Chytridiomycosis is an emerging infectious disease of amphibians caused by a chytrid fungus, Batrachochytrium dendrobatidis. This panzootic does not equally affect all amphibian species within an assemblage; some populations decline, others persist. Little is known about the factors that affect disease resistance. Differences in behavior, life history, biogeography, or immune function may impact survival. We found that an innate immune defense, antimicrobial skin peptides, varied significantly among species within a rainforest stream amphibian assemblage that has not been exposed to B. dendrobatidis. If exposed, all amphibian species at this central Panamanian site are at risk of population declines. In vitro pathogen growth inhibition by peptides from Panamanian species compared with species with known resistance (Rana pipiens and Xenopus laevis) or susceptibility (Bufo boreas) suggests that of the nine species examined, two species (Centrolene prosoblepon and Phyllomedusa lemur) may demonstrate strong resistance, and the other species will have a higher risk of disease-associated population declines. We found little variation among geographically distinct B. dendrobatidis isolates in sensitivity to an amphibian skin peptide mixture. This supports the hypothesis that B. dendrobatidis is a generalist pathogen and that species possessing an innate immunologic defense at the time of disease emergence are more likely to survive.

It has been difficult to perform cytokine studies for many wildlife and nontraditional species because of a lack of immunologic reagents at the protein level. Recently, simple and rapid assays for quantifying mRNA expression by real-time reverse transcription–polymerase chain reaction (RT-PCR) have been used for analysis of cytokine profiles in humans and other mammalian species. This report describes the development and application of real time RT-PCR to measure the expression of several important elk (Cervus elaphus) cytokine mRNAs, including interleukin (IL)-2, IL-4, IL-10, IL-12p40, interferon-gamma, tumor necrosis factor (TNF)- α, and the enzyme-inducible nitric oxide synthase, all of which are involved in immune responses and regulation. For the broadest potential application of the assay, primers and probes were designed using consensus sequences from several species of interest. To obtain standardized quantitative results, external controls consisting of a DNA template for each target gene were used to generate linear standard curves over a 6 to 8 log range with detection of as few as 10 copies of amplicon per reaction. Sample-to-sample variation in the efficiency of the RT, as well as in the quantity and quality of the starting RNA, was compensated for by normalizing the results to the endogenous housekeeping gene β₂ microglobulin. The assay was evaluated by monitoring the kinetics of cytokine mRNA synthesis induced by mitogenic and antigenic stimulation of peripheral blood mononuclear cells (PBMCs) from Mycobacterium bovis–infected elk. Concanavalin A–stimulated PBMCs demonstrated a rapid but transient increase in cytokine mRNA expression following in vitro mitogenic activation with optimal mRNA induction observed after 4 to 16 hr. The PBMCs stimulated with the mycobacterial recall antigen, bovine-purified protein derivative (PPD-bovis), demonstrated variable mRNA induction kinetics for each cytokine. Whereas PPD-bovis optimally induced IL-2 mRNA after 8 hr of in vitro stimulation, longer in vitro stimulation times were necessary for the optimal induction of IL-4 and TNF- α mRNA (up to 48 hr). We demonstrate real-time RT-PCR to be a rapid, sensitive, and reproducible technique, which will make it a valuable tool in the study of immunologic responses and cytokine profiles of cervids and other nontraditional livestock and wildlife species.

Feline immunodeficiency virus (FIV) is a lentivirus related to human immunodeficiency virus (HIV) that causes feline AIDS in the domestic cat (Felis catus). Serological surveys indicate that at least 25 other species of cat possess antibodies that cross-react with domestic cat FIV. Most infected nondomestic cat species are without major symptoms of disease. Long-term studies of FIV genome variation and pathogenesis reveal patterns consistent with coadaptation of virus and host in free-ranging FIV-Ple–infected African lions (Panthera leo) and FIV-Pco–infected pumas (Puma concolor) populations. This report examined correlates of immunodeficiency in wild and captive lions and pumas by quantifying CD5 , CD4 , and CD8 T-cell subsets. Free-ranging FIV-Ple–infected lions had immunofluorescence flow cytometry (IFC) profiles marked by a dramatic decline in CD4 subsets, a reduction of the CD4 /CD8 ratio, reduction of CD8 β ^high cells, and expansion of the CD8 β ^low subset relative to uninfected lions. An overall significant depletion in CD5 T-cells in seropositive lions was linked with a compensatory increase in total CD5− lymphocytes. The IFC profiles were altered significantly in 50% of the seropositive individuals examined. The FIV-Pco–infected pumas had a more generalized response of lymphopenia expressed as a significant decline in total lymphocytes, CD5 T-cells, and CD5− lymphocytes as well as a significant reduction in CD4 T-cells. Like lions, seropositive pumas had a significant decline in CD8 β ^high cells but differed by not having compensatory expansion of CD8 β ^low cells relative to controls. Results from FIV-infected lions and pumas parallel human and Asian monkey CD4 diminution in HIV and SIV infection, respectively, and suggest there may be unrecognized immunological consequences of FIV infection in these two species of large cats.

