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The skin microbiome is an important part of amphibian immune defenses and protects against pathogens such as the chytrid fungus Batrachochytrium dendrobatidis (Bd), which causes the skin disease chytridiomycosis. Alteration of the microbiome by anthropogenic factors, like pesticides, can impact this protective trait, disrupting its functionality. Chlorothalonil is a widely used fungicide that has been recognized as having an impact on amphibians, but so far, no studies have investigated its effects on amphibian microbial communities. In the present study, we used the amphibian Lithobates vibicarius from the montane forest of Costa Rica, which now appears to persist despite ongoing Bd-exposure, as an experimental model organism. We used 16S rRNA amplicon sequencing to investigate the effect of chlorothalonil on tadpoles’ skin microbiome. We found that exposure to chlorothalonil changes bacterial community composition, with more significant changes at a higher concentration. We also found that a larger number of bacteria were reduced on tadpoles’ skin when exposed to the higher concentration of chlorothalonil. We detected four presumed Bd-inhibitory bacteria being suppressed on tadpoles exposed to the fungicide. Our results suggest that exposure to a widely used fungicide could be impacting host-associated bacterial communities, potentially disrupting an amphibian protective trait against pathogens.

Universidad Nacional, Costa Rica Universidad de Costa Rica, Costa Rica Universidade de São Paulo, Brasil Red Mesoamericana y del Caribe para la Conservación de Anfbios y Reptiles Instituto Internacional en Conservación y Manejo de Vida Silvestre

In this study, the effect of four anaesthetic protocols that included the combination of xylazine (X) and ketamine (K) with acepromazine (A) and opioids (methadone (Me), morphine (Mo) or tramadol (T)) was evaluated in laboratory rats of both sexes. Ultrasonic vocalization (USV) was used as an indicator of pain during the recovery period. The objective was to evaluate the physiological parameters and the analgesic effect of each protocol to determine which protocol was the safest and fulfil the requirements of a balanced anaesthesia. The better protocols were the XKA protocol for both sexes and the XKMe protocol for females because the combinations achieve surgical plane of anaesthesia in rats. However, pain assessment during the formalin test revealed that rats anaesthetized with XKA produced more numbers of USV, suggesting that it is not a good protocol for the control of immediate postoperative pain. All protocols produced depression in body temperature and respiratory and heart rates, and had important effects, such as micturition and maintenance of open eyes. Only rats anaesthetized with XKA protocol did not present piloerection. These results demonstrated that good monitoring and care during anaesthesia must be included to prevent complications that compromise the life of the animal and to ensure a good recovery. The inclusion of analgesia in anaesthesia protocols must be used routinely, ensuring minimal presence of pain and thus more reliable results in the experimental procedures.

Amphibians are globally threatened by emerging infectious diseases, and ranaviruses are among the most concerning pathogens to threaten species in the wild. We sampled for ranaviruses in wild amphibians at 8 sites in Costa Rica, spanning broad climatic zones and taxonomic associations. Seven of these sites are inhabited by highly threatened amphibian species that persist at low global population sizes after population declines due to amphibian chytridiomycosis. One of the surveyed sites is occupied by an introduced amphibian species, which is relatively rare in Central America but may be an important pathway for long-distance transport of ranaviruses. We detected ranavirus using quantitative polymerase chain reaction in 16.3% of the 243 individuals and among 5 of our 8 sites, but not at the site with the introduced species. Infection prevalence varied among species and sites, but not with mean annual temperature or mean annual precipitation. Infection intensity did not vary with species, site, temperature, or precipitation. Our results show that ranavirus infection is spatially widespread in Costa Rica, affecting a broad range of host species, and occurs across climatic zones—though we encountered no mortality or morbidity in our sampled species. Ranaviruses are known to cause intermittent mass mortality in amphibian populations, and the threatened species sampled here are likely vulnerable to population impacts from emerging ranaviruses. Therefore, we believe the potential impacts of ranaviruses on amphibian populations in tropical regions have likely been underestimated, and that they should be viewed as a potential major stressor to threatened amphibians in tropical regions.

