The fungus Aphanoascella galapagosensis affects bacterial diversity of Galapagos giant tortoise carapaces.

AIMS: This study aimed to describe the bacterial microbiome associated with the carapace of three species of Galapagos giant tortoises (Chelonoidis porteri, Chelonoidis donfaustoi, and Chelonoidis vandenburghi) and determine the potential effect of the whitish lesions caused by the fungus Aphanoascella galapagosensis. METHODS AND RESULTS: We used Oxford Nanopore's MinION to evaluate the external bacterial microbiome associated with the carapaces from the aforementioned species. Taxonomic assignment was carried out by Bugseq and the bacterial communities were compared between carapaces with and without lesions using a NMDS with Bray-Curtis as the dissimilarity index. We found four genera of bacteria that were ubiquitous throughout all individuals, suggesting the presence of shared taxa. The results also displayed a significant difference in the microbiome between carapaces with and without lesions, and for species-carapace interaction, but not among species. CONCLUSIONS: This study establishes a baseline of the bacterial diversity of the carapace within three Galapagos giant tortoise species, showcasing the presence of a distinctive microbial community. Furthermore, our findings suggest a significant influence of the fungus Aphanoascella galapagosensis on the bacterial populations inhabiting the carapace of these reptiles.

Haematology and plasma biochemistry reference intervals of Española, San Cristobal and Eastern Santa Cruz Galapagos tortoise species.

Normal reference intervals (RI) of hematologic and biochemical parameters are important for assessing and monitoring the health status of captive and free-living chelonians; however, such information is not available for most wildlife species. Giant Galapagos tortoises are one of the most iconic animals on earth and health information can make an important contribution to their conservation and management. This study provides formal RI of haematology and plasma biochemistry parameters and describes cell morphology along with morphometrics of free-living Eastern Santa Cruz (Chelonoidis donfaustoi), Española (Chelonoidis hoodensis) and San Cristóbal tortoises (Chelonoidis chathamensis). We explored differences in blood parameters between sexes, across the tortoise species in this study and with previously published RI of the Western Santa Cruz tortoise (Chelonoidis porteri). Biochemistry parameters of both Santa Cruz species were overall more similar to each other than to Española and San Cristobal tortoises. This research constitutes the first RI for these three Galapagos tortoise species and may be of value for advising captive-breeding and conservation plans. We recommend further research to establish RI in additional tortoise species so we may better understand and interpret haematology and biochemistry parameters as a valuable conservation tool for species of this critically endangered taxon.

ESBL-Producing Enterobacterales at the Human-Domestic Animal-Wildlife Interface: A One Health Approach to Antimicrobial Resistance in Piauí, Northeastern Brazil.

The use, misuse, and overuse of antimicrobials is one of the main public health threats of the 21st century. We investigated the risk factor of the presence of extended-spectrum, cephalosporin-resistant Enterobacterales in feces of non-domestic and domestic birds and other domestic animals in Piauí State, northeast Brazil. We collected a total of 387 cloacal and rectal swab samples of free-living birds, domestic birds, and domestic mammals in five municipalities: Amarante, Água Branca, Lagoa Alegre, Parnaíba, and Teresina. A total of 59/387 (15.2%) of these samples harbored extended spectrum beta-lactamase (ESBL)-producing Enterobacterales. Using the MALDI-TOF technique, we identified fifty-seven samples as Escherichia coli and two samples as Klebsiella pneumoniae. Teresina and Parnaíba had the highest prevalence of animals with resistant bacteria (32.1% and 27.1%, respectively) and highest exposure risk factor (OR of 16.06 and 8.58, respectively, and p < 0.001 for all). Multidrug-resistant, ESBL-producing Enterobacterales were observed in 72.8% of the samples (43/59). For the free-living birds, the positive samples belonged to a great kiskadee (Pitangus sulphuratus) and a semipalmated sandpiper (Calidris pusilla) in migratory and resident species, respectively. For domestic animals, the swine samples showed the highest prevalence of antimicrobial resistance. The lack of access to veterinary care and information regarding antimicrobial therapy, along with the easy access to antimicrobials without medical prescription, favors the inadequate use of antimicrobials in Piauí.