During an 18-mo period (May 2002–November 2003), 10 animals in a herd of 19 reindeer (Rangifer tarandus) at the National Animal Disease Center (NADC) experienced episodes of anemia. Affected animals had histories of weight loss, unthriftiness, occasionally edema of dependent parts and moderate anemia characterized by microcytosis or macrocytosis, hypochromasia, schistocytosis, keratocytosis, acanthocytosis, and dacryocytosis. Numerous basophilic punctate to ring-shaped bodies, measuring less than 1.0 μm, were found on the surface of red blood cells and were often observed encircling the outer margins of the cells. Based on cytologic findings, DNA preparations from selected affected animals in the NADC herd and one animal from a private herd experiencing similar episodes of anemia were assayed by polymerase chain reaction (PCR) for the presence of hemotropic bacteria using primers targeting the 16S rRNA genes of Mycoplasma (Eperythrozoon) suis, Mycoplasma (Haemobartonella) haemofelis, Anaplasma marginale, Anaplasma spp., and Ehrlichia spp. Amplification products were detected from four of the affected animals using primers specific for the 16S rRNA gene of M. haemofelis and Mycoplasma haemocanis. Product from one of the animals was sequenced and internal primers were designed from the resulting sequence to perform a nested PCR assay. Samples from 10 reindeer were positive using the nested PCR reaction and products from seven animals were sequenced; BLAST searches and phylogenetic analysis were performed on the resulting sequences. Sequence data from six animals revealed homology to an organism most closely related to Mycoplasma ovis, Mycoplasma wenyonii, and Mycoplasma haemolamae; sequence from a single animal was most closely related to M. haemofelis and M. haemocanis. This represents the first identification of a haemomycoplasma species in reindeer. Although several animals were also infected with abomasal nematodes, the presence of this newly described haemomycoplasma may have contributed to the anemic syndrome.

During austral summers 1999–2000 and 2000–01, two outbreaks of avian cholera occurred in the Hope Bay area (63°24′S, 56°59′W), located on the tip of the Antarctic Peninsula. Eighty-six dead birds were found: five kelp gulls (Larus dominicanus), 36 skuas (Stercorarius sp.), and 45 Adelie penguins (Pygoscelis adeliae). The carcasses were studied using clinical, pathological, and microbiological criteria. Water samples from ponds where birds were settled and samples from 90 healthy birds also were analyzed during the second outbreak. Pasteurella multocida isolates were identified by biochemical tests, capsular type, somatic serotype, and susceptibility to nine antibiotics. Molecular subtyping was performed by ApaI and SmaI pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC-PCR). In February 2000, mortality in skuas was 16% and 2% in kelp gulls. In the 2000–01 breeding season, mortality in south polar skuas was 47%, 24% in brown skuas, 1.4% in kelp gulls, and 0.01% in Adelie penguins. All birds had lesions of avian cholera. In kelp gulls the presentation was chronic, whereas skuas and penguins suffered subacute and acute disease, respectively. Fifty-five isolates recovered from dead birds and one from water were identified as P. multocida gallicida, type A:1. The strains presented a unique molecular pattern by PFGE and ERIC-PCR. A possible hypothesis to explain the origin of the outbreaks was that nonbreeder kelp gulls carried P. multocida gallicida to Hope Bay, and avian cholera was transmitted through water to skuas and penguins. This study reports avian cholera in new bird species, their potential role in the transmission of the disease, and the different responses of these species to the disease.

Brucellosis occurs in free-ranging elk (Cervus elaphus) and bison (Bison bison) in the Greater Yellowstone Area, which includes portions of Idaho, Wyoming, and Montana. Brucella abortus was first detected in elk in Idaho in 1998, and from 1998 to 2002, serologic surveillance of hunter-killed elk was conducted in northeastern and southeastern Idaho. Prevalence of antibodies in these elk varied annually, but averaged between 2% and 3%. Elk were also trapped in northeastern Idaho from 1998–2002 and tested for brucellosis using serology and tissue culture. In areas where artificial feeding of elk was done, antibody prevalence ranged from 12% to 80% depending on site, age, and sex. At one feeding site (Rainey Creek), a decline in the prevalence of antibodies (from 56.8% in 1999 to 13.5% in 2002) was detected after the removal of seropositive elk over 4 yr. Seropositive elk removed from two artificial winter feeding sites (Rainey Creek and Conant Creek) were euthanized and sampled or held in captivity and allowed to calve prior to euthanasia and necropsy. At necropsy, B. abortus biovar 1 and B. abortus biovar 4 were isolated from both cows and calves; however, biovar 4 was predominant. A dual infection with both biovars was found in one calf born to a seropositive cow from which biovar 4 was isolated. Abortions (16%), stillbirths (8%), and weak calves (4%) were observed in these elk. These findings confirm the presence of brucellosis in elk in eastern Idaho and provide information on disease management options.

