A Severe Ranavirus Outbreak in Captive, Wild-Caught Box Turtles.

A Ranavirus outbreak in a captive population of wild-caught individuals was monitored using clinical evaluations and real-time PCR in 317 wild box turtles held in captivity during translocation. During the 2-year study period, the population experienced 71.6% mortality, suggesting that ranaviruses can rapidly attenuate populations. Wide variation in infection rate (7-94% per sampling period) was observed, which may have been driven by clearing and reinfection, adaptive immunity, or imperfect detection using noninvasive samples. Only nasal clinical signs were significantly related to infection status, and agreement among sample types was low. Subsequent to the initial outbreak, low mortality but high real-time PCR prevalence of Ranavirus was observed, suggesting that surviving individuals might be tolerant.

A cross sectional study evaluating the prevalence of Coxiella burnetii, potential risk factors for infection, and agreement between diagnostic methods in goats in Indiana.

Coxiella burnetii is the etiologic agent of the zoonotic disease Q fever and is considered to be endemic in domestic ruminants. Small ruminants in particular are important reservoirs for human infection. Serologic and molecular methods are both available for diagnosis of infection with C. burnetii, but there has been little research evaluating the prevalence of this organism in small ruminants outside of the context of clinical disease outbreaks. The objectives of this study were to estimate seroprevalence of C. burnetii and the prevalence of shedding of C. burnetii DNA in milk by goats in Indiana, USA, to evaluate potential risk factors for association with C. burnetii exposure and shedding, and to assess the level of agreement between the enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR) tests used to estimate prevalence. A total of 649 does over 1 year of age and not pregnant at the time of sampling were included in the study. Serum samples were collected from 608 does representing 89 farms. Milk samples were collected from 387 does representing 85 farms. Both milk and serum samples were collected from 356 does representing 80 farms. The estimated individual seroprevalence and shedding prevalence in milk adjusted for clustering were 3.1% (n=23/608, 95% CI: 1.2-7.0%) and 2.5% (n=9/387, 9.5% CI: 1.0-5.6%) respectively. Estimated adjusted herd level C. burnetii seroprevalence and herd level shedding prevalence were 11.5% (n=10/89, 95% CI: 6.4-20.1%) and 7.0% (n=6/85, 95% CI: 3.3-14.6%) respectively. Based on a generalized estimating equation model (GEE), meat breeds of goat had 7.0 times increased odds of shedding C. burnetii DNA in milk samples as compared to dairy breeds. Agreement between tests as determined by Cohen's kappa was poor at both the individual (kappa=0.04, 95% CI: -0.1 to 0.2) and herd (kappa=0.2, 95% CI: -0.1 to 0.5) levels. This indicates that serologic screening alone is unlikely to prevent the introduction of does shedding C. burnetii into herds.

Estimated herd prevalence and sequence types of Coxiella burnetii in bulk tank milk samples from commercial dairies in Indiana.

BACKGROUND: Coxiella burnetii is the etiologic agent of Q fever, a zoonotic disease causing influenza-like illness, pregnancy loss, cardiovascular disease and chronic fatigue syndrome in people. C. burnetii is considered to be enzootic in ruminants, but clinical signs of infection do not always manifest. National studies have documented the presence of C. burnetii in dairy herds in Indiana. This represents an opportunity to better characterize the distribution and prevalence of C. burnetii infection at the state scale, allowing evaluation of the need for surveillance and response planning to occur at this level. A cross-sectional study was conducted to estimate the herd prevalence of C. burnetii in commercial cattle dairies in Indiana and characterize the strains of C. burnetii within these dairies. RESULTS: Bulk tank milk samples were collected between June and August of 2011 by the Indiana State Board of Animal Health (ISBOAH). A total of 316 of these samples were tested for the IS1111 transposon of C. burnetii using quantitative real time polymerase chain reaction (PCR). Single nucleotide polymorphism (SNP) genotyping was used to identify the multispacer sequence genotypes (ST) present in samples where the IS1111 transposon was identified. The geographic distribution of dairies testing positive for C. burnetii DNA and the identified STs were also evaluated. The estimated overall herd prevalence for C. burnetii DNA was 61.1 % (95 % CI 55.6-66.3 %). The highest estimated regional prevalence was 70.2 % in the Central region of Indiana. An ST was identifiable in 74 of the positive 178 samples (41.6 %) and none of the 10 negative samples tested. Of these samples, 71 (95.9 %) were identified as ST20, 2 (2.7 %) as ST8 and a combination of ST20 and ST8 was identified in a single sample. CONCLUSIONS: C. burnetii is present in dairy herds throughout Indiana. Indiana follows national trends with ST20 most commonly identified. The presence of multiple STs in a single bulk tank sample indicates that multiple strains of C. burnetii can circulate within a herd. This supports potential transmission of C. burnetii between goats and cattle, presenting the potential for a switch in the dominant genotype found in a given species.

