Upconverting phosphor technology-based lateral flow assay for the rapid and sensitive detection of anti-Trichinella spiralis IgG antibodies in pig serum.

BACKGROUND: Trichinella spiralis is a zoonotic food-borne parasite. A disease caused by infection with T. spiralis is called trichinellosis in humans. It is important to investigate the epidemic situation and the surveillance of herds and then prevent infection in humans. Therefore, this study is to develop a rapid and sensitive diagnostic method for on-site test in domestic and wild animals. METHODS: Upconverting phosphor nanoparticles (UCNPs), an excellent optical label, were conjugated with the excretory-secretory (ES) antigens from T. spiralis muscle larvae (ML) or goat anti-rabbit IgG, and a lateral flow (LF) assay based on these probes (UCNPs-ES/goat anti-rabbit IgG) was developed for the rapid and sensitive detection of anti-T. spiralis IgG antibodies in pig serum. The assay is named the UPT-LF-ES assay. In addition, the probes were characterized, and the assay was optimized. A cut-off threshold of the assay was also identified by using 169 known negative pig samples. Performance of the assay to T. spiralis with different infective numbers, cross-reactivity with other parasitic infections, the single-blinded experiment, and coincidence were evaluated with the assay. RESULTS: The UPT-LF-ES assay was successfully constructed and optimized based on the probes of UCNPs-ES/goat anti-rabbit IgG. In the pigs infected with 100, 1000, and 10,000 ML, positive results were first presented at 35 days post-infection (dpi), 30 dpi, and 25 dpi, respectively. The assay had no cross-reaction with other parasitic infections. A single-blinded experiment indicated that the sensitivity and specificity of the UPT-LF-ES assay were 100% and 100%, respectively, the area under the receiver operating characteristic (ROC) curve was 1.000. In addition, the value detected by the UPT-LF-ES assay was significantly different between positive and negative samples. Moreover, compared with the "gold standard" magnetic stirrer method, the coincidence rate of the UPT-LF-ES assay was 87.27%, and the kappa (K) coefficient was 0.7454, showing a substantial agreement. CONCLUSIONS: The UPT-LF-ES assay is a useful point-of-care test (POCT) with T. spiralis in the detection of pig, which contributes to preventing human trichinellosis.

Prevalence and associated risk factors of Leishmania infection among immunocompetent hosts, a community-based study in Chiang Rai, Thailand.

BACKGROUND: Leishmaniasis is an emerging infectious disease reported in the north and south of Thailand of which patients with HIV/AIDS are a high risk group for acquiring the infection. A lack of information regarding prevalence, and the risk association of Leishmania infection among asymptomatic immunocompetent hosts needs further investigation. Information on potential vectors and animal reservoirs in the affected areas is also important to control disease transmission. METHODS: An outbreak investigation and a cross-sectional study were conducted following one index case of cutaneous leishmaniasis (CL) caused by L. martiniquensis in an immunocompetent male patient reported in August 2015, Chiang Rai Province, Thailand. From September to November 2015, a total of 392 participants at two study areas who were related to the index case, 130 students at a semi-boarding vocational school and 262 hill tribe villagers in the patient's hometown, were recruited in this study. The nested internal transcribed spacer 1-PCR (ITS1-PCR) was performed to detect Leishmania DNA in buffy coat, and nucleotide sequencing was used to identify species. Antibody screening in plasma was performed using the Direct Agglutination Test (DAT), and associated risk factors were analyzed using a standardized questionnaire. Captured sandflies within the study areas were identified and detected for Leishmania DNA using nested ITS1-PCR. Moreover, the animal reservoirs in the study areas were also explored for Leishmania infection. RESULTS: Of 392 participants, 28 (7.1%) were positive for Leishmania infection of which 1 (4.8%) was L. martiniquensis, 12 (57.1%) were L. orientalis and 8 (38.1%) were Leishmania spp. Of 28, 15 (53.6%) were DAT positive. None showed any symptoms of CL or visceral leishmaniasis. Risk factors were associated with being female (adjusted odds ratio, AOR 2.52, 95%CI 1.01-6.26), increasing age (AOR 1.05, 95%CI 1.02-1.08), having an animal enclosure in a housing area (AOR 3.04, 95%CI 1.13-8.22), being exposed to termite mounds (AOR 3.74, 95%CI 1.11-12.58) and having domestic animals in a housing area (AOR 7.11, 95%CI 2.08-24.37). At the semi-boarding vocational school, six Sergentomyia gemmea samples were PCR positive for DNA of L. orientalis and one S. gemmea was PCR positive for DNA of L. donovani/L. infantum. Additionally, one Phlebotomus stantoni was PCR positive for DNA of L. martiniquensis, and one black rat (Rattus rattus) was PCR positive for DNA of L. martiniquensis. CONCLUSION: This information could be useful for monitoring Leishmania infection among immunocompetent hosts in affected areas and also setting up strategies for prevention and control. A follow-up study of asymptomatic individuals with seropositive results as well as those with positive PCR results is recommended.