Clinical signs of upper respiratory tract disease-like syndrome (URTD-LS) were observed in free-ranging eastern box turtles (Terrapene carolina carolina) from Virginia, USA (May 2001–August 2003), some of which also had aural abscesses. After a Mycoplasma sp. was detected by polymerase chain reaction (PCR), a study was undertaken to better define the range of clinical signs of disease and to distinguish mycoplasma-associated URTD-LS from other suspected causes of URTD-LS and aural abscessation in box turtles. Nasal and/or ocular swabs (from turtles possessing URTD-LS) or nasal washes (from asymptomatic turtles) were collected from turtles May 2001–August 2003; samples were assayed for Mycoplasma spp., chelonian herpesvirus, and iridoviruses by PCR testing. A partial DNA sequence (933 bases) of the small ribosomal subunit (16S rRNA) of the box turtle Mycoplasma sp. was analyzed to determine its phylogenetic relatedness to other Mycoplasma spp. of veterinary interest. Mycoplasma sp. was detected in seven (six with clinical signs of URTD-LS; one asymptomatic) of 23 fortuitously collected animals from six of 11 Virginia counties. Clinical signs in Mycoplasma sp.–infected animals included unilateral to bilateral serous to mucopurulent nasal discharge, epiphora, ocular edema, and conjunctival injection. Five Mycoplasma sp.–positive animals possessed aural abscesses; two did not. Analysis of the mycoplasma 16S rRNA gene sequence from one asymptomatic and three symptomatic animals representing four counties revealed a consensus Mycoplasma sp. sequence closely related to, but distinct from, M. agassizii. None of the samples collected contained viral DNA of chelonian herpesviruses or invertebrate and vertebrate (including FV3) iridoviruses. In conclusion, a new Mycoplasma sp. was associated with URTD-LS in native box turtles from Virginia that was not codetected with other suspected causes of chelonian upper respiratory disease; there was no proof of a direct relationship between aural abscessation and the Mycoplasma sp.

Harbor seals (Phoca vitulina richardsi) were captured in the coastal regions of Southeast Alaska, Gulf of Alaska, Prince William Sound (PWS), and Kodiak Island during 1976–1999. Blood was collected from 286 seals. Sera were tested for evidence of exposure to Brucella spp., phocid herpesvirus-1 (PhoHV-1), phocid herpesvirus-2 (PhHV-2), and phocine distemper virus (PDV). Antibody prevalence rates were 46% (46/100) for Brucella spp., 93% (225/243) for PhoHV-1, 0% (0/286) for PhHV-2, and 1% (2/160) for PDV. Antibody prevalence for Brucella spp. was directly related to host age. Antibody prevalence for PhoHV-1 was higher in PWS as compared to the other three regions. No evidence of mortality attributable to these four agents was observed during the course of this study. Based on the results of this survey, none of these agents is considered a significant mortality factor in harbor seals from the four regions of coastal Alaska included in the study.

Batrachochytrium dendrobatidis is a fungal pathogen that has been implicated in amphibian declines worldwide. Histopathologic techniques have been used to diagnose the disease, but their sensitivity has not been determined. It is also unclear whether the probability of detection varies between skin samples derived from different body parts. We examined 24 Fitzinger's rainfrogs (Eleutherodactylus fitzingeri) with chytridiomycosis. This is a common frog species with a broad range and high abundance throughout most of Costa Rica. We sampled 12 different body parts from each animal, and alternated the staining between a routinely used stain (hematoxylin and eosin [H&E]), and a more fungus-specific stain (periodic acid–Schiff [PAS]). The pelvic patch and the innermost finger of the hand were consistently the best places to detect the disease, although significant differences were found only with the gular area, the abdomen, and toes four and five. We found more positive samples using PAS than using H&E in all body parts, although significant differences were detected only in samples derived from the pelvic patch. Using the best combination of factors (stain and body part) and animals with the lightest infections (to test the sensitivity of the technique), we calculated that at least 17 sections are needed in order to reach 95% confidence that a frog is or is not infected. We conclude that the choice of stain and body part can significantly alter estimates of prevalence of B. dendrobatidis.

Members of the genus Ranavirus (family Iridoviridae) can cause catastrophic mortality of pond-breeding amphibians and are associated with an emerging infectious disease that may be contributing to amphibian declines. We conducted three experiments to examine factors that may affect transmission both within and between local breeding populations of the wood frog (Rana sylvatica). In a laboratory study, when exposed to moribund tadpoles collected during a local ranaviral die-off, uninfected tadpoles died as soon as 4 days after exposure. The onset of death was accelerated when tadpoles were allowed to scavenge on carcasses of infected tadpoles. In a mesocosm experiment that was conducted in outdoor wading pools, die-offs of tadpoles began approximately 19 days after infected tadpoles were added to pools containing uninfected tadpoles. Mass die-offs with greater than 98% mortality occurred in all pools, regardless of the initial tadpole density. In a second mesocosm experiment, the addition of water and bottom sediments that were collected from a pond during a ranaviral die-off did not result in lower tadpole survival or growth relative to controls. Only a small percentage of tadpoles appeared to be sick, and most tadpoles survived until the first individuals began metamorphosing within a pool. However, tests for ranavirus using polymerase chain reaction were positive for most pools that received contaminated sediment, suggesting that some infections were sublethal. Our results indicate that transmission within ponds is enhanced by scavenging and that spread between local ponds could occur via the transport of contaminated sediment by animals or humans.

Tissue and blood samples were collected from 152 wild boars (Sus scrofa) from the Maremma area (Grosseto district, Central Italy) between November 2002 and January 2003. The presence of pseudorabies virus (PRV) antibodies, antigen, and DNA were confirmed by an enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and polymerase chain reaction (PCR), respectively. Of 152 animals, 62 (41%) were positive for viral antigen in tonsillar tissue. Of the 80 serum samples that were suitable for testing, 41 (51%) were positive for PRV antibodies. Positive immunohistochemistry results were confirmed by PCR. A significantly higher prevalence of PRV antigen and seroprevalence was detected in older animals. No differences were detected between males and females or for animals coming from different areas sampled. Results confirm that PRV is endemic in this wild boar population with a high prevalence of infection. The results of immunohistochemistry investigations demonstrated that a large number of wild boars harbor PRV in tonsillar tissues and should be considered as an important reservoir of PRV.