Novel herpesvirus in the critically endangered Galapagos pink land iguana.

Virus surveillance in wildlife is important to understanding ecosystem health, taxonomy, and evolution. Nevertheless, viruses in reptiles, and specifically in squamates, continue to be understudied. Herein, we conducted a health assessment on the critically endangered Galapagos pink land iguana (Conolophus marthae) and the vulnerable Galapagos land iguana (Conolophus subcristatus). We collected oral and/or cloacal swabs from 47 clinically healthy iguanas and tested for adenovirus (cloacal swabs, n = 47) and herpesvirus (oral swabs, n = 45) using broad-spectrum PCRs. Two out of 38 (5.3 %) Galapagos pink land iguanas tested positive for herpesvirus, while no herpesvirus was detected in all Galapagos land iguanas (n = 7). Both herpesviral sequences were identical between them and divergent (61.9 % amino acid identity) when compared to the closest herpesvirus sequences available in GenBank/EMBL/DDBJ. The genetic distance between this and other herpesviruses is consistent with its classification as a novel virus species. All iguanas were negative for adenovirus. This is the first description of a herpesvirus in iguanas of the Galapagos islands, and the first report of a potential pathogen for the iconic Galapagos pink land iguana. Further research is needed to understand the implications of this virus in the conservation and management of one of the most endangered iguana species in the world.

Ranavirus (Frog Virus 3) Infection in Free-Living Three-Toed Box Turtles (Terrapene mexicana triunguis) in Missouri, USA.

Frog virus 3 (FV3) and related ranaviruses are emerging infectious disease threats to ectothermic vertebrate species globally. Although the impact of these viruses on amphibian health is relatively well studied, less is understood about their effects on reptile health. We report two cases of FV3 infection, 11 mo apart, in three-toed box turtles (Terrapene mexicana triunguis) from a wildlife rehabilitation center. Case 1 had upper respiratory signs upon intake but had no clinical signs at the time of euthanasia 1 mo later. Case 2 presented for vehicular trauma, had ulcerative pharyngitis and glossitis, and died overnight. In case 1, we detected FV3 nucleic acid with qPCR in oral swabs, kidney, liver, spleen, and tongue. In case 2, we detected FV3 in an oral swab, an oral plaque, heart, kidney, lung, liver, spleen, and tongue. We also detected FV3 nucleic acid with in situ hybridization for case 2. For both cases, FV3 was isolated in cell culture and identified with DNA sequencing. Histopathologic examination of postmortem tissue from case 1 was unremarkable, whereas acute hemorrhagic pneumonia and splenic necrosis were noted in case 2. The difference in clinical signs between the two cases may have been due to differences in the temporal course of FV3 disease at the time of necropsy. Failure to detect this infection previously in Missouri reptiles may be due to lack of surveillance, although cases may also represent a novel spillover to box turtles in Missouri. Our findings reiterate previous suggestions that the range of FV3 infection may be greater than previously documented and that infection may occur in host species yet to be tested.

HEMATOLOGY AND BLOOD CHEMISTRY VALUES IN CUBAN CROCODILES (CROCODYLUS RHOMBIFER) HOUSED AT THE ZAPATA SWAMP CROCODILE FARM, CUBA.