Amphibians have declined around the world in recent years, in parallel with the emergence of an epidermal disease called chytridiomycosis, caused by the chytrid fungus

Amphibians have declined around the world in recent years, in parallel with the emergence of an epidermal disease called chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). This disease has been associated with mass mortality in amphibians worldwide, including in Costa Rica, and Bd is considered an important contributor to the disappearance of this group of vertebrates. While many species are susceptible to the disease, others show tolerance and manage to survive infection with the pathogen. We evaluated the pathogen Bd circulating in Costa Rica and the capacity of amphibian skin bacteria to inhibit the growth of the pathogen in vitro. We isolated and characterized – genetically and morphologically – several Bd isolates from areas with declining populations of amphibians. We determined that the circulating chytrid fungus in Costa Rica belongs to the virulent strain Bd-GPL-2, which has been related to massive amphibian deaths worldwide; however, the isolates obtained showed genetic and morphological variation. Furthermore, we isolated epidermal bacteria from 12 amphibian species of surviving populations, some in danger of extinction, and evaluated their inhibitory activity against the collection of chytrid isolates. Through bioassays we confirmed the presence of chytrid-inhibitory bacterial genera in Costa Rican amphibians. However, we observed that the inhibition varied between different isolates of the same bacterial genus, and each bacterial isolation inhibited fungal isolation differently. In total, 14 bacterial isolates belonging to the genera Stenotrophomonas , Streptomyces , Enterobacter , Pseudomonas and Klebsiella showed inhibitory activity against all Bd isolates. Given the observed variation both in the pathogen and in the bacterial inhibition capacity, it is highly relevant to include local isolates and to consider the origin of the microorganisms when performing in vivo infection tests aimed at developing and implementing mitigation strategies for chytridiomycosis.

Global amphibian biodiversity has declined dramatically in the past 4 decades, and many amphibian species have declined to near extinction as a result of emergence of the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). However, persistent or recovering populations of several amphibian species have recently been rediscovered, and such populations may illustrate how amphibian species that are highly susceptible to chytridiomycosis may survive in the presence of Bd. We conducted field surveys for Bd infection in 7 species of Costa Rican amphibians (all species that have declined to near extinction but for which isolated populations persist) to characterize infection profiles in highly Bd-susceptible amphibians post-decline. We found highly variable patterns in infection, with some species showing low prevalence (~10%) and low infection intensity and others showing high infection prevalence (>80%) and either low or high infection intensity. Across sites, infection rates were negatively associated with mean annual precipitation, and infection intensity across sites was negatively associated with mean average temperatures. Our results illustrate that even the most Bd-susceptible amphibians can persist in Bd-enzootic ecosystems, and that multiple ecological or evolutionary mechanisms likely exist for host-pathogen co-existence between Bd and the most Bd-susceptible amphibian species. Continued monitoring of these populations is necessary to evaluate population trends (continuing decline, stability, or population growth). These results should inform efforts to mitigate impacts of Bd on amphibians in the field.

Simple Summary Through a systematic review of reports where swine were used as animal biomodels for testing or researching new surgical techniques, we sought to determine the quality of the report of the methodologies carried out on the basis of the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) in a total of 108 studies from 2013 to 2018. In a large percentage of the articles, the information presented in the methodology of the studies showed incomplete data according to the ARRIVE guidelines recommendations for reporting the use of animals. There was a strong focus on descriptions of surgical techniques; however, sample size calculation, description of maintenance conditions, animal handling, and anesthetic and pain management protocols used were not very detailed. This could lead to the inability of others to replicate the described experiments. For this reason, we encourage authors to implement the ARRIVE guidelines to improve the quality of scientific reports and ensure animal welfare. Abstract Over the last two decades, pigs have become animal biomodels widely used for the investigation and practice of surgical techniques because of their great physiological and anatomical similarities to humans. Even though many of these studies must be carried out later in humans, the description of basic information is limited, making exact repetitions of the reported experimental methods impossible. In this review, 108 studies from 2013 to 2018 were considered to determine the quality of adherence to the ARRIVE guidelines in the reports of the methodologies. The majority of the studies lacked the details recommended in the ARRIVE guidelines regarding data directly related to the welfare of animals undergoing surgery and those about anesthetic protocols and analgesics. Information related to sample size calculation and housing and husbandry conditions was also very limited. We believe that the ARRIVE guidelines are an excellent tool for good-quality reporting. We encourage scientists to consistently use them as a tool to improve the quality of their scientific reports and, consequently, ensure animal welfare.