The Spanish ibex (Capra pyrenaica hispanica) population of southern Spain was surveyed for potential pathogens associated with the conjunctiva, external ear canal, as well as reproductive and upper respiratory tracts. We sampled 321 ibex (131 adult males, 100 adult females, and 90 yearlings); these included 271 apparently healthy animals and 50 that were naturally infected with Sarcoptes scabiei. A total of 688 bacterial isolates were identified (377 gram-negatives, 225 gram-positives, and 86 Mycoplasma spp.); sex, age, location, infection with S. scabiei, and disposition of the animal (free-ranging versus captive) were evaluated as risk factors for infection. Infections with Mycoplasma agalactiae and Mycoplasma arginini were associated with age, having a higher frequency of isolation in young animals. With Escherichia coli, Mannheimia haemolytica, Pasteurella multocida biotype A, and Staphylococcus aureus, significantly higher isolation rates were associated with adults. The isolation frequency for E. coli was higher in females, whereas Moraxella bovis isolations were mostly associated with males. The presence of mange increased the risk of infection with both Streptococcus equi subsp. zooepidemicus and M. haemolytica. The geographic origin of sampled animals was related to the isolation of Branhamella ovis, M. agalactiae, and all Pasteurella sp. Isolations of M. haemolytica, P. multocida biotype A, E. coli, and B. ovis were more prevalent in samples from free-ranging rather than captive animals. Of the gram-positive bacteria, S. aureus represented the predominant species isolated from nasal, vaginal, and ocular samples. Mycoplasma agalactiae and M. arginini were the predominant Mycoplasma spp., and both were associated most often with the external ear canal. The most frequently isolated gram-negative bacteria included E. coli, M. haemolytica, P. multocida biotype A, and B. ovis. Isolation rates of gram-negative species varied by source. In nasal samples, M. haemolytica and P. multocida biotype A were isolated most frequently, whereas in ocular and vaginal samples, B. ovis and E. coli, respectively, were most frequently isolated.

Sixty-one birds of prey admitted to The Wildlife Center of Virginia (WCV; Waynesboro, Virginia, USA) from June to November 2003 were tested for West Nile virus (WNV) infection. Choanal and/or cloacal swabs were obtained and submitted to Virginia's Division of Consolidated Laboratory Services (Richmond, Virginia, USA) for analysis with real-time reverse transcriptase polymerase chain reaction (RT-PCR). Forty birds of prey were positive for WNV by RT-PCR. Five avian families and nine species of raptors were represented, with great horned owls (Bubo virginianus) and red-tailed hawks (Buteo jamaicensis) most frequently affected. Presenting clinical signs were consistent with previous reports of WNV infection in raptors; however, these differed between species. Of WNV positive birds, nonspecific signs of illness were the most common clinical findings, particularly in red-tailed hawks; signs included dehydration (n=20), emaciation (n=18), and depression (n=15). Neurologic abnormalities were frequently identified, especially in great horned owls, and included head tremors (n=17), ataxia (n=13), head incoordination (n=7), torticollis (n=3), nystagmus (n=3), and head tilt (n=3). Great horned owls exhibited anemia and leukocytosis with heterophilia, eosinophilia, and monocytosis consistent with chronic inflammation. Red-tailed hawks were anemic with a heterophilic leukocytosis and regenerative left shift. The majority of WNV cases occurred during August and September; there was a marked increase in the number of raptors admittedto WCV during these months followed by a marked decrease during October, November, and December. This pattern differed from mean monthly admissions during the previous 10 years and suggests a negative impact on local raptor populations. The effects of WNV on avian populations are largely unknown; however, because of their ecological importance, further investigation of the effects of WNV on raptor populations is warranted.

An observational study was conducted to determine the proportionate mortality of wild trumpeter (Cygnus buccinator) and tundra (Cygnus columbianus columbianus) swans that died during the winters of 2000–02 in northwestern Washington State, USA. Among 400 swans necropsied, 81% were lead poisoned (302/365 trumpeter swans; 20/35 tundra swans). Mortality started in mid-November and peaked from late December through mid-February; swan mortality that was not associated with lead poisoning was uniformly lower throughout the winter months. Lead poisoning was 24 times more likely to be the cause of death in swans found in Whatcom County compared to swans found in other locations in northwestern Washington State (95% CI: 12.7, 47.0). Mortality attributable to lead poisoning was twice as likely in adults as in juveniles (95% CI: 1.0, 4.2). Aspergillosis was documented in 62 trumpeter and two tundra swans, including 37 swans in which mortality was caused by lead poisoning. Males were twice as likely as females to have aspergillosis (95% CI: 1.1, 3.8). Traumatic injuries were documented in 37 trumpeter and seven tundra swans, including seven trumpeter swans with concurrent lead poisoning. Dead swans found outside Whatcom County were four times more likely to have traumatic injuries compared to those found in Whatcom County (95% CI: 1.6, 10.0). Overall, lead-poisoned swans were significantly less likely to have concurrent aspergillosis or traumatic injuries. There was no apparent association between grit ingestion (total mass or mass categorized by size) and lead poisoning or number of lead shot. Not surprisingly, lead-poisoned swans were more likely to have one or more lead shot compared to swans that died from other causes (OR 294; 95% CI: 92, 1,005); lead-poisoned swans were also more likely to have one or more nontoxic shot compared to swans that were not poisoned (OR 63; 95% CI: 19, 318). The source(s) of shot are unknown but likely are in or near Whatcom County, Washington.

