Comparative analysis of the blood transcriptomes between wolves and dogs.

Dogs were domesticated by human and originated from wolves. Their evolutionary relationships have attracted much scientific interest due to their genetic affinity but different habitats. To identify the differences between dogs and wolves associated with domestication, we analysed the blood transcriptomes of wolves and dogs by RNA-Seq. We obtained a total of 30.87 Gb of raw reads from two dogs and three wolves using RNA-Seq technology. Comparisons of the wolf and dog transcriptomes revealed 524 genes differentially expressed genes between them. We found that some genes related to immune function (DCK, ICAM4, GAPDH and BSG) and aerobic capacity (HBA1, HBA2 and HBB) were more highly expressed in the wolf. Six differentially expressed genes related to the innate immune response (CCL23, TRIM10, DUSP10, RAB27A, CLEC5A and GCH1) were found in the wolf by a Gene Ontology enrichment analysis. Immune system development was also enriched only in the wolf group. The ALAS2, HMBS and FECH genes, shown to be enriched by the Kyoto Encyclopedia of Genes and Genomes analysis, were associated with the higher aerobic capacity and hypoxia endurance of the wolf. The results suggest that the wolf might have greater resistance to pathogens, hypoxia endurance and aerobic capacity than dogs do.

Characterization of the peripheral blood transcriptome and adaptive evolution of the MHC I and TLR gene families in the wolf (Canis lupus).

BACKGROUND: The wolf (Canis lupus) is one of the most widely distributed terrestrial mammals, because it is well adapted to various ecological niches and their corresponding pathogen environments. Immunological competence is a crucial factor involved in adapting to a changing environment and fighting pathogen infection in animals. In this study, the peripheral blood transcriptome of wolves was generated via RNA-seq to advance understanding of the wolf immunome, with a special focus on the major histocompatibility complex class I (MHC I) and toll-like receptor (TLR) gene families, which are involved in pathogen recognition and defense. RESULTS: The blood transcriptomic libraries of eight wolves originating from Tibet and Inner Mongolia were sequenced, and approximately 383 million reads were generated. Using a genome-guided assembly strategy, we obtained 123,851 unigenes, with a mean length of 845 bp and an N50 length of 1121 bp. On the basis of BLAST searches against the NCBI non-redundant protein database (Nr), a total of 36,192 (29.22%) unigenes were annotated. For functional classification, 24,663 unigenes were assigned to 13,016 Gene Ontology (GO) terms belonging to 51 sub-categories of the three main GO categories. Additionally, 7682 unigenes were classified into 6 Kyoto Encyclopedia of Genes and Genomes (KEGG) categories, in which the most represented functional sub-categories were signal transduction and the immune system, and 16,238 unigenes were functionally classified into 25 Eukaryotic Orthologous Groups (KOG) categories. We observed an overall higher ω (d N/d S) value at antigen-binding sites (ABSs) than at non-ABS regions as well as clear evidence of intergenic/intragenic recombination events at wolf MHC I loci. Additionally, our analysis revealed that carnivorous TLRs were dominated by purifying selection, with mean ω values at each TLR locus ranging from 0.173 to 0.527. However, we also found significant instances of positive selection that acted on several codons in pathogen recognition domains and were linked to species-specific differences in pathogen recognition. CONCLUSIONS: This study represents the first attempt to characterize the blood transcriptome of the wolf and to highlight the value of investigating the immune system. Balancing selection and recombination have contributed to the historical evolution of wolf MHC I genes. Moreover, TLRs in carnivores have undergone adaptive evolution against the background of purifying selection, and a high level of adaptive evolution was detected in the wolf TLR system.

Seasonal Cycles in Hematology and Body Mass in Free-Ranging Gray Wolves (Canis lupus) from Northeastern Minnesota, USA.

Studies of captive gray wolves (Canis lupus) showed seasonal cycles in hematologic values and female body mass. We used a remotely controlled recapture collar to determine whether nine female and five male free-ranging wolves handled four to 17 times in NE Minnesota, US showed similar cycles. Hematocrit, hemoglobin, red blood cell count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and body mass increased from summer toward a winter peak and then decreased again toward summer. Several hematologic values differed considerably from those of captive wolves, and the ranges in free-ranging wolves were much greater than those of captives.