Emerging infectious diseases are a growing threat to biodiversity worldwide. Outbreaks of the infectious disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), have caused the decline and extinction of numerous amphibian species. In Costa Rica, a major decline event occurred in 1987, more than two decades before this pathogen was discovered. The loss of many species in Costa Rica is assumed to be due to Bd-epizootics, but there are few studies that provide data from amphibians in the time leading up to the proposed epizootics. In this study, we provide new data on Bd infection rates of amphibians collected throughout Costa Rica, in the decades prior to the epizootics. We used a quantitative PCR assay to test for Bd infection in 1016 specimens collected throughout Costa Rica. We found Bd-infected hosts collected as early as 1964, and a infection prevalence average per decade of just 4%. The infection prevalence remained relatively low and geographically constrained until the 1980s when epizootics are hypothesized to have occurred. After that time, infection prevalence increased three-fold and Bd-infected hosts we collected throughout the entire country. Our results, suggest that Bd may either have invaded Costa Rica earlier than previously known, and spread more slowly than previously reported, or that an endemic lineage of the pathogen may exists. To help visualize areas where future studies should take place, we provide a Bd habitat suitability model trained with local data. Studies that provide information on genetic lineages of Bd are needed to determine whether an endemic lineage of Bd or the Global Panzootic Lineage (identified from mass die off sites globally) was present in Costa Rica and responsible for the epizootics that caused amphibian communities to collapse.

Declines of amphibian populations have been a worldwide issue of concern for the scientific community during the last several decades. Efforts are being carried out to elucidate factors related to this phenomenon. Among these factors, pathogens, climate change, and environmental pollution have been suggested as possible causes. Regarding environmental pollutants, some pesticides are persistent in the environment and capable of being transported long distances from their release point. In Costa Rica, some pesticides have been detected in protected areas, at locations where amphibian populations have declined. Information about toxicity of pesticides used in Costa Rican agriculture to amphibians is still scarce, particularly for native species.Toxicity tests with chlorothalonil, a fungicide intensively used in Costa Rica, were carried out exposing tadpoles of three Costa Rican native species: Agalychnis callidryas, Isthmohyla pseudopuma, and Smilisca baudinii in order to evaluate acute and chronic toxicity as well as the biomarkers cholinesterase activity (ChE), glutathione-S transferase activity (GST), and lipid peroxidation (LPO).96-h LC50: 26.6 (18.9-35.8) μg/L to A. callidryas, 25.5 (21.3-29.7) μg/L to I pseudopuma and 32.3 (26.3-39.7) μg/L to S. baudinii were determined for chlorothalonil. These three species of anurans are among the most sensitive to chlorothalonil according to the literature. Besides, GST was induced in S. baudinii after exposure to sub-lethal concentrations of chlorothalonil while evisceration occurred in S. baudinii and A. callidryas tadpoles exposed to lethal concentrations of the fungicide. Chronic exposure to sub-lethal concentrations accelerated development in S. baudinii and caused lesions in tail of S. baudinii and I. pseudopuma tadpoles. Our results demonstrate that chlorothalonil is highly toxic to native amphibian species and that low concentrations can cause biochemical responses related to phase II of biotransformation and effects on development.

We report the rediscovery of a declared extinct frog,Craugastor escoces, at the edge of a protected forest in the Juan Castro Blanco National Park, Alajuela, Costa Rica. This species, which is endemic to Costa Rica, had not been observed since 1986. The rediscovery of this species comes after 30 years without being seen and 12 years of being declared extinct. The site where we found the species is located in the west of the central mountain region of Costa Rica, which is >15 km away from its known distribution. Our finding adds another species to the list of the few rediscovered frogs declared extinct. Knowing thatC. escocescan still be found triggers the need for conservation strategies to help the species survive.