Carcasses of 26 wolves were collected during the 2000/2001 and 2003/2004 hunting seasons and examined for helminths. Thirteen helminth species were recorded: one trematode (Alaria alata), seven cestodes (Diphyllobothrium latum, Mesocestoides lineatus, Taenia hydatigena, Taenia multiceps, Taenia ovis, Taenia pisiformis, and Echinococcus granulosus), and five nematode species (Uncinaria stenocephala, Toxascaris leonina, Toxocara canis, Trichinella nativa, and Trichinella britovi). The most common species were A. alata and U. stenocephala. Mature Echinococcus granulosus was found and described for the first time in Estonia, and its identity verified using PCR-RFLP analysis. Sequencing a fragment of the mitochondrial DNA NADH dehydrogenase 1 (mtND1) gene showed that the E. granulosus strain from Estonia was identical to strain G10, recently characterized in reindeer and moose in Finland.

Piroplasms, morphologically indistinguishable from Cytauxzoon felis, previously were detected in 36% of cougars in Florida. We utilized a nested 18S rRNA assay, which amplifies DNA from all piroplasms, to screen blood samples collected from 41 cougars from Florida (39 native Florida panthers [Puma concolor coryi] and two translocated Texas cougars [P. c. stanleyana]) from 1989–2005. Thirty-nine of the 41 cougars (95%) were positive for piroplasms; however, sequence analysis and restriction enzyme digestion revealed that only five were positive for C. felis. Samples from 32 cougars were positive for a Babesia sp. Two cougars were co-infected with both C. felis and the Babesia sp. Phylogenetic analysis of 18S rRNA gene sequence indicated that the Florida panther Babesia sp. was most closely related to a Babesia sp. reported from Ixodes ovatus from Japan, Babesia divergens, and Babesia odocoilei. This study indicates that Florida panthers harbor two distinct piroplasms, C. felis and a Babesia sp., and that some individuals are infected with both organisms. The infectivity and pathogenicity of this Babesia sp. for domestic cats is unknown. This represents the first report of a feline Babesia sp. in North America.

Piroplasmosis was first described in raccoons (Procyon lotor) in 1926, and the official description of a small piroplasm as Babesia lotori was done in 1981. Babesia microti-like gene sequences have been characterized in raccoons in both North American and Japan. It is well documented that the microscopic appearance of piroplasms does not always accurately predict the genotype and phylogenetic classification. Discrepancies using phenotype to predict genotype have been reported most frequently when evaluating small piroplasms. We amplified and sequenced the full-length 18S rRNA gene from a small piroplasm identified in a raccoon and used this sequence for phylogenetic analyses. Based on these analyses, the organism was placed in the Babesia sensu stricto clade, confirming that it is a true Babesia sp. This documents that at least two Babesia spp. can infect raccoons. The data generated in this study can be used to design molecular diagnostic tests for detection of this Babesia sp., which will be useful for epidemiologic and comparative phylogenetic studies. As piroplasmosis has been documented with increased frequency in humans in recent years, the results of this study will aid in the recognition of zoonotic babesiosis.

Wildlife reservoir species and genetic diversity of Anaplasma ovis (Rickettsiales: Anaplasmataceae) have been poorly characterized. Bighorn sheep (Ovis canadensis), captured in Montana from December 2004 to January 2005, were tested for antibodies to Anaplasma spp.; the presence of A. ovis was determined by the characterization of major surface protein msp4 sequences. Anaplasma antibodies were detected in 25/180 (14%) sampled bighorn sheep and A. ovis msp4 sequences were amplified by polymerase chain reaction (PCR) and sequenced from 9/23 (39%) of seropositive animals. All animals were negative by PCR for the related pathogens, Anaplasma phagocytophilum and Anaplasma marginale. All msp4 sequences identified in the bighorn sheep were identical and corresponded to a single A. ovis genotype that was identical to a sheep isolate reported previously from Idaho. The finding of a single genotype of A. ovis in this wild herd of bighorn sheep was in contrast to the genetic diversity reported for A. marginale in cattle herds in the western United States and worldwide. These results demonstrated that bighorn sheep may be a wildlife reservoir of A. ovis in Montana.

In searching for an invertebrate host for Hepatozoon sp. infecting the montane vole (Microtus montanus), we collected fleas, ticks, and mites from live-trapped voles and searched squash preparations for Hepatozoon oocysts. From 1989 through 1996, we identified six species of fleas in Grand Teton National Park: Megabothris abantis, Megabothris asio megacolpus, Aetheca wagneri, Peromyscopsylla selenis, Peromyscopsylla. hesperomys, and Hystrichopsylla dippiei dippiei. We found Hepatozoon oocysts only in M. abantis; we found no oocysts in mites or ticks. We conclude that M. abantis is an invertebrate host of Hepatozoon sp. and is likely to be the definitive host for the Hepatozoon spp. of M. montanus.