We report hematology and biochemistry reference intervals (RI) for the critically endangered Cuban crocodile (Crocodylus rhombifer). In November 2019, we sampled 43 adult crocodiles (6 male, 37 female) under human care at the Zapata Swamp Crocodile Farm in Matanzas, Cuba. These crocodiles are part of a breeding program for the species registered by the Convention on International Trade in Endangered Species (CITES). Visual health evaluations were performed immediately after manual restraint, and blood was collected from the postoccipital sinus. We performed packed cell volume (PCV), total solids (TS), complete blood counts (CBC), and biochemistry profiles for each crocodile on the day of sampling. Mean PCV (n = 42) was 21.1 ± 5.0% and TS (n = 42) 7.3 ± 1.2 mg/dl, respectively. Absolute white blood cell (WBC) (n = 40) was 9.6 ± 5.7 ｘ 109/L. Similar to other crocodilian species, the dominant leukocyte was lymphocytes (70.7 ± 10.4%), followed by heterophils (18.7 ± 9.7%). Two of the crocodiles had a high heterophil:lymphocyte ratio (0.87 and 0.74), although on visual exam they were both considered healthy. The range of creatine kinase was 41-1,482 U/L, and the higher values may be a reflection of muscle exertion at time of handling. Limitations to the study included skewed sex ratios and high lipemia and hemolysis in most samples collected. These are the first reference intervals reported for this species, including the first descriptions of WBC morphology. These data are valuable for the management of animals at the Zapata Swamp Crocodile Farm, for comparison with free-living Cuban crocodiles in Cuba, and for comparison with those managed under human care outside of Cuba.

Health assessments uncover novel viral sequences in five species of Galapagos tortoises.

Emerging infectious diseases (EIDs) have been reported as causes of morbidity and mortality in free-living animal populations, including turtles and tortoises, and they have even resulted in species extinctions, with human activities contributing to the spread of many of these diseases. In the Galapagos, giant tortoises are endangered due to habitat change, invasive species, and other human impacts; however, the impact of EIDs on Galapagos tortoise conservation remains understudied. To fill this gap, we conducted health assessments of five tortoise species from the islands of Santa Cruz, Isabela and Española. We performed health evaluations of 454 animals and PCR testing for pathogens known to be relevant in other tortoise species. We identified two novel sequences of adenoviruses and four of herpesviruses. Based on alignments of the DNA polymerase gene and maximum likelihood phylogenetic analyses, we found both novel adenoviruses to be most closely related to red footed tortoise adenovirus 2, by nucleotide sequence and red footed tortoise adenovirus 1, based on amino acid sequence. Three of the herpesvirus sequences translated into the same deduced amino acid sequence; therefore, they may be considered the same viral species, closely related to terrapene herpesvirus 2. The fourth herpesvirus sequence was highly divergent from any sequence previously detected and is related to an eagle owl herpesvirus based on nucleotide sequence and to loggerhead oro-cutaneous herpesvirus based on amino acids. These novel viruses may be pathogenic for giant tortoises under specific conditions (e.g., stress). Continued screening is crucial to determine if these viruses play a role in tortoise fitness, morbidity and survival. This information allows us to provide recommendations to the Galapagos National Park Directorate and other institutions to improve the management of these unique species both in Galapagos and globally, and for tortoise reintroduction plans throughout the archipelago.

Antimicrobial resistance in Galapagos tortoises as an indicator of the growing human footprint.

Antimicrobial resistance has become one of the main public health threats worldwide with anthropogenic activities driving the spread of resistance. Understanding and combatting the spread of resistant bacteria is a top priority for global health institutions, and it is included as one of the main goals of the One Health initiative. Giant tortoises (Chelonoidis spp.), some of the most iconic species on Earth, are widely distributed across the Galapagos archipelago and are thus perfect candidates to test the hypothesis that wildlife species in the Galapagos carry antimicrobial resistant genes (ARGs) associated with human activities. We sampled a total of 200 free-living Galapagos tortoises from western Santa Cruz Island (C. porteri), the most human-populated island of the archipelago, and 70 tortoises (C. vandenburghi) from the isolated Alcedo Volcano on Isabela Island, a natural area with minimal human presence. Fecal samples were analyzed by quantitative PCR for a panel of 21 ARGs conferring resistance for eight antimicrobial classes. We found ARGs in both Santa Cruz and Alcedo Volcano giant tortoises; however, both qualitative and quantitative results showed higher loads of ARGs in tortoises inhabiting the human modified environments of Santa Cruz. Moreover, Santa Cruz tortoises sampled in higher human-modified landscapes (i.e., farmlands and urban areas) presented a higher number of ARGs, antimicrobial classes, and multi-resistant microbiomes than those from less anthropized areas within the same island. Our findings suggest that human activities in Galapagos have a negative impact on ecosystem health through ARG dispersal. This research highlights a new threat for the health and conservation of the unique wildlife of the Galapagos, their ecosystems, and the humans inhabiting this World Heritage Site. Our recommendation to local policy makers is to control and reduce the use of antibiotics in both human and animal health, thus helping enforce antimicrobial regulations.