Licania platypus se distribuye ampliamente en países tropicales. Tradicionalmente, en América Central se ha utilizado para diversos problemas de salud, especialmente gastrointestinales. Objetivo: Investigar la actividad antiulcerosa gástrica y el efecto sobre la motilidad gastrointestinal del extracto acuoso de hojas de Licania platypus. Métodos: El daño gástrico fue inducido con los modelos de etanol e indometacina y se evaluaron otros parámetros en el modelo de ligadura pilórica. Además, se analizó el efecto sobre la motilidad gastrointestinal utilizando rojo de fenol. Resultados: La administración oral del extracto acuosos (1000mg/kg) redujo significativamente el área de las lesiones gástricas (p

Mariculture of Florida pompano Trachinotus carolinus in Central America has increased over the last few decades and it is now a highly valued food fish. High feed costs and infectious diseases are significant impediments to the expansion of mariculture. Members of the genus Megalocytivirus (MCV), subfamily Alphairidovirinae, within the family Iridoviridae, are emerging pathogens that negatively impact Asian mariculture. A significant mortality event in Florida pompano fingerlings cultured in Central America occurred in October 2014. Affected fish presented with abdominal distension, darkening of the skin, and periocular hemorrhages. Microscopic lesions included cytomegalic 'inclusion body-bearing cells' characterized by basophilic granular cytoplasmic inclusions in multiple organs. Transmission electron microscopy revealed arrays of hexagonal virions (155-180 nm in diameter) with electron-dense cores within the cytoplasm of cytomegalic cells. Pathological findings were suggestive of an MCV infection, and the diagnosis was later confirmed by partial PCR amplification and sequencing of the viral gene encoding the myristylated membrane protein. The viral sequence revealed that the fingerlings were infected with an MCV genotype, red seabream iridovirus (RSIV), previously reported only from epizootics in Asian mariculture. This case underscores the threat RSIV poses to global mariculture, including the production of Florida pompano in Central America.

Emerging infectious diseases are a growing threat to biodiversity worldwide. Outbreaks of the infectious disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), are implicated in the decline and extinction of numerous amphibian species. In Costa Rica, a major decline event occurred in 1987, more than two decades before this pathogen was discovered. The loss of many species in Costa Rica is assumed to be due to Bd-epizootics, but there are few studies that provide data from amphibians in the time leading up to the proposed epizootics. In this study, we provide new data on Bd infection rates of amphibians collected throughout Costa Rica, in the decades prior to the epizootics. We used a quantitative PCR assay to test for Bd presence in 1016 anuran museum specimens collected throughout Costa Rica. The earliest specimen that tested positive for Bd was collected in 1964. Across all time periods, we found an overall infection rate (defined as the proportion of Bd-positive individuals) of 4%. The number of infected individuals remained relatively low across all species tested and the range of Bd-positive specimens was shown to be geographically constrained up until the 1980s; when epizootics are hypothesized to have occurred. After that time, infection rate increased three-fold, and the range of specimens tested positive for Bd increased, with Bd-positive specimens collected across the entire country. Our results suggest that Bd dynamics in Costa Rica are more complicated than previously thought. The discovery of Bd's presence in the country preceding massive declines leads to a number of different hypotheses: 1) Bd invaded Costa Rica earlier than previously known, and spread more slowly than previously reported; 2) Bd invaded multiple times and faded out; 3) an endemic Bd lineage existed; 4) an earlier Bd lineage evolved into the current Bd lineage or hybridized with an invasive lineage; or 5) an earlier Bd lineage went extinct and a new invasion event occurred causing epizootics. To help visualize areas where future studies should take place, we provide a Bd habitat suitability model trained with local data. Studies that provide information on genetic lineages of Bd are needed to determine the most plausible spatial-temporal, host-pathogen dynamics that could best explain the epizootics resulting in amphibian declines in Costa Rica and throughout Central America.