Deer keds, Lipoptena mazamae (Diptera: Hippoboscidae), were collected from white-tailed deer (Odocoileus virginianus) and humans in Georgia and South Carolina, USA (1 October 2001–6 January 2005) and screened for the presence of DNA from Bartonella spp. Forty deer keds were screened for Bartonella spp. by polymerase chain reaction using primers specific to the riboflavin synthase gene (ribC) of Bartonella. Bartonella species closely related to Bartonella schoenbuchensis and to the etiologic agent of cat-scratch disease (Bartonella henselae) were detected in 10 keds and one ked, respectively.

The proventriculi of 11 Eurasian cranes (Grus grus) from central Iran were examined for the existence of parasitic helminths. Preliminary reports suggested that the death of these birds was related to untimely cold weather. Nine proventriculi (82%) were heavily infected by the nematode Tetrameres grusi. Glandular structure of the infected proventriculi was replaced by epithelial atrophy but significant inflammatory reactions were not observed in any of the infected organs. In serious infections, the nematode produced vast structural and functional changes, causing organ dysfunction and glandular necrosis. The coincidence of heavy helminth infection at times of environmental stress may lead to debilitation, wasting, and perhaps mortality in migratory cranes.

Thirty-five Florida panthers (Puma concolor coryi [Bangs, 1899]) collected from six counties in southern Florida between 1978 and 2003 were examined at necropsy for gastrointestinal helminths. The panthers were placed into two groups: 1) treated with anthelmintics (n=17), and 2) untreated (n=18). Nine species of helminths (one trematode, six nematodes, and two cestodes) were identified in the untreated panthers. The most prevalent helminths were Alaria marcianae (LaRue, 1917) (100%), Spirometra mansonoides (Mueller, 1935) (91%), and Ancylostoma pluridentatum (Alessandrini, 1905) (89%). Ancylostoma caninum (Ercolani, 1859) is reported from the Florida panther for the first time. The intensities of helminths with prevalences >10% did not differ between untreated panthers collected in 1978–1983 and 1996–2003. Treated panthers had helminth faunas similar to those of untreated panthers. The current anthelmintic treatment being used reduced the intensity of both A. marcianae and A. pluridentatum in panthers ≤6 mo posttreatment (PT); however, treated panthers between 6 and 9 mo PT, and >9 mo PT were similar to untreated panthers. Treatment was less effective on S. mansonoides and Taenia omissa Lühe, 1910. Treated panthers had slightly lower intensities of S. mansonoides at ≤6 mo PT; however, between 6 and 9 mo PT and >9 mo PT they had significantly higher intensities than untreated panthers. At all periods PT, the intensity of T. omissa for the treated panthers was similar to that of untreated panthers. We suggest that Mesocestoides sp. may not be present in the Florida panther population as reported earlier by Forrester et al. (1985), due to parasite misidentification by those authors.

An infectious keratoconjunctivitis (IKC) epizootic in bighorn sheep (Ovis canadensis) occurred in the Silver Bell Mountains, Arizona, USA, from 1 December 2003 to 31 March 2004. We used standard culture methods and polymerase chain reaction (PCR) amplification of the 16S rRNA gene to test for the causative agents of IKC and other diseases reported to be associated with bighorn sheep populations. All bighorn sheep and domestic goat test results were negative except for Mycoplasma spp. and Branhamella spp. The culture and PCR results differed. Conjunctival swabs from four of 19 IKC-affected bighorn sheep tested by culture were positive for Mycoplasma spp., whereas 22 of 22 bighorn sheep samples tested by PCR were positive for Mycoplasma spp. None of 13 domestic goats tested positive by culture for Mycoplasma spp., whereas five of 16 tested positive by PCR. Three of 16 domestic goats and seven of 24 IKC-affected bighorn sheep tested positive for Branhamella spp. by culture. Bighorn sheep began showing clinical signs of IKC between 21 and 28 days following initial detection of domestic goats in bighorn sheep habitat. The IKC epizootic lasted 122 days, and individual bighorn sheep were blind for an average of 38.4 days. Given the clear potential for disease transmission to bighorn sheep, we recommend that land managers not allow the pasturing of domestic goats near occupied bighorn sheep habitat.

Brucella spp. have been recently isolated from a variety of marine mammals. Serum samples from 58 Pacific bottlenose dolphins (Tursipa aduncus) from the Solomon Islands were tested for antibodies to Brucella spp. by the tube agglutination test (TAT), enzymed-linked immunosorbent assay (ELISA), and immunoblotting. Anti-Brucella spp. antibodies were detected by TAT and ELISA in 31 and 40 of 58 samples, respectively. These results suggest that Pacific bottlenose dolphins from the Solomon Islands are infected with Brucella spp. or a Brucella-like organism.