Hematology and blood chemistry parameters differ in free-ranging maned wolves (Chrysocyon brachyurus) living in the Serra da Canastra National Park versus adjacent farmlands, Brazil.

There has been growing interest in the specific impacts of anthropogenic factors on the health of wildlife. This study examined hematology and serum chemistry status of a prominent carnivore, the maned wolf (Chrysocyon brachyurus), living in, on the boundaries to, or on adjacent farmlands to the Serra da Canastra National Park, Brazil. Twenty-eighty wolves were captured, and values were compared 1) between subadults (n=8 animals) and adults (n=20 animals), 2) males (n=12 animals) and females (n=16 animals), and 3) among wolves living inside the park (n=11), near the park border (n=11 animals), and in neighboring farming areas (n=6 animals). Age, gender, and wolf locations influenced (P<0.05) hematology and serum biochemistry values. Specifically, adults had lower (P<0.05) circulating phosphorus than subadults. Males had lower (P<0.05) serum glucose, creatinine phosphokinase, and cholesterol and higher (P<0.05) potassium than females. Erythrocyte count and serum cholinesterase were lower (P<0.05) in wolves living within the park compared with near the park border or on farmlands. Mean corpuscular volume was lower (P<0.05) in wolves living near the park border than those ranging within the park and on farmlands. Aspartate transaminase and chloride were higher (P<0.05) in wolves living inside the park compared with those ranging near the park border. Creatinine phosphokinase was lower (P<0.05) in wolves living on farmland compared with the other two locations. These results clearly reveal a relationship between age and gender on hematology and serum biochemistry values in free-living maned wolves. More importantly, certain traits indicative of health are potentially compromised in wolves living in areas under anthropogenic pressure. These data lay a foundation for examining the influence of farming and local domestic species on disease susceptibility and fitness in the maned wolf.

Serologic survey for canine distemper virus and canine parvovirus in free-ranging wild carnivores from Portugal.

A serologic survey for Canine distemper virus (CDV) and canine parvovirus (CPV) was performed on serum and lung extract from an opportunistic sample of 120 free-ranging wild carnivores (13 species) from Portugal, collected from 1995 to 2006. Antibodies to CDV were detected in wolf (Canis lupus; 3/27) and red fox (Vulpes vulpes; 2/22). Antibodies to CPV were detected in wolf (9/28), red fox (2/14), wildcat (Felis silvestris;1/8), genet (Genetta genetta; 17/18), and stone marten (Martes foina; 3/17). Antibodies to CPV were detected throughout the study, whereas for CDV antibodies were detected in 3 of 10 yr and only during winter. The extremely high CPV antibody prevalence in genets is unprecedented. Although based on a limited sample, these data suggest widespread exposure of free-ranging Iberian carnivores to CDV and CPV.

Hematology and serum clinical chemistry reference intervals for free-ranging Scandinavian gray wolves (Canis lupus).

BACKGROUND: Scandinavian free-ranging wolves (Canis lupus) are endangered, such that laboratory data to assess their health status is increasingly important. Although wolves have been studied for decades, most biological information comes from captive animals. OBJECTIVES: The objective of the present study was to establish reference intervals for 30 clinical chemical and 8 hematologic analytes in Scandinavian free-ranging wolves. METHODS: All wolves were tracked and chemically immobilized from a helicopter before examination and blood sampling in the winter of 7 consecutive years (1998-2004). Seventy-nine blood samples were collected from 57 gray wolves, including 24 juveniles (24 samples), 17 adult females (25 samples), and 16 adult males (30 samples). Whole blood and serum samples were stored at refrigeration temperature for 1-3 days before hematologic analyses and for 1-5 days before serum biochemical analyses. Reference intervals were calculated as 95% confidence intervals except for juveniles where the minimum and maximum values were used. RESULTS: Significant differences were observed between adult and juvenile wolves for RBC parameters, alkaline phosphatase and amylase activities, and total protein, albumin, gamma-globulins, cholesterol, creatinine, calcium, chloride, magnesium, phosphate, and sodium concentrations. CONCLUSION: Compared with published reference values for captive wolves, reference intervals for free-ranging wolves reflected exercise activity associated with capture (higher creatine kinase activity, higher glucose concentration), and differences in nutritional status (higher urea concentration).