BLOOD FATTY ACID PROFILES OF NERITIC JUVENILE WILD GREEN TURTLES (CHELONIA MYDAS) AND KEMP'S RIDLEYS (LEPIDOCHELYS KEMPII).

Blood fatty acid profiles can indicate an animal's wild-type diet composition and fatty acid status, but have not been reported in sea turtles. Newer technologies allow for fatty acid profiles from very small (less than three drops) samples of whole blood. This study examined whole blood fatty acid profiles of presumably healthy, neritic, juvenile, wild green (Chelonia mydas) (n = 9; 6 males, 3 females) and Kemp's ridley (Lepidochelys kempii) (n = 8; 6 males, 2 females) turtles from North Carolina, USA. Saturated fatty acids, which can be synthesized de novo, consisted primarily of 16:0, although green turtle blood had a higher proportion of 18:0 (P < 0.001) than Kemp's ridleys, while Kemp's ridley blood had higher proportions of 17:0 (P = 0.007), 20:0 (P = 0.03), 22:0 (P= 0.002), and 24:0 (P < 0.001) as compared with green turtles. Total monounsaturated w7 fatty acids, which can be synthesized de novo or may be diet derived, were higher in Kemp's ridleys and predominantly in the form of 16:1 and 18:1w7 fatty acids. Kemp's ridley blood had more than double the relative proportion of 16:1w7 as compared with green turtles (P= 0.03). Green turtles had higher levels of 18:2w6 than Kemp's ridleys (P= 0.02). In both turtle species, 20:4w6 was detected, despite predicted low dietary proportions, suggesting bioconversion from precursors. Finally, green turtles had higher levels of 18:3w3 compared with Kemp's ridleys, while Kemp's ridleys had higher proportions of 20:5w3 compared with green turtles (P < 0.001, = 0.007, respectively). Whole blood fatty acid profiles generally correlate to previous work with lipid depots, supporting the use of this less invasive methodology to advance the understanding of fatty acid nutrition of sea turtles. These data can be used to assess and guide nutrition and health programs for sea turtles under human care.

Morphological and molecular diagnosis of diseases of free-ranging crab-eating foxes (Cerdocyon thous) / [Diagnóstico morfológico e molecular de doenças de cachorro-do-mato (Cerdocyon thous) de vida livre]

The aim of this study was to investigate the occurrence of diseases in free-ranging wild canids that were roadkill on highways in the State of Espírito Santo, Brazil. PCR tests were performed for the detection of Brucella sp., Babesia sp., Rangelia sp., and Hepatozoon sp. in the spleen. Morphological evaluation and identification of parasites was performed in the liver and lung. Twenty specimens of C. thous were necropsied at the Animal Pathology Sector of the Veterinary Hospital of the Universidade Federal do Espírito Santo. Tissue samples were processed for histopathological examination and polymerase chain reaction (PCR) analysis. There was no PCR amplification of genomic DNA sequences of Brucella sp., Babesia sp., Rangelia sp., and Hepatozoon sp. using DNA extracted from the spleen as template. Histologically, lesions associated with parasitism by Platynosomum sp. and Angiostrongylus sp. were observed in the liver and lung, respectively. This is the first report of Platynosomum sp. and Angiostrongylus sp. parasitism in C. thous in the state of Espírito Santo, Brazil. Therefore, this study demonstrated parasitism of crab-eating foxes by Platynosomum sp. and Angiostrongylus sp. Importantly, no evidence of infection with Brucella sp., Babesia sp., Rangelia sp., and Hepatozoon sp. was obtained by PCR analysis.(AU)