To determine whether swift foxes (Vulpes velox) could facilitate transmission of Yersinia pestis to uninfected black-tailed prairie dog (Cynomys ludovicianus) colonies by acquiring infected fleas, ectoparasite and sero-logic samples were collected from swift foxes living adjacent to prairie dog towns during a 2004 plague epizootic in northwestern Texas, USA. A previous study (1999–2001) indicated that these swift foxes were infested almost exclusively with the flea Pulex irritans. Black-tailed prairie dogs examined from the study area harbored only Pulex simulans and Oropsylla hirsuta. Although P. irritans was most common, P. simulans and O. hirsuta were collected from six swift foxes and a single coyote (Canis latrans) following the plague epizootic. Thus, both of these canids could act as transport hosts (at least temporarily) of prairie dog fleas following the loss of their normal hosts during a plague die-off. All six adult swift foxes tested positive for antibodies to Y. pestis. All 107 fleas from swift foxes tested negative for Y. pestis by mouse inoculation. Although swift foxes could potentially carry Y. pestis to un-infected prairie dog colonies, we believe they play only a minor role in plague epidemiology, considering that they harbored just a few uninfected prairie dog fleas (P. simulans and O. hirsuta).

Mycoplasma gallisepticum (MG) conjunctivitis emerged in 1994 as a disease of free-ranging house finches (Carpodacus mexicanus) in North America and has also been isolated from other songbirds with conjunctivitis. Random amplification of polymorphic DNA (RAPD) of house finch and other songbird isolates has suggested that a single ‘strain’ initiated this outbreak. To explore the possibility of genomic variability among house finch isolates of MG and to evaluate the utility of a second technique for MG genotyping, we selected samples from our archive of reference strains and wild songbird isolates to analyze using both RAPD and amplified-fragment length polymorphism (AFLP); this is a newer technique that has been successfully used to explore the genomic variability of several Mycoplasma species. Both RAPD and AFLP results confirmed previous observations that during the initial stages of the MG epidemic in songbirds, isolates from different geographic locations and songbird species had genotypes that appeared to be highly similar, further supporting a single point source of origin. One 2001 isolate from New York was clearly different from the other songbird samples and clustered together with the vaccine and reference strains, indicating that substantial molecular evolution or a separate introduction has occurred.

Mycoplasma gallisepticum, an important pathogen of poultry, especially chickens and turkeys, emerged in 1994 as the cause of conjunctivitis in house finches (Carpodacus mexicanus) in their eastern range of North America. The resulting epidemic of M. gallisepticum conjunctivitis severely decreased house finch abundance and the continuing endemic disease in the eastern range has been associated with repeating seasonal peaks of conjunctivitis and limitation of host populations. Mycoplasma gallisepticum conjunctivitis was first confirmed in the western native range of house finches in 2002 in a Missoula, Montana, population. Herein, we report further western expansion of M. gallisepticum conjunctivitis in the native range of house finches based on positive polymerase chain reaction results with samples from birds captured in 2004 and 2005 near Portland, Oregon.

A combination of medetomidinetiletamine-zolazepam was used to conduct six immobilizations of free-ranging lions (Panthera leo) in Waza National Park, Cameroon, during 1999 and 2000. Drugs were administered by dart injection at 0.07±0.01 (mean±SD) mg/kg of medetomidine and 1.8±0.5 mg/kg of tiletamine-zolazepam. Chemical immobilization was characterized by smooth inductions (14.1±6 min), satisfactory analgesia, and muscle relaxation. One animal was treated for bradypnea. No major alterations of physiologic parameters (heart and respiratory rates, rectal temperature) were seen during immobilization in the other lions. Relative arterial oxygen saturation was measured in two animals and revealed mild hypoxemia. The animals received atipamezole at 0.3±0.1 mg/kg intramuscularly for reversal of anesthesia. Recoveries were uneventful. All animals were radiocollared, and no mortalities occurred during an 18-mo follow-up period. Use of medetomidine-tileta-mine-zolazepam for anesthesia and reversal of anesthesia with atipamezole appear to be useful for reversible immobilization of free-ranging lions.

Four medetomidine/ketamine (M/K) doses (30 μg/kg/3 mg/kg; 40/4; 50/5; 60/6), administered by intramuscular injection, were evaluated for short-term immobilization of adult male variable flying foxes (Pteropus hypomelanus). The highest dose (60 μg/kg/6 mg/kg) produced a significantly faster induction (31±46 sec) than the lowest dose (30/3) (125±62 sec). The highest dose levels (50/5, 60/6) produced significantly longer immobilization times (52.5±25.7 min and 60.6± 20.8 min, respectively) than did the lower doses (30/3, 40/4) (18.8±8.7 min and 31.0±14.3 min, respectively). The dose at which 50% of the bats were immobilized for ≥30 min (ED₅₀) was approximately 40 μg/kg/4 mg/kg. This dose produced a mean immobilization time of 31±14 min, bradypnea and bradycardia. In conclusion, a M/K dose of 50 ug/kg/5 mg/kg is recommended for greater than 30 min of relaxed immobilization in free-living variable flying foxes and is sufficient for safe collection of samples.

