodium, chloride, urea, and potassium. The physiological effects of salinity mirror those of turtles incubated in drier media with low water potential. Salinisation of river banks has the potential to reduce hatching success and fitness of nesting reptiles.