O objetivo deste estudo foi investigar a ocorrência de doenças em canídeos silvestres de vida livre que foram atropelados em rodovias no estado do Espírito Santo, Brasil. Testes de PCR foram realizados para a detecção de Brucella sp., Babesia sp., Rangelia sp. e Hepatozoon sp. no baço. A avaliação morfológica e a identificação de parasitas foram realizadaa no fígado e no pulmão. Vinte espécimes de C. thous foram necropsiados no Setor de Patologia Animal do Hospital Veterinário da Universidade Federal do Espírito Santo. Amostras de tecido foram processadas para exame histopatológico e análise de reação em cadeia da polimerase (PCR). Não houve amplificação por PCR das sequências de DNA genômico de Brucella sp., Babesia sp., Rangelia sp. e Hepatozoon sp. usando-se DNA extraído do baço como modelo. Histologicamente, lesões associadas ao parasitismo por Platynosomum sp. e Angiostrongylus sp. foram observadas no fígado e no pulmão, respectivamente. Este é o primeiro relato de Platynosomum sp. e Angiostrongylus sp. parasitismo em C. thous no estado do Espírito Santo, Brasil. Portanto, este estudo demonstrou parasitismo de cachorro-do-mato por Platynosomum sp. e Angiostrongylus sp. É importante detacar que não há evidências de infecção por Brucella sp., Babesia sp., Rangelia sp. e Hepatozoon sp. por análise de PCR.(AU)

Seroprevalence of Hepatitis E Virus in Moose (Alces alces), Reindeer (Rangifer tarandus), Red Deer (Cervus elaphus), Roe Deer (Capreolus capreolus), and Muskoxen (Ovibos moschatus) from Norway.

Hepatitis E virus (HEV), a major cause of viral hepatitis worldwide, is considered an emerging foodborne zoonosis in Europe. Pigs (Sus scrofa domestica) and wild boars (S. scrofa) are recognized as important HEV reservoirs. Additionally, HEV infection and exposure have been described in cervids. In Norway, HEV has been identified in pigs and humans; however, little is known regarding its presence in wild ungulates in the country. We used a species-independent double-antigen sandwich ELISA to detect antibodies against HEV in the sera of 715 wild ungulates from Norway, including 164 moose (Alces alces), 186 wild Eurasian tundra reindeer (Rangifer tarandus tarandus), 177 red deer (Cervus elaphus), 86 European roe deer (Capreolus capreolus), and 102 muskoxen (Ovibos moschatus). The overall seroprevalence was 12.3% (88/715). Wild reindeer had the highest seropositivity (23.1%, 43/186), followed by moose (19.5%, 32/164), muskoxen (5.9%, 6/102), and red deer (4%, 7/177). All roe deer were negative. According to our results, HEV is circulating in wild ungulates in Norway. The high seroprevalence observed in wild reindeer and moose indicates that these species may be potential reservoirs of HEV. To the authors' knowledge, this is the first report of HEV exposure in reindeer from Europe and in muskoxen worldwide.

Reference values for biochemical analytes in feral sheep from Socorro Island, Revillagigedo Archipelago, Mexico / [Valores de referência para analitos bioquímicos em carneiros selvagens na Ilha Socorro, Arquipélago de Revillagigedo, México]

To establish reference values for biochemical analytes related to freshwater shortage adaptation, a total of 376 blood samples were collected from feral sheep at Socorro Island, Revillagigedo Archipelago. Year-round variation was assessed by sampling at the beginning of each season defined by the March equinox, June solstice, September equinox, and December solstice. The resulting data set was analyzed using Gaussian distribution and descriptive statistics. Confidence intervals of 95% were established. Analysis of variance was used to compare the mean values of each season. Total cholesterol, triglycerides, urea, albumin, total protein, sodium ion, anion gap, creatine kinase, arginine vasopressin, and aldosterone showed concentrations above the reference range for domestic sheep. Triglycerides, urea, albumin, sodium ion, and aldosterone showed concentrations within the reference range for domestic goats. Most biochemical analytes showed differences (P<0.05) between seasons, with the highest values occurring during winter, and the lowest during spring. Results could help improve the accuracy of metabolic profiles used as a tool for evaluating dehydration indicators, and to describe the physiological mechanisms employed by feral sheep to cope with seasonal availability of freshwater.(AU)