Haptoglobin (Hp) levels were measured in blood serum from 185 apparently healthy ringed seals (Pusa hispida) from Svalbard (age range, 1–30 yr) collected during the spring seasons of 2002 through 2004. The Hp concentration was 0.26±0.02 g/l (mean±SE; range, 0.0–1.5 g/l). Maturity status, body condition index (CI), interactions between CI and maturity status, and sex and maturity status all had significant influences on Hp levels. Mature males had significantly higher Hp concentrations compared with mature females (0.30±0.03 vs. 0.17±0.01 g/l, F_1,16=14.9, P<0.01). No differences were found between Hp levels in immature male and female seals (0.33±0.06 g/l). The Hp levels increased significantly in immature seals when the CI decreased. Despite the fact that mature males had a significantly lower CI than mature females, no direct correlation was found between Hp concentration and CI among adults. The elevated Hp values found in mature males might be related to injuries inflicted among males while fighting during the mating season or to high stress levels related to mating competition. The Hp concentrations of ringed seals measured in this study are useful baseline data for development of a diagnostic tool for future monitoring of the general health of this and other ringed seal populations.

The objective of this study was to establish reference baseline data for hematologic, biochemical, and cytologic findings in apparently healthy Atlantic bottlenose dolphins (Tursiops truncatus) inhabiting the Indian River Lagoon, Florida, USA. Sixty-two dolphins were captured, examined, and released during June 2003 and June 2004. Mean, standard deviation, and range were calculated for each parameter, and values for which published data were available, were close to or within the ranges previously reported for free-ranging bottlenose dolphins. No pathologic abnormalities were found in fecal and blowhole cytologic specimens. However, 24% (7/29) of the dolphins examined in 2003 had evidence of gastritis, which was graded as severe in 14% (4/29) of the cases. In 2004, only 4% (1/24) of dolphins sampled had evidence of mild or moderate gastritis; no severe inflammation was present. Dolphins with evidence of gastritis were 8 yr of age or older and predominantly male. Several statistically significant differences were found between males and females, between pregnant and nonpregnant animals, and between juveniles (<6 yr) and adults (≥6 yr). However, the values remained within the established ranges for this species, and the differences were not likely to be of clinical significance.

A blocking enzyme-linked immunosorbent assay was used to test 97 serum samples from big brown bats (Eptesicus fuscus) captured in six counties in Illinois between May 2002 and February 2004 for West Nile virus (WNV) antibodies. One female big brown bat tested positive for WNV antibodies. Samples of kidney, liver, and heart tissue were collected from 312 bats of seven species that were submitted to the Illinois (USA) Department of Public Health or the Illinois Department of Agriculture diagnostic laboratories between January 2001 and December 2003. Tissue samples were tested for WNV using TaqMan reverse transcriptase polymerase chain reaction and all were negative. Prevalence of WNV antibodies in the bats (1%) was lower than previously reported for other flaviviruses, but similar to the prevalence (2%) of WNV antibodies reported in bats from New Jersey and New York, USA. Additional research is needed to determine potential impact of WNV infections on bats and to determine whether they play a role in the WNV transmission cycle.

Red foxes (Vulpes vulpes) are the main reservoir of rabies in Slovenia, whereas cases of rabies in other wildlife species occur sporadically. In 1995, a program of oral vaccination of wildlife in Slovenia was initiated; baits with oral vaccine were distributed by air at a density of 20 baits/km². During 1995, when the oral vaccination program was started, 1,089 cases of rabies (including both wild and domestic animals) were reported. Five years later (1999), only six positive animals were detected among 1,195 tested (0.5%). Despite an increase in bait density (25 baits/km²) during the years 2000 and 2001, reported rabies cases increased to 115 and 135, respectively. In 2003, following initiation of a new bait-dropping strategy, which incorporated perpendicular rather than parallel flight lines, the number of rabies cases decreased to eight.

The collection of blood samples for serological studies is often stressful for the focus animal. Recently, the use of bloodsucking bugs, such as Dipetalogaster maximus or Triatoma infestans (Reduviidae; Triatominae; Heteroptera), has been suggested as a new and less invasive method for blood collection. To evaluate this technique, we collected paired blood samples from 20 domestic rabbits (Oryctolagus cuniculus) during a study of rabbit hemorrhagic disease virus (RHDV). For each rabbit, blood samples were collected by the conventional method (needle and syringe from the vena auricularis) and through feeding by D. maximus. Samples were tested for RHDV antibodies using standard test kits at three different dilutions. Antibody titers were identical for 56 paired samples and differed in only four cases. The simple matching indices were 1 for the 1:10 dilution and 0.9 for the 1:100 and 1:1000 dilutions. The major advantages of the new technique are 1) the possibility to obtain blood from animals where veins are inaccessible and 2) the fact that anesthesia of focus animals may not be necessary.

Serum samples from 18 pumas (Puma concolor), one ocelot (Leopardus pardalis), and two little spotted cats (Leopardus tigrinus) collected from free-ranging animals in Brazil between 1998 and 2004 were tested by indirect immunofluorescence (IFA) for antibodies to feline herpesvirus 1 (FHV 1), calicivirus (FCV), coronavirus (FCoV), parvovirus (FPV), Ehrlichia canis, Anaplasma phagocytophilum, and Bartonella henselae. Serum samples also were tested, by Western blot and ELISA, for feline leukemia virus (FeLV) specific antibodies and antigen, respectively, by Western blot for antibodies to feline immunodeficiency virus (FIV), and by indirect ELISA for antibodies to puma lentivirus (PLV). Antibodies to FHV 1, FCV, FCoV, FPV, FeLV, FIV, PLV or related viruses, and to B. henselae were detected. Furthermore, high-titered antibodies to E. canis or a closely related agent were detected in a puma for the first time.