Haematology and plasma biochemistry reference intervals for the critically endangered western Santa Cruz Galapagos tortoise (Chelonoidis porteri).

Reference intervals (RIs) are an increasingly valuable tool for monitoring captive and free-living wildlife populations. Galapagos tortoises are one of the most emblematic species on Earth with 9 of the 12 extant species considered endangered due to human activities. As part of a long-term health assessment within the Galapagos Tortoise Movement Ecology Programme, we sampled a total of 210 free-living Santa Cruz Galapagos tortoises (Chelonoidis porteri). We collected blood from the brachial vein and performed packed cell volume (PCV), total solids (TS), morphological evaluation, white blood cell (WBC) count estimates and differentials and a VetScan biochemistry panel for each individual. We calculated 95% RIs and 90% confidence intervals (CIs) using the Reference Value Advisor tool and following international standard guidelines. Tortoises were categorized by estimated age and sex, with RI and CI reported here for 164 adult tortoises and 46 sub-adult tortoises. We found significant differences between sexes, with adult females presenting a lower PCV and higher values for both calcium and potassium. Among age groups, adult tortoises presented higher PCV, TS and albumin and lower WBC counts, aspartate aminotransferase and creatine kinase than sub-adult tortoises. We also found that tortoises presented higher numbers of lymphocytes during the dry season, but higher basophils, eosinophils, phosphorus, potassium and TS during the humid season. The heterophil:lymphocyte ratio did not differ between groups. To the authors' knowledge, this is the first report of formal plasma biochemistry and haematology RI for free-living Galapagos tortoises. With the present study we provide an important diagnostic tool for captive-breeding programs in the Galapagos and zoological institutions globally that care for giant tortoises. The ultimate goal of this study is to contribute to the understanding of giant tortoise baseline health parameters and to inform local management decisions that help to conserve these emblematic species.

Field Anesthesia of the Maned Wolf (Chrysocyon brachyurus) in Bolivia.

Fifteen maned wolves (Chrysocyon brachyurus) were anesthetized a total of 43 times as part of a long-term ecology and health study in a remote region of northeastern Bolivia. We administered tiletamine-zolazepam (TZ) to wolves in box traps or free-ranging, from blinds or on foot, at a mean dosage of 4.6 mg/kg intramuscularly. Detailed anesthetic information was recorded in 24 of these events in 11 wolves (six males, five females), and wolves were monitored closely post procedure with very high frequency or global positioning system telemetry collars. Anesthetic induction was smooth and rapid in all cases, with a mean 6.4 min from injection to recumbency. Vital parameters were stable during the majority of procedures. As expected with this drug combination, recovery was long (mean time to standing 163 min [range: 80-235 min]) but smooth, and animals were monitored in most cases in box traps until stable for release. One case of apnea and prolonged recovery is reported. In two cases, wolves recovered normally but were found to move minimally in the 2.5-4 d postprocedure before resuming normal movements. Overall, TZ provided safe, stable immobilization of free-ranging maned wolves in remote and extreme field conditions, although postanesthesia monitoring via telemetry is recommended.

Individual, household and environmental factors associated with arboviruses in rural human populations, Brazil.