Para estabelecer valores de referência para analitos bioquímicos relacionados à adaptação da escassez de água doce, um total de 376 amostras de sangue foram coletadas de carneiros selvagens na ilha de Socorro, no arquipélago de Revillagigedo. A variação durante todo o ano foi avaliada por amostragem no início de cada estação definida pelo equinócio de março, solstício de junho, equinócio de setembro e solstício de dezembro. O conjunto de dados resultante foi analisado usando distribuição Gaussiana e estatística descritiva. Intervalos de confiança de 95% foram estabelecidos. A análise de variância foi usada para comparar os valores médios de cada estação. O colesterol total, triglicerídeos, ureia, albumina, proteína total, íon sódio, hiato aniônico, creatina quinase, arginina vasopressina e aldosterona apresentaram concentrações acima do intervalo de referência para carneiros domésticos. Triglicerídeos, ureia, albumina, íon sódio e aldosterona apresentaram concentrações dentro da faixa de referência para cabras domésticas. A maioria dos analitos bioquímicos apresentou diferenças (P<0,05) entre as estações, com os maiores valores ocorrendo no inverno e os menores na primavera. Os resultados podem ajudar a melhorar a precisão dos perfis metabólicos usados como uma ferramenta para avaliar os indicadores de desidratação e descrever os mecanismos fisiológicos empregados pelas carneiros selvagens para lidar com a disponibilidade sazonal de água doce.(AU)

Hematology and Clinical Biochemistry Profiles in Antillean Manatee Trichechus manatus manatus from Different Types of Captivity and Free Living in Northeast Brazil.

The Antillean manatee Trichechus manatus manatus can be found along the northern and northeastern coasts of Brazil. Previous studies on the clinical biochemistry of these animals were conducted in North America and the Caribbean, whereas little is known regarding these parameters in South American manatee populations. Accordingly, the objective of the present study was to examine the hematology and clinical biochemistry of Antillean manatees of different sexes and from different environments in northeast Brazil. Whole-blood and serum samples were obtained from healthy individuals. The hemogram analysis was performed and the levels of blood biochemical components were determined using an automated platform. The only statistically significant difference observed in the hemogram was a higher number of heterophils in manatees that were screened during the dry season of the year. Clinical biochemistry profiling revealed that free-ranging manatees presented lower levels of creatinine. Albumin was detected in higher concentrations in animals from rehabilitation captivity, and amylase presented higher levels in manatees that were kept in acclimation captivity. Free-ranging manatees showed higher serum aspartate aminotransferase levels than manatees in rehabilitation captivity. These results can aid veterinarians and conservation professionals in the development of better captive management procedures and in the clinical approach to manatees.

Haematology and blood chemistry in free-ranging quokkas (Setonix brachyurus): Reference intervals and assessing the effects of site, sampling time, and infectious agents.

Quokkas (Setonix brachyurus) are small macropodid marsupials from Western Australia, which are identified as of conservation concern. Studies on their blood analytes exist but involve small sample sizes and are associated with very little information concerning the health of the animals. Blood was collected from free-ranging quokkas from Rottnest Island (n = 113) and mainland (n = 37) Western Australia, between September 2010 and December 2011, to establish haematology and blood chemistry reference intervals. Differences in haematology and blood chemistry between sites (Rottnest Island v mainland) were significant for haematology (HMT, p = 0.003), blood chemistry (BLC, p = 0.001) and peripheral blood cell morphology (PBCM, p = 0.001). Except for alkaline phosphatase, all blood chemistry analytes were higher in mainland animals. There were also differences with time of year in HMT (p = 0.001), BLC (p = 0.001) and PBCM (p = 0.001) for Rottnest Island quokkas. A small sample of captive animals (n = 8) were opportunistically sampled for plasma concentrations of vitamin E and were found to be deficient compared with wild-caught animals. Fifty-eight of the 150 quokkas were also tested for the presence of Salmonella, microfilariae, Macropodid herpesvirus-6, Theileria spp., Babesia spp., trypanosomes, Cryptococcus spp. and other saprophytic fungi. All eight infectious agents were detected in this study. Infectious agents were detected in 24 of these 58 quokkas (41%), with more than one infectious agent detected for all 24 individuals. Salmonella were detected concurrently with microfilariae in 8 of these 24 quokkas, and this mixed infection was associated with lower values across all haematological analytes, with Salmonella having the greater involvement in the decreased haematological values (p < 0.05). There was no evidence for an effect of sex on HMT, BLC and PBCM. Our data provide important haematological and blood chemistry reference intervals for free-ranging quokkas. We applied novel methods of analyses to HMT and BLC that can be used more broadly, aiding identification of potential disease in wildlife.