La especie Licania platypus, localmente conocida como “sonzapote” se encuentra ampliamente distribuida en países tropicales. Tradicionalmente, en América Central se han utilizado diferentes partes de la planta para diversos problemas de salud, especialmente gastrointestinales. Objetivo: Investigar la posible actividad antiulcerosa gástrica del extracto etanólico de hojas de Licania platypus. Métodos: Las lesiones gástricas fueron inducidas por los modelos de etanol e indometacina. También, se evaluó el extracto con el modelo modificado de Shay. El extracto se administró por vía oral y se midió el área dañada. Además, se analizaron otros parámetros en el modelo de ligadura pilórica. Resultados: La administración oral de una dosis única del extracto (500 mg/kg) redujo significativamente el área de las lesiones gástricas (p < 0.05) en los modelos de inducción de etanol e indometacina. Además, se observó un incremento significativo en la secreción de moco gástrico en el modelo modificado de Shay (p < 0.05). Conclusión: El extracto etanólico de Licania platypus ejerce un efecto gastroprotector en diferentes modelos de inducción de úlceras agudas. Este trabajo brinda la primera evidencia de especies pertenecientes al género Licania con actividad gastroprotectora y permite promover la realización de estudios para elucidar un mecanismo de acción específico.

Anfíbios são indicadores ambientais potencialmente confiáveis e eficientes. Estudos referentes a morfologia de leucócitos de anuros são limitados, com poucos estudos morfometricos disponíveis em literatura. O presente estudo empregou técnicas morfometricas para caracterizar leucócitos de anuros Neotropicais brasileiros selecionados e compara-los com a espécie exótica rã-touro (Lithobates catesbeianus), família Ranidae. Esfregaços sanguíneos de 28 espécimes pertencentes a seis gêneros diferentes (Hyla, Phyllomedusa, Hypsiboas, Scinax, Physalaemus e Proceratophrys) foram comparados com amostras de esfregacos de L. catesbeianus. A média do diâmetro dos leucócitos foi calculada por um software de análise de imagens. One-way e teste de Bonferroni foram utilizados para avaliação estatística. Linfócitos, neutrófilos, eosinófilos e basófilos mostraram-se significativamente menores que os valores de referência reportados em outros gêneros de anfíbios, incluindo Lithobathes; por outro lado, a média do diâmetro dos monócitos não demonstrou variação significativa entre os gêneros. Esse e o primeiro estudo de avaliação morfometrica de leucócitos em espécies de anuros brasileiros. Nossos resultados sugerem que a separação geográfica possivelmente influencia a morfometria leucocitaria. Amphibians are potentially reliable and efficient bioindicators. Existing anuran white blood cell morphology studies are limited, with only a few morphometric studies available. We employed morphometric techniques to characterize leukocytes of selected Neotropical anurans from Brazil and compare our findings with the exotic American Bullfrog (Lithobates catesbeianus), genus Ranidae. We compared blood smears of 28 specimens from six different genera (Hyla, Phyllomedusa, Hypsiboas, Scinax, Physalaemus, and Proceratophrys) with samples from L. catesbeianus. Leukocyte average diameter was calculated by an image analysis software. One-way analyses of variance and Bonferroni tests were used on statistical analyses. Lymphocytes, neutrophils, eosinophils, and basophils were significantly smaller than the reference ranges reported for other amphibian genera, including Lithobathes, whereas monocyte diameters did not differ significantly between genera. This is the first study to evaluate leukocyte morphometrics of Brazilian anuran species. Our findings suggest that geographical separation could possibly influence leukocyte morphometry.

The eco-epidemiology of American cutaneous leishmaniasis (ACL) is driven by animal reservoir species that are a source of infection for sand flies that serve as vectors infecting humans with Leishmania spp parasites. The emergence and re-emergence of this disease across Latin America calls for further studies to identify reservoir species associated with enzootic transmission. Here, we present results from a survey of 52 individuals from 13 wild mammal species at endemic sites in Costa Rica and Panama where ACL mammal hosts have not been previously studied. For Leishmania spp. diagnostics we employed a novel PCR technique using blood samples collected on filter paper. We only found Leishmania spp parasites in one host, the two-toed sloth, Choloepus hoffmanni. Our findings add further support to the role of two-toed sloths as an important ACL reservoir in Central America., Tropical Medicine and Health, 43(1), pp.75-78; 2015

El estudio muestra, por primera vez, la detección de Hepatozoon procyonis en prociónidos, Procyon lotor de Costa Rica. Para caracterizar los gametocitos, se tomó muestras de sangre de mapaches infectados naturalmente. Los gametocitos concuerdan con Hepatozoon procyonis, los cuales fueron observados en neutrófilos del citoplasma, resultando su morfología similar a la reportada en mapaches de todas las Américas.