Landscape change is one of the foremost drivers of the emergence of infectious diseases. Exploring demographic, household and environmental conditions under which infectious diseases occur may inform strategies to prevent disease emergence in human populations. We collected blood samples from 523 humans and explore factors for arbovirus emergence in Bahia, Brazil. The overall arbovirus seroprevalence was 65.2%, with the genus Flavivirus most prevalent (64.4%). Based on monotypic reactions, the population had contact with five arbovirus: Dengue 3, Ilheus, Oropouche, Caraparu and Eastern equine encephalitis virus. To our knowledge, this is the first study reporting exposure to Oropouche, Caraparu and Eastern equine encephalitis virus in human populations in Bahia, Northeast of Brazil. The best model fit demonstrated that household and environmental variables were more predictive of the risk of arbovirus exposure than demographic variables. The presence of forest and free-living monkeys in the areas close to the communities had a protective effect for the human population (i.e. lower seroprevalence). The dilution effect is considered as one explanation for this finding. These results highlight the important ecological role of wildlife-friendly agriculture.

Circulating nutrient concentrations in free-ranging Humboldt penguins (Spheniscus humboldti) in Punta San Juan, Peru.

Free-ranging Humboldt penguin (HP, Spheniscus humboldti) populations are under pressure from resource competition with industrial fisheries, habitat loss, and El Niño Southern Oscillation events. Foraging patterns for this top marine predator change during periods of aberrant oceanographic conditions and scarce fish stock numbers. These radical dietary fluctuations can lead to poor fertility, early embryonic death, poor hatchability, suppressed immune function, high chick mortality, and illness. To understand the variability of nutrient status in reproductive seasons, we measured select circulating nutrient concentrations (fat-soluble vitamins A, D, E, and carotenoids, fatty acids, amino acids, minerals, and electrolytes) of 105 HP at Punta San Juan, Peru during the first reproductive seasons of 2007 and 2008. We determined significant differences in nutrient status between sexes, years of sampling, and reproductive stages. Males (4.5 ± 0.38 kg) weighed more than females (4.0 ± 0.29 kg) and exhibited higher concentrations of vitamin A (0.71 ± 0.11 vs. 0.61 ± 0.12 µg/ml) and docosahexaenoic acid (6.70 ± 1.61 vs. 5.65 ± 1.59%). Males also displayed lower concentrations of ß-carotene (0.01 ± 0.01 vs. 0.012 ± 0.001 µg/ml) and phosphorus (3.43 ± 0.83 vs. 4.40 ± 1.66 mg/dl). Comparison between the 2 years showed most circulating amino acid concentrations were higher in 2007. Significant differences in circulating amino acids and vitamins were also noted between different reproductive stages. These results demonstrate concentrations of nutrients can vary due to the physiological state of the animal, as well as the overall dynamics of their marine ecosystem habitat.

Mortality in Three-Toed Box Turtles (Terrapene mexicana triunguis) at Two Sites in Missouri.

Once ubiquitous, North American box turtles are experiencing reductions in abundance and range, but the magnitude of these losses is largely unknown. In Missouri, native box turtles (Terrapene mexicana triunguis and Terrapene ornata ornata) are declining across the state due to anthropogenic disturbances such as urbanization, habitat fragmentation, and vehicle collisions. Through radio-tracking over a period of 7 years, we documented the survival of adult three-toed box turtles at two sites in Missouri: Forest Park (urban park) and Tyson Research Center (TRC) (a protected rural forest). Estimated annual survival of adult turtles in Forest Park was 79% (95% CI: 0.68-0.87) while at TRC annual survival was 93% (95% CI: 0.83-0.97). The odds of annual survival for a turtle at TRC were 3.5 times that of a turtle living in Forest Park. "Winter kill," which refers to box turtles found dead on the surface during brumation or within 2 weeks of emergence, was the most frequently documented category of mortality in Forest Park. At TRC, winter kill was not documented; however, the reasons for most deaths were unknown. These data raise questions about the potential of large urban parks as refuges for box turtles, which we may answer by future studies that compare box turtles living in multiple urban and rural settings. Our preliminary data suggest that even the largest urban parks may not be able to sustain populations of box turtles which has severe implications as urbanization continues to degrade and eliminate box turtle habitat throughout their range.

Antimicrobial resistance genes present in the faecal microbiota of free-living Galapagos tortoises (Chelonoidis porteri).