Desordens hematológicas em onças-pardas (Puma concolor; Linnaeus, 1771) infectadas por Citauxzoon felis / [Hematological disorders in Brown jaguars (Puma concolor; Linnaeus 1771) infected by Citauxzoon felis]

Este estudo objetivou descrever o aspecto hematológico de seis onças-pardas (Puma concolor) infectadas pelo Cytauxzoon felis. Os seis casos de infecção foram identificados durante o manejo sanitário de 11 animais de um centro de reabilitação de animais silvestres. Estruturas compatíveis com piroplasmídeos foram observadas durante a avaliação do esfregaço sanguíneo e confirmadas como Cytauxzoon felis pela técnica de PCR. A análise estatística demonstrou diferença significativa (P<0,05) no número absoluto dos linfócitos entre os grupos dos animais infectados e não infectados. Assim, expressivas alterações hematológicas e bioquímicas entre os grupos investigados alertam para a dificuldade de identificação de onças-pardas infectadas por C. felis, apoiada apenas em exames de rotina, bem como para o risco, sobretudo, da reintrodução desses animais na natureza.(AU)

This Cytauxzoon felis by the PCR technique. Statistical analysis showed a significant difference is study aimed to describe the hematological appearance of six puma (puma concolor) infected with cytauxzoon felis. The six cases of infection were identified during the sanitary management of 11 animals from a wild animal rehabilitation center. Piroplasmid compatible structures were observed during the blood smear evaluation and confirmed as (P<0.05) in the absolute number of lymphocytes between the groups of infected and uninfected animals. Thus expressive hematological and biochemical alterations between the groups investigated alert to the difficulty of identifying infected brown jaguars by C. felis, supported only by routine examinations, and the risk especially when aiming at the reintroduction of these animals in the wild.(AU)

ESTABLISHMENT OF ACUTE-PHASE PROTEIN AND SERUM PROTEIN ELECTROPHORESIS PRELIMINARY REFERENCE VALUES FOR PRONGHORN (ANTILOCAPRA AMERICANA).

Pronghorn (Antilocapra americana) are native to western North America and are found in 24 Association of Zoos and Aquariums (AZA)-accredited institutions. Acute-phase proteins (APP) are a broad class of proteins that are stimulated in response to inflammation and have been shown to be a sensitive measure of inflammation in equids and ruminants. In this study, blood samples from clinically normal free-ranging and captive populations of pronghorn were analyzed using assays for protein electrophoresis (EPH) and APP, including serum amyloid A (SAA) and haptoglobin (HP), to develop preliminary ranges to gauge potential differences between these populations. Additional samples were taken from clinically abnormal captive pronghorn with facial abscesses. By EPH measurements, albumin: globulin ratio mean and SE were significantly different (P <0.05) with 1.02 (0.08) for captive populations and 1.91 (0.05) for free-ranging populations. Total protein mean and SE were significantly different (P <0.05) for captive and free-ranging populations, respectively 5.6 (0.3) g/dl and 6.9 (0.1) g/dl. Mean and SD of SAA for captive pronghorn were 1.4 (3.2) mg/L, and were significantly different from the free-ranging population, which was below the limits of detection for (P <0.05). There was no difference in HP levels between these groups. In a case study of a pronghorn with facial abscesses, elevated levels of HP, but not SAA, suggested that HP maybe useful in certain disease states. Future studies should explore the use of these biomarkers as tools to monitor general health, prognosis, and subclinical disease.

SERUM BIOCHEMICAL AND HEMATOLOGIC REFERENCE INTERVALS FOR WEANLING NORTHWEST ATLANTIC GRAY SEALS (HALICHOERUS GRYPUS).

Baseline health parameters are limited in the primary literature for gray seals (Halichoerus grypus) in the northwest Atlantic. Accurate normal physiologic reference ranges for both species and specific geographic populations are vital tools for assessing the health of individuals and understanding the health of the entire population. This study developed comprehensive reference intervals for biochemical and hematologic parameters of recently weaned gray seal pups on Cape Cod, Massachusetts from samples collected in 2013, 2016, and 2017. Reference ranges were developed using methodology outlined by the American Society of Clinical Veterinary Pathology. By establishing more comprehensive biochemical and hematologic reference ranges for this population based on a robust sample size, this study provides a new tool for clinicians, researchers, and rehabilitation organizations to improve individual patient care and population research.

Blood meal sources and bacterial microbiome diversity in wild-caught tsetse flies.