High plasma IgE levels within the Scandinavian wolf population, and its implications for mammalian IgE homeostasis.

Immunoglobulin E (IgE) serves as an important link between innate and adaptive immunity through its ability to bind high affinity receptors on mast cells and basophils. Large differences in IgE levels may here affect this important link, and IgE levels in natural non-domestic animal populations may therefore be very informative concerning the levels of IgE that this system have been balanced against during recent mammalian evolution. However, very few such studies have been performed. Here, we present an analysis of total IgE levels in 65 Scandinavian wolves: 57 free living (wild), and 8 wolves in captivity (Zoo). The 57 wild wolves correspond to approximately 30% of the entire wolf population in Sweden and Norway and thus represent a large fraction of the entire population, making this a unique sample from a wild canine population. The median IgE level in these wolves was 67 microg/ml, which is approximately twice the level seen in domestic dogs and more than 100 times the levels in non-atopic humans. The collected information from domestic and wild populations now indicate that the very low IgE levels observed in man and laboratory rodents are most likely an effect of a life in a relatively parasite free environment, and that total IgE levels under maximally stimulatory (normal) conditions may reach 100-200 microg/ml.

Prevalence of antibodies against canine distemper virus and canine parvovirus among foxes and wolves from Spain.

Viral diseases can influence the population dynamics of wild carnivores and can have effects on carnivore conservation. Hence, a serologic survey was conducted in an opportunistic sample of 137 foxes (Vulpes vulpes) and 37 wolves (Canis lupus) in Spain for 1997-2007 to detect antibodies against canine distemper virus (CDV) and against canine parvovirus (CPV) by indirect ELISA. Antibodies against CDV were detected in 18.7% of the analyzed animals and antibodies against CPV in 17.2%. There was no difference in antibody prevalence to CDV between both species, even in the same region (P>0.05), but there was a significant difference in antibody prevalence to CPV between foxes (5.1%) and wolves (62.2%) (P<0.05). In fox populations there was a significant difference in antibody prevalence to CDV between geographic areas (Aragón 26.4%, La Mancha 7.8%, P<0.05). In wolf populations there was significantly higher antibody prevalence against CPV (P<0.05) in Castilla y León (100%) than in the Cantabric region (53.3%). There was no significant sex or age-related difference in the antibody prevalence against CDV or CPV in foxes. These results indicate that contact with CDV is widespread among wild canid populations in Spain and that CPV is endemic in the Iberian wolf population. The implications of these results are briefly discussed.

Detection of Leishmania infantum in captive wolves from Southwestern Europe.

The aim of the present study was to determine the prevalence of Leishmania infantum infection in a wild reservoir host (Canis lupus) throughout an endemic area for the disease (Southern Europe). For that reason, the serum and peripheral blood samples of 33 captive wolves from the European Breeding of Endangered Species Programme (EEP) were analyzed using the enzyme-linked immunosorbent assay (ELISA) and real-time quantitative PCR (qPCR). L. infantum was detected in three samples from Central Portugal and Central and Northern Spain. Even though L. infantum infection in positive samples was low, surveillance of zoonotic leishmaniosis in this population is recommended as the parasite load could be higher in other tissues due to parasite tropism and most of the EEP institutions studied are located in endemic areas for canine leishmaniosis in Europe.

Quantification of sexual steroid hormones in faeces of Iberian wolf (Canis lupus signatus): a non-invasive sex typing method.

The determination of gender in wild animals is essential for behavioural and ecological studies, and also for conservation. The objectives of this study were (i) the determination of gender in faecal samples of Iberian wolf based on the differential concentrations of sexual steroid hormones (SSH) and (ii) to analyse the profiles of SSH in males and females (considering the gender determination carried out previously) during the non-reproductive and reproductive periods. The quantification of androgens (testosterone, T), progestin (progesterone, P) and oestrogen (oestradiol, E) was conducted by means of enzyme immunoassay. The k-means conglomerate analysis showed that the 59 faecal samples grouped into three different conglomerates, considering SSH levels. Groups 1 and 2 showed higher levels of T than group 3. Therefore, the faecal samples included in groups 1 and 2 (17 samples) corresponded to males and those of group 3 (42 samples) to females. The levels of T + P + E and T/P were higher in the group of males than in the group of females. The results of this study also showed that levels of T in males were higher during the reproductive period than in the non-reproductive period. However, the concentrations of P and E turned out to be higher during the non-reproductive season. In females, the levels of the three hormones (T, P and E) were higher during the reproductive period.