Antimicrobial resistance (AMR), encoded by plasmid-mediated AMR genes (ARGs), is an increasing global public health threat. Wildlife play a fundamental role as sentinels, reservoirs and potential vectors of ARGs. For the first time in Galapagos, we have identified and quantified the presence of ARGs in free-living giant tortoises (Chelonoidis porteri). We performed ARG analyses by quantitative PCR of faeces collected from the cloaca of 30 tortoises widely distributed across Santa Cruz Island. Validated samples (n = 28) were analysed by a panel of up to 21 different ARGs and all 28 tortoise samples were positive to one or more genes encoding resistance. Thirteen of 21 tested ARGs were present in at least one sample, and 10 tortoises (35.7%) had a multi-resistant pattern. We recommend additional research so we may more fully understand resistance patterns across taxa and geographical locations throughout the Galapagos archipelago, and the implications of ARGs for the health of wildlife, domestic animals, and humans. In this study, we found 100% of sampled giant tortoises had ARGs present in their faeces, suggesting a large-scale distribution of these genes within the archipelago.

Diversity patterns of hematophagous insects in Atlantic forest fragments and human-modified areas of southern Bahia, Brazil.

There have been several important outbreaks of mosquito-borne diseases in the Neotropics in recent years, particularly in Brazil. Some taxa are also considered to be indicators of environmental health. Despite the importance of understanding insect abundance and distribution to the understanding of disease dynamics and design strategies to manage them, very little is known about their ecology in many tropical regions. We studied the abundance and diversity of mosquitoes and sand flies in the Bahia State of Brazil, a point of origin for arbovirus outbreaks, including Zika and Chikungunya fever. During 2009-2014, 51 mosquito taxa were identified, belonging to three dipteran families, Ceratopogonidae, Culicidae, and Psychodidae. The family Culicidae, including the Sabethini tribe, were the most abundant (81.5%) and most taxa-rich (n=45). While season (winter and summer) was a strong factor determinant of the occurrence of the most abundant taxa, the stratification level in the forest (ground or tree level) had a strong effect and the dominant taxa at ground level were completely different from the dominant species collected at tree level. We suggest that sites with a mix of forest and agroforestry systems support the highest biodiversity of hematophagous insects as compared to highly disturbed landscapes.

FIRST RECORD OF HEMATOLOGIC VALUES IN FREE-LIVING AND CAPTIVE MANED SLOTHS (BRADYPUS TORQUATUS; XENARTHA, BRADYPODIDAE).

Bradypus torquatus is a rare and endemic sloth species from the Atlantic Forest, Brazil. Due to a lack of medical information including hematologic reference parameters for the species, hematologic baseline values were determined using samples from 14 clinically healthy B. torquatus , under captive (n = 7) and free-living (n = 7) conditions in Bahia State, Brazil. Additionally, the morphology of the blood cells is presented, with a demonstration that the Barr body chromosome may assist with sex determination of the species. The Barr body chromosome was present in all seven females and absent in all males. Many erythrocytes were approximately the size of small lymphocytes, with red blood cells exhibiting anisocystosis, normochromia, and apparent macrocytosis, compared with domestic animals. This study provides the first published hematologic values and cell morphology for B. torquatus . However, further studies are suggested using an automated hematology analyzer with larger sample sizes so that reference intervals may be established and hematologic values better understood for sex, habitat type, and age cohorts.

Occurrence of Prosthenorchis elegans in Free-living Primates from the Atlantic Forest of Southern Bahia, Brazil.

Parasite prevalence and abundance are important factors affecting species' conservation. During necropsies on a free-living golden-headed lion tamarin ( Leontopithecus chrysomelas ) and two Wied's marmosets ( Callithrix kuhlii ) in the Atlantic Forest of southern Bahia, Brazil, we collected a large number of adult intestinal parasites that we identified as Prosthenorchis elegans. This parasite is pathogenic for neotropical primates. Prosthenorchis spp. infestation is influenced by diet with increased risk of exposure from ingesting invertebrate intermediate hosts. The biological similarities and sympatric nature of these two nonhuman primates support that they may harbor similar infectious and parasitic agents.