Tsetse flies are the vectors of African trypanosomiasis affecting 36 sub-Saharan countries. Both wild and domestic animals play a crucial role in maintaining the disease-causing parasites (trypanosomes). Thus, the identification of animal reservoirs of trypanosomes is vital for the effective control of African trypanosomiasis. Additionally, the biotic and abiotic factors that drive gut microbiome diversity in tsetse flies are primarily unresolved, especially under natural, field conditions. In this study, we present a comprehensive DNA metabarcoding approach for individual tsetse fly analysis in the identification of mammalian blood meal sources and fly bacterial microbiome composition. We analyzed samples from two endemic foci, Kafue, Zambia collected in June 2017, and Hurungwe, Zimbabwe sampled in April 2014 (pilot study) and detected DNA of various mammals including humans, wild animals, domestic animals and small mammals (rat and bat). The bacterial diversity was relatively similar in flies with different mammalian species DNA, trypanosome infected and uninfected flies, and female and male flies. This study is the first report on bat DNA detection in wild tsetse flies. This study reveals that small mammals such as bats and rats are among the opportunistic blood meal sources for tsetse flies in the wild, and the implication on tsetse biology and ecology needs to be studied.

Wild Bird Surveillance for Avian Influenza Virus.

Avian influenza (AI) viruses have been routinely isolated from a wide diversity of free-living avian species, representing numerous taxonomic orders. Birds in orders Anseriformes and Charadriiformes are considered the natural reservoirs for all AI viruses; it is from these orders that AI viruses have been most frequently isolated. Since first recognized in the late 1800s, AI viruses have been an important cause of disease in poultry and, occasionally, in non-gallinaceous birds and mammals. While AI viruses tend to be of low pathogenicity (LP) in wild birds, the 2014-2015 incursion of highly pathogenic avian influenza (HPAI) clade 2.3.4.4 H5Nx viruses into North America and the recent circulation of HPAI H5 viruses in European wild birds highlight the need for targeted, thorough, and continuous surveillance programs in the wild bird reservoir. Such programs are crucial to understanding the potential risk for the incursion of AI into human and domestic animal populations. The aim of this chapter is to provide general concepts and guidelines for the planning and implementation of surveillance plans for AI viruses in wild birds.

Haematological and biochemical parameters of wild capuchin monkeys in Brasília, Federal District-Brazil.

BACKGROUND: Wild capuchin monkeys (Sapajus libidinosus) usually are found in conserved forests near the zoo and the urban areas of Brasília city, Brazil. In this study, some capuchin monkeys were captured using traps, followed by safe biological procedures for their overall health analysis, based on specific haematological and biochemical tests of blood samples. METHODS: Blood was collected from a total of 17 monkeys for the determination of parameters, namely packed cell volume (PCV), leucocytes, erythrocytes, platelets and triglycerides. Statistical analyses for average values, median, standard deviation and range were performed. RESULTS: These parameters were set based on the minimum and maximum values obtained from the blood tests. Data are presented in tabulated form. CONCLUSIONS: Capture procedures were based on animal safety analysis for free-living animals and would help future studies on wild animals. The collected samples used in this study suggested the animals to be apparently healthy in their habitat.

Arboviruses and Blood Meal Sources in Zoophilic Mosquitoes at Human-Wildlife Interfaces in Kenya.

Background: Zoophilic mosquitoes play an important role in the transmission of arboviruses of medical importance at human-wildlife interfaces, yet arbovirus surveillance efforts have been focused mostly on anthropophilic mosquitoes. Understanding the diversity of zoophilic mosquitoes and their associated feeding patterns and arboviruses can inform better vector control strategies. Materials and Methods: We morphologically identified mosquitoes collected from two game reserves in Kenya, the Maasai Mara National Reserve (MMNR) and locations near the Shimba Hills National Reserve (SHNR). Representative mosquitoes were also identified by cytochrome c oxidase subunit 1 (COI) barcode sequencing. In addition, we identified the vertebrate hosts of mosquito blood meals from the contents of each mosquito's abdomen by high-resolution melting (HRM) analysis and sequencing of COI, 16S ribosomal RNA, and cytochrome b gene PCR products. Similarly, mosquito arbovirus infections were identified by HRM analysis and sequencing of Alphavirus- and Flavivirus-specific RT-PCR products. Results: Of 2858 mosquitoes collected, 51 were engorged with blood meals from seven different vertebrate hosts, including humans, birds, domestic, and peridomestic animals and wildlife. Culex was the most abundant mosquito genus, with Culex pipiens being the most abundant species in both study regions. Among MMNR samples, we detected dengue serotype-2 virus (DENV-2) for the first time in Aedes tarsalis and Aedes tricholabis, as well as Sindbis virus in male Cx. pipiens. We also detected DENV-2 in Aedes aegypti sampled from locations near the SHNR. Human and diverse wildlife blood meals were identified, including bushbuck blood in the dengue-infected Ae. tarsalis and both human and hippopotamus blood in a single Eretmapodites chrysogaster mosquito. Conclusions: Our findings highlight the potential risk of sylvatic dengue and Sindbis transmission to humans by zoophilic mosquitoes at human-wildlife interfaces in Africa. Of specific importance, we provide evidence of sylvatic DENV-2 in Ae. tarsalis and Ae. tricholabis, representing potential new dengue vectors.