Ultrasonographic characteristics of the reproductive tract and serum progesterone and estradiol concentrations in captive female red wolves (Canis rufus) with and without reproductive tract disease.

OBJECTIVE To describe ultrasonographic characteristics of the reproductive tract and serum progesterone and estradiol concentrations in captive female red wolves (Canis rufus) with and without reproductive tract disease. DESIGN Prospective study. ANIMALS 13 adult female red wolves. PROCEDURES Wolves with varying parity and history of contraceptive treatment were anesthetized to facilitate ultrasonographic examination and measurement of the reproductive tract and blood collection for determination of serum progesterone and estradiol concentrations in December 2011 and June 2012. Additionally, during the December evaluation, fine-needle aspirate samples of the uterus were obtained for cytologic evaluation. Measurements were compared between wolves with and without reproductive tract disease and between wolves that had and had not received a contraceptive. RESULTS 7 of 13 wolves had or developed reproductive tract disease during the study. Ranges for measurements of reproductive tract structures overlapped between ultrasonographically normal and abnormal tracts, but measurements for abnormal tracts were generally greater than those for normal tracts. The ultrasonographic diagnosis was consistent with the histologic diagnosis for reproductive tracts obtained from wolves that were sterilized, were euthanized, or died during the study. Cytologic results for fine-needle aspirate samples of the uterus and serum progesterone and estradiol concentrations were unable to distinguish wolves with and without reproductive tract disease. Reproductive tract disease was not associated with parity or contraceptive administration. CONCLUSIONS AND CLINICAL RELEVANCE The ultrasonographic images, reproductive tract measurements, and descriptions of reproductive tract lesions provided in this study can be used as diagnostic guidelines for the treatment and management of red wolves with reproductive tract disease.

A SEROSURVEY OF DISEASES OF FREE-RANGING GRAY WOLVES (CANIS LUPUS) IN MINNESOTA, USA.

We tested serum samples from 387 free-ranging wolves ( Canis lupus ) from 2007 to 2013 for exposure to eight canid pathogens to establish baseline data on disease prevalence and spatial distribution in Minnesota's wolf population. We found high exposure to canine adenoviruses 1 and 2 (88% adults, 45% pups), canine parvovirus (82% adults, 24% pups), and Lyme disease (76% adults, 39% pups). Sixty-six percent of adults and 36% of pups exhibited exposure to the protozoan parasite Neospora caninum . Exposure to arboviruses was confirmed, including West Nile virus (37% adults, 18% pups) and eastern equine encephalitis (3% adults). Exposure rates were lower for canine distemper (19% adults, 5% pups) and heartworm (7% adults, 3% pups). Significant spatial trends were observed in wolves exposed to canine parvovirus and Lyme disease. Serologic data do not confirm clinical disease, but better understanding of disease ecology of wolves can provide valuable insight into wildlife population dynamics and improve management of these species.

IgA deficiency in wolves from Canada and Scandinavia.

Immunoglobulin A deficiency (IgAD) is the most common primary immunodeficiency in both humans and selected breeds of domestic dogs. In both species, IgAD is associated with recurrent infections and immune mediated diseases. Previous results imply that IgAD is also common in the wild ancestor of domestic dogs, the gray wolf. Here, we report that serum IgA concentrations are significantly different in Scandinavian and Canadian wolves (p = 3.252e-15) with an increased prevalence for IgAD in Scandinavian wolves (60%), which is as high as those found in high-risk dog breeds.

Serologic survey for selected disease agents in wolves (Canis lupus) from Alaska and the Yukon Territory, 1984-2000.