Reports on ectoparasites on dogs in Central America are scarce. The aim of this study was to identify flea, louse and tick species infesting dogs in home environments on the Caribbean slope of Costa Rica, and determine their frequency and coexistence. Ectoparasites were collected from dogs in 83 rural homes at five study sites. Specimens were identified and separated according to species. Fleas were the most common ectoparasite (G = 22,217, DF = 8, p = 0.004). Ctenocephalides felis and Pulex simulans were found in 83% and 55% of the homes with ectoparasites, respectively. Trichodectes canis (13%), Heterodoxus spiniger (10%), Rhipicephalus sanguineus (18%), Rhipicephalus (Boophilus) microplus (5%) and Amblyomma ovale (8%) were also present. More than one species was collected in most cases (66%), and the most common combination was C. felis and P. simulans (59% of homes with fleas). The high frequency of P. simulans emphasizes the need for adequate identification. This was the first study involving different ectoparasites of dogs in Costa Rica, as well as the first report of T. canis in this country. The relative frequency and coexistence of these ectoparasites in the home environment may have implications for animal and human health. Relatos de ectoparasitos em cães da América Central são escassos. O objetivo deste estudo foi identificar espécies de pulgas, piolhos e carrapatos que infestam os cães no ambiente doméstico da vertente Caribe da Costa Rica, determinando sua frequência e coexistência. Foram coletados ctoparasitos de cães em 83 casas rurais de cinco sítios do estudo. Os indivíduos foram separados e identificados. Os ectoparasitos mais comuns foram as pulgas (G = 22.217, DF = 8, p = 0.004). Ctenocephalides felis e Pulex simulans foram observados em 83% e 55% das moradias com ectoparasitos, respectivamente. Da mesma forma também foram observados Trichodectes canis (13%), Heterodoxus spiniger (10%), Rhipicephalus sanguineus (18%), Rhipicephalus (Boophilus) microplus (5%) e Amblyomma ovale (8%). Na maioria dos casos, mais de uma espécie foi achada (66%), sendo que a combinação mais comum foi C. felis e P. simulans (59% de moradias com pulgas). A frequência alta de P. simulans ressalta a necessidade de sua adequada identificação. Este é o primeiro estudo que envolve diferentes ectoparasitas de cães na Costa Rica, da mesma forma, é o primeiro relato de T. canis no país. A frequência relativa e coexistência destes ectoparasitos no ambiente domiciliar poderiam ter implicações na saúde animal e humana. Los informes sobre ectoparásitos en perros en América Central son escasos. El objetivo de este estudio fue identificar las especies de pulgas, piojos y garrapatas que infestan a los perros en los entornos domésticos de la vertiente caribeña de Costa Rica, y determinar su frecuencia y coexistencia. Se recogieron ectoparásitos de los perros en 83 hogares rurales de cinco lugares de estudio. Se identificaron los ejemplares y se separaron según las especies. Las pulgas fueron el ectoparásito más común (G = 22.217, DF = 8, p = 0.004). Se encontraron Ctenocephalides felis y Pulex simulans en el 83% y el 55% de los hogares con ectoparásitos, respectivamente. También estaban presentes Trichodectes canis (13%), Heterodoxus spiniger (10%), Rhipicephalus sanguineus (18%), Rhipicephalus (Boophilus) microplus (5%) y Amblyomma ovale (8%). En la mayoría de los casos se recogió más de una especie (66%), y la combinación más común fue C. felis y P. simulans (59% de los hogares con pulgas). La alta frecuencia de P. simulans pone de relieve la necesidad de una identificación adecuada. Este fue el primer estudio sobre diferentes ectoparásitos de perros en Costa Rica, así como el primer informe sobre T. canis en este país. La relativa frecuencia y coexistencia de estos ectoparásitos en el entorno doméstico puede tener implicaciones para la salud animal y humana. Universidad Nacional, Costa Rica Escuela de Medicina Veterinaria