The buffy coat method: a tool for detection of blood parasites without staining procedures.

BACKGROUND: Blood parasites belonging to the Apicomplexa, Trypanosomatidae and Filarioidea are widespread in birds and have been studied extensively. Microscopical examination (ME) of stained blood films remains the gold standard method for the detection of these infections in birds, particularly because co-infections predominate in wildlife. None of the available molecular tools can detect all co-infections at the same time, but ME provides opportunities for this to be achieved. However, fixation, drying and staining of blood films as well as their ME are relatively time-consuming. This limits the detection of infected hosts during fieldwork when captured animals should be released soon after sampling. It is an obstacle for quick selection of donor hosts for parasite experimental, histological and other investigations in the field. This study modified, tested and described the buffy coat method (BCM) for quick diagnostics (~ 20 min/sample) of avian blood parasites. METHODS: Blood of 345 birds belonging to 42 species was collected, and each sample was examined using ME of stained blood films and the buffy coat, which was examined after centrifugation in capillary tubes and after being transferred to objective glass slides. Parasite detection using these methods was compared using sensitivity, specificity, positive and negative predictive values and Cohen's kappa index. RESULTS: Haemoproteus, Leucocytozoon, Plasmodium, microfilariae, Trypanosoma and Lankesterella parasites were detected. BCM had a high sensitivity (> 90%) and specificity (> 90%) for detection of Haemoproteus and microfilariae infections. It was of moderate sensitivity (57%) and high specificity (> 90%) for Lankesterella infections, but of low sensitivity (20%) and high specificity (> 90%) for Leucocytozoon infections. Trypanosoma and Plasmodium parasites were detected only by BCM and ME, respectively. According to Cohen's kappa index, the agreement between two diagnostic tools was substantial for Haemoproteus (0.80), moderate for Lankesterella (0.46) and fair for microfilariae and Leucocytozoon (0.28) infections. CONCLUSIONS: BCM is sensitive and recommended as a quick and reliable tool to detect Haemoproteus, Trypanosoma and microfilariae parasites during fieldwork. However, it is not suitable for detection of species of Leucocytozoon and Plasmodium. BCM is a useful tool for diagnostics of blood parasite co-infections. Its application might be extended to studies of blood parasites in other vertebrates during field studies.

Venous hematology, biochemistry, and blood gas analysis of free-ranging Eastern Copperheads (Agkistrodon contortrix) and Eastern Ratsnakes (Pantherophis alleghaniensis).

Hematology, plasma biochemistry, and blood gas analysis were performed on venous samples obtained from free-ranging Eastern Copperheads (Agkistrodon contortrix) and Eastern Ratsnakes (Pantherophis alleghaniensis) in central North Carolina during a mark-recapture study conducted from April to October 2015 at the North Carolina Zoo. Blood samples were collected from 31 (15 male and 16 female) free-ranging copperheads and 34 (20 male and 14 female) free-ranging ratsnakes at the beginning and end of restraint. Restraint was performed for morphometric measurements, sex determination, and identification via placement of intracelomic passive integrated transponder (PIT) tags and marking of ventral scutes with a handheld electrocautery unit. Blood gas analytes were measured at the beginning of restraint and compared to analytes measured at the end to evaluate for changes secondary to handling. Total restraint time prior to the first blood sampling was 1.4 ± 0.4 mins (mean ± SD) and 1.0 ± 0.2 mins (mean ± SD) and restraint time prior to second blood sampling was 12.5 ± 2.4 mins (mean ± SD) and 13.5 ± 3.4 mins (mean ± SD) for copperheads and ratsnakes, respectively. Blood lactate concentrations at the beginning of restraint were similar for both species. Lactate concentrations increased significantly and pH decreased significantly for both species at the end of restraint when compared to the beginning of restraint. Furthermore, lactate concentrations at the end of restraint were significantly elevated in ratsnakes compared to copperheads. This study provides guidelines for interpretation of venous hematology, plasma biochemistry, and blood gas values for free-ranging copperheads and ratsnakes in central North Carolina and demonstrates the physiological response to venous blood gas analytes secondary to capture and restraint.