Wolves (Canis lupus) were captured in several geographic areas of Alaska (USA) and the Yukon Territory (Canada) during 1984-2000. Blood was collected from 1,122 animals. Sera were tested for antibodies against infectious canine hepatitis virus (ICH), canine distemper virus (CDV), canine parvovirus (CPV), Francisella tularensis, and serovars of Leptospira interrogans. Antibody prevalence for ICH was >84% for all areas. Area-specific prevalences of antibodies ranged from 12% to 70% for CPV, from 0% to 41% for CDV, and from 4% to 21% for F. tularensis. There was no evidence of CDV exposure at the two southernmost locations in Alaska. Prevalence of antibodies for ICH increased slightly during the 16-yr course of the survey. There was essentially no evidence of exposure to L. interrogans. Prevalences of antibodies for both CPV and CDV were age-specific, with higher values in the adult cohort compared with the pup cohort. There were no sex-specific differences in prevalence of antibodies for any of the five disease agents.

Serum biochemistry of captive and free-ranging gray wolves (Canis lupus).

Normal serum biochemistry values are frequently obtained from studies of captive sedentary (zoo) or free-ranging (wild) animals. It is frequently assumed that values obtained from these two populations are directly referable to each other. We tested this assumption using 20 captive gray wolves (Canis lupus) in Minnesota, USA, and 11 free-ranging gray wolves in Alaska, USA. Free-ranging wolves had significantly (P < 0.05) lower sodium, chloride, and creatinine concentrations and significantly higher potassium and blood urea nitrogen (BUN) concentrations; BUN to creatinine ratios; and alanine aminotransferase, aspartate aminotransferase, and creatine kinase activities relative to captive wolves. Corticosteroid-induced alkaline phosphatase activity (a marker of stress in domestic dogs) was detected in 3 of 11 free-ranging wolves and in 0 of 20 captive wolves (P = 0.037). This study provides clear evidence that serum biochemical differences can exist between captive and free-ranging populations of one species. Accordingly, evaluation of the health status of an animal should incorporate an understanding of the potential confounding effect that nutrition, activity level, and environmental stress could have on the factor(s) being measured.

Serum lipid concentrations in six canid and four ursid species in four zoos.

Serum lipid levels were measured in healthy captive wild canids and ursids, and the values were compared with previously published data. Serum lipid levels were evaluated in blood samples collected from eight African wild dogs (Lycaon pictus), three arctic foxes (Alopex lagopus), nine gray wolves (Canis lupus), four maned wolves (Chrysocyon brachyurus), two Mexican wolves (Canis lupus baleiyi), nine red wolves (Canis rufus), two brown bears (Ursus arctos), six polar bears (Ursus maritimus), six spectacled bears (Tremarctos ornatus), and five sun bears (Ursus malayanus). Samples were analyzed for total cholesterol, triacylglycerides, high-density lipoprotein-cholesterol, and low-density lipoprotein-cholesterol. Although the results showed a great variation among species, circulating lipids appeared especially high, sometimes extremely so, in the spectacled bears, polar bears, sun bears, and maned wolves compared with all other species sampled. The study provides a substantial basis for comparing lipid levels in presumed healthy animals and indicates a need for controlled study of the effects of diet on circulating lipid levels.

Diseases and parasites in wolves of the Riding Mountain National Park region, Manitoba, Canada.

We examined wolf (Canis lupus) blood and fecal samples from the Riding Mountain National Park (RMNP) region of Manitoba, Canada. In 601 fecal samples collected during two study periods in RMNP and the Duck Mountain Provincial Park and Forest (DMPPF) we found gastrointestinal helminth eggs from Alaria sp. (15.5%), Capillaria sp. (1.0%), taeniid tapeworms (30.8%), Toxascaris sp. (1.7%), Toxocara sp. (0.2%), Trichuris sp. (2.2%), and Moniezia sp. (0.5%). In addition, we found Demodex sp. (0.2%) and the protozoal cysts/oocysts of Sarcocystis sp. (37.3%), Cryptosporidium sp. (1.2%), coccidia (Isospora sp. or Eimeria sp.) (1.7%), and Giardia sp. (29.5%). No fecal shedding of canine parvovirus (CPV, n=387) was detected. All 18 blood samples collected in RMNP showed CPV exposure and eight of 18 blood samples indicated canine distemper virus (CDV) exposure. One wolf died from CDV. Our results are consistent with previous findings on pathogens affecting wolves and with high Giardia sp. prevalence in wolves inhabiting agricultural regions.