COMPARISON OF TOTAL LEUKOCYTE QUANTIFICATION METHODS IN FREE-LIVING GALAPAGOS TORTOISES (CHELONOIDIS SPP.).

Reptile hematologic data provide important health information for conservation efforts of vulnerable wildlife species such as the Galapagos tortoise (Chelonoidis spp.). Given the reported discrepancies between manual leukocyte counts for nonmammalian species, two manual leukocyte quantification methods, the Natt and Herrick's (NH) and the Eopette (EO), were compared to white blood cell (WBC) estimates from blood films of 42 free-living, clinically healthy, adult female Galapagos tortoises. To investigate the effects of delay in sample processing, estimated WBC counts and leukocyte differentials were compared for blood films prepared at time of collection under field conditions (T0) to blood films prepared from samples that were stored for 18-23 hr at 4°C in the laboratory (T1). Passing-Bablok regression analysis revealed no constant or proportional error between the NH and WBC estimates (T0 and T1) with slopes of 1.1 and 0.9, respectively. However both constant and proportional errors were present between EO and WBC estimates (T0 and T1) with slopes of 3.1 and 2.7, respectively. Bland Altman plots also showed agreement between the NH and WBC estimates where the points fell within the confidence-interval limit lines and were evenly distributed about the mean. In contrast, the EO and WBC estimate comparisons showed numerous points above the upper limit line, especially at higher concentrations. WBC estimates obtained from T0 and T1 films were in agreement, whereas heterophil and monocyte percentages based on differentials were not. Cell morphology and preservation were superior in T0 blood films because thrombocytes exhibited swelling after storage, becoming difficult to differentiate from lymphocytes. In this study, the highest quality and most reliable hematologic data in Galapagos tortoises were obtained by combining immediate blood film preparation with the NH leukocyte quantification method and a confirmatory WBC estimate from the blood film.

Attempted detection of Toxoplasma gondii oocysts in environmental waters using a simple approach to evaluate the potential for waterborne transmission in the Galápagos Islands, Ecuador.

Toxoplasmosis is a health concern for wildlife and humans, particularly in island ecosystems. In the Galápagos Islands, exposure to Toxoplasma gondii has been found in marine avifauna on islands with and without domestic cats. To evaluate potential waterborne transmission of T. gondii, we attempted to use filtration and epifluorescent microscopy to detect autofluorescent T. gondii oocysts in fresh and estuarine surface water samples. T. gondii oocyst-like structures were microscopically visualized but were not confirmed by polymerase chain reaction and sequence analyses. Further research is needed to refine environmental pathogen screening techniques and to evaluate disease risk of waterborne zoonoses such as T. gondii for wildlife and humans, particularly in the Galápagos and other naive island ecosystems.

Seroprevalence of malarial antibodies in Galapagos penguins (Spheniscus mendiculus).

A parasite species of the genus Plasmodium has recently been documented in the endangered Galapagos penguin (Spheniscus mendiculus). Avian malaria causes high mortality in several species after initial exposure and there is great concern for the conservation of the endemic Galapagos penguin. Using a Plasmodium spp. circumsporozoite protein antigen, we standardized an enzyme-linked immunosorbent assay to test the level of exposure in this small population, as indicated by seroprevalence. Sera from adult and juvenile Galapagos penguins collected between 2004 and 2009 on the Galapagos archipelago were tested for the presence of anti- Plasmodium spp. antibodies. Penguins were also tested for the prevalence of avian malaria parasite DNA using a polymerase chain reaction (PCR) screening. Total seroprevalence of malarial antibodies in this sample group was 97.2%, which suggests high exposure to the parasite and low Plasmodium-induced mortality. However, total prevalence of Plasmodium parasite DNA by PCR screening was 9.2%, and this suggests that parasite prevalence may be under-detected through PCR screening. Multiple detection methods may be necessary to measure the real extent of Plasmodium exposure on the archipelago.