Species-specific differences in Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti seroprevalence in Namibian wildlife.

BACKGROUND: Knowledge about parasitic infections is crucial information for animal health, particularly of free-ranging species that might come into contact with livestock and humans. METHODS: We investigated the seroprevalence of three tissue-cyst-forming apicomplexan parasites (Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti) in 506 individuals of 12 wildlife species in Namibia using in-house enzyme linked immunosorbent assays (indirect ELISAs applying purified antigens) for screening and immunoblots as confirmatory tests. We included six species of the suborder Feliformia, four species of the suborder Caniformia and two species of the suborder Ruminantia. For the two species for which we had most samples and life-history information, i.e. cheetahs (Acinonyx jubatus, n = 250) and leopards (Panthera pardus, n = 58), we investigated T. gondii seroprevalence in relation to age class, sex, sociality (solitary, mother-offspring group, independent sibling group, coalition group) and site (natural habitat vs farmland). RESULTS: All but one carnivore species (bat-eared fox Otocyon megalotis, n = 4) were seropositive to T. gondii, with a seroprevalence ranging from 52.4% (131/250) in cheetahs to 93.2% (55/59) in African lions (Panthera leo). We also detected antibodies to T. gondii in 10.0% (2/20) of blue wildebeest (Connochaetes taurinus). Adult cheetahs and leopards were more likely to be seropositive to T. gondii than subadult conspecifics, whereas seroprevalence did not vary with sex, sociality and site. Furthermore, we measured antibodies to N. caninum in 15.4% (2/13) of brown hyenas (Hyaena brunnea) and 2.6% (1/39) of black-backed jackals (Canis mesomelas). Antibodies to B. besnoiti were detected in 3.4% (2/59) of African lions and 20.0% (4/20) of blue wildebeest. CONCLUSIONS: Our results demonstrate that Namibian wildlife species were exposed to apicomplexan parasites at different prevalences, depending on parasite and host species. In addition to serological work, molecular work is also needed to better understand the sylvatic cycle and the clear role of wildlife in the epidemiology of these parasites in southern Africa.

Under the weather: Corticosterone levels in wild nestlings are associated with ambient temperature and wind.

Animals time reproductive events to overlap with periods of favorable environmental conditions. However, weather conditions can be unpredictable. Young animals may be particularly susceptible to extreme weather during sensitive developmental periods. Here, we investigated the effects of adverse weather conditions on corticosterone levels (a hormone linked to the avian stress response) and body condition of wild nestling zebra finches (Taeniopygia guttata). We sought to tease apart the direct versus indirect (i.e. parental) effects of weather on nestling physiology and condition by increasing parental work load with a clutch manipulation experiment. We found that high temperatures were associated with lower levels of restraint-induced corticosterone and high wind speeds were associated with higher levels of baseline corticosterone. We found no associations between weather and nestling body condition. However, clutch manipulation did affect body condition, with nestlings from experimentally enlarged clutches in worse condition compared to nestlings from experimentally reduced clutches. Our findings suggest that weather can directly affect wild nestlings via changes in corticosterone levels. Further research is needed to understand how changes in corticosterone levels affect phenotype and survival in wild nestlings. Understanding how developing animals respond to changes in environmental predictability and extreme weather is vital for understanding the potential for rapid adaptation in the face of changing climatic conditions.

Values of plasma total proteins of free-living marmosets (Callithrix jacchus) in an urban forest.

BACKGROUND: Callithrix jacchus is a small primate widely used in experimentation, but data on plasma total protein (PTP) values of free-living animals inhabiting its own endemism region are non-existent. METHODS: Marmosets belonging to two free-living groups were captured for marking and obtaining biological material. The collected blood was centrifuged to obtain the plasma and thus to determine the concentration of PTP. RESULTS AND CONCLUSIONS: Females and adults had the highest mean PTP. Mean found were higher than other values described for other neotropical primates, and the high PTP values presented by the animals indicate dehydration. Due to the importance of total plasma proteins for health and management, obtaining these values as a reference is relevant for the free-living Callithrix genus and other neotropical primates.