Comparison of serum hormone levels of captive and free-living maned wolves Chrysocyon brachyurus.

Serum hormone levels were compared between captive and free-living maned wolves and seasonal variations of sex hormones were studied. Blood samples were collected from 16 male and 26 female adult animals from Brazilian zoos, and from 30 male and 24 female free-living adults to determine serum progesterone and testosterone by radioimmunoassay. Serum testosterone concentrations varied (P < 0.05) across seasons for 16 captive males, being higher in autumn (2184.7 +/- 355.1 pg/mL) than in summer (1080.7 +/- 205.4 pg/mL), winter (1270.1 +/- 276.6 pg/mL) and spring (963.9 +/- 248.1 pg/mL), although they did not differ between summer, winter and spring. Testosterone concentration of 30 free-living males differed (P < 0.05) between autumn (824.1 +/- 512.2 pg/mL), winter (14.4 +/- 8.0 pg/mL) and spring (151.9 +/- 90.5 pg/mL). Comparison between captive and free-living animals showed no difference in autumn (P > 0.05). Sixteen captive males showed higher testosterone concentration during winter and spring compared with 30 free-living animals (P < 0.05). Progesterone concentration varied among seasons in 26 captive females (P < 0.05), being higher in autumn (15.3 +/- 3.1 ng/mL) than in summer (6.6 +/- 1.5 ng/mL), winter (5.3 +/- 3.1 ng/mL) and spring (4.3 +/- 0.7 ng/mL). Progesterone concentration of 24 free-living females varied between autumn (17.1 +/- 6.0 ng/mL) and winter (1.7 +/- 0.3 ng/mL) (P < 0.05), but we could not obtain data for spring or summer. No difference in progesterone levels was observed between captive and free-living females in autumn and winter.

Crystallization, preliminary X-ray diffraction analysis and Patterson search of oxyhaemoglobin I from the wolf (Chrysocyon brachiurus).

Oxyhaemoglobin I isolated from the Brazilian wolf Chrysocyon brachiurus has been crystallized and X-ray diffraction data has been collected to 2.06 A resolution using a synchrotron-radiation source. Crystals were determined to belong to the space group P2(1)2(1)2(1) and preliminary structural analysis revealed the presence of one tetramer in the asymmetric unit. The structure was determined using standard molecular-replacement techniques and is currently being refined using maximum-likelihood protocols. This is the first haemoglobin isolated from a member of the Canidae family to be crystallized and it will provide further insights in the comparative biochemistry of vertebrate haemoglobins.

Comparison of serum hormone levels of captive and free-living maned wolves Chrysocyon brachyurus

Serum hormone levels were compared between captive and free-living maned wolves and seasonal variations of sex hormones were studied. Blood samples were collected from 16 male and 26 female adult animals from Brazilian zoos, and from 30 male and 24 female free-living adults to determine serum progesterone and testosterone by radioimmunoassay. Serum testosterone concentrations varied (P < 0.05) across seasons for 16 captive males, being higher in autumn (2184.7 ± 355.1 pg/mL) than in summer (1080.7 ± 205.4 pg/mL), winter (1270.1 ± 276.6 pg/mL) and spring (963.9 ± 248.1 pg/mL), although they did not differ between summer, winter and spring. Testosterone concentration of 30 free-living males differed (P < 0.05) between autumn (824.1 ± 512.2 pg/mL), winter (14.4 ± 8.0 pg/mL) and spring (151.9 ± 90.5 pg/mL). Comparison between captive and free-living animals showed no difference in autumn (P > 0.05). Sixteen captive males showed higher testosterone concentration during winter and spring compared with 30 free-living animals (P < 0.05). Progesterone concentration varied among seasons in 26 captive females (P < 0.05), being higher in autumn (15.3 ± 3.1 ng/mL) than in summer (6.6 ± 1.5 ng/mL), winter (5.3 ± 3.1 ng/mL) and spring (4.3 ± 0.7 ng/mL). Progesterone concentration of 24 free-living females varied between autumn (17.1 ± 6.0 ng/mL) and winter (1.7 ± 0.3 ng/mL) (P < 0.05), but we could not obtain data for spring or summer. No difference in progesterone levels was observed between captive and free-living females in autumn and winter.