Mosquitoes as a Potential Vector of Ranavirus Transmission in Terrestrial Turtles.

Ranaviruses are significant pathogens of amphibians, reptiles, and fishes, contributing to mass mortality events worldwide. Despite an increasing focus on ranavirus ecology, our understanding of ranavirus transmission, especially among reptilian hosts, remains limited. For example, experimental evidence for oral transmission of the virus in chelonians is mixed. Consequently, vector-borne transmission has been hypothesized in terrestrial turtle species. To test this hypothesis, mosquitoes captured during a 2012/2013 ranavirus outbreak in box turtles from southwestern Indiana were pooled by genus and tested for ranavirus DNA using qPCR. Two of 30 pools tested positive for ranavirus. Additionally, an individual Aedes sp. mosquito observed engorging on a box turtle also tested positive for ranavirus. Although our approach does not rule out the possibility that the sequenced ranavirus was simply from virus in bloodmeal, it does suggests that mosquitoes may be involved in virus transmission as a mechanical or biological vector among ectothermic vertebrates. While additional studies are needed to elucidate the exact role of mosquitoes in ranavirus ecology, our study suggests that a greater focus on vector-borne transmission may be necessary to fully understand ranaviral disease dynamics in herpetofauna.

Low usage of government healthcare facilities for acute respiratory infections in guatemala: implications for influenza surveillance.

BACKGROUND: Sentinel surveillance for severe acute respiratory infections in hospitals and influenza-like illness in ambulatory clinics is recommended to assist in global pandemic influenza preparedness. Healthcare utilization patterns will affect the generalizability of data from sentinel sites and the potential to use them to estimate burden of disease. The objective of this study was to measure healthcare utilization patterns in Guatemala to inform the establishment of a sentinel surveillance system for influenza and other respiratory infections, and allow estimation of disease burden. METHODS: We used a stratified, two-stage cluster survey sample to select 1200 households from the Department of Santa Rosa. Trained interviewers screened household residents for self-reported pneumonia in the last year and influenza-like illness (ILI) in the last month and asked about healthcare utilization for each illness episode. RESULTS: We surveyed 1131 (94%) households and 5449 residents between October and December 2006 and identified 323 (6%) cases of pneumonia and 628 (13%) cases of ILI. Treatment for pneumonia outside the home was sought by 92% of the children <5 years old and 73% of the persons aged five years and older. For both children <5 years old (53%) and persons aged five years and older (31%) who reported pneumonia, private clinics were the most frequently reported source of care. For ILI, treatment was sought outside the home by 81% of children <5 years old and 65% of persons aged five years and older. Government ambulatory clinics were the most frequently sought source of care for ILI both for children <5 years old (41%) and persons aged five years and older (36%). CONCLUSIONS: Sentinel surveillance for influenza and other respiratory infections based in government health facilities in Guatemala will significantly underestimate the burden of disease. Adjustment for healthcare utilization practices will permit more accurate estimation of the incidence of influenza and other respiratory pathogens in the community.

Survey for the pathogenic chytrid fungus Batrachochytrium dendrobatidis in southwestern North Carolina salamander populations.

Batrachochytrium dendrobatidis is a fungal pathogen responsible for a potentially fatal disease of amphibians. We conducted a survey for B. dendrobatidis in the Appalachian Mountains of southwestern North Carolina, USA, from 10 June to 23 July 23 2009. Ventral skin swabs were collected from plethodontid salamanders (n=278) and real-time PCR was performed to test for the presence of B. dendrobatidis. We found no evidence of B. dendrobatidis, suggesting that B. dendrobatidis is absent or present in such low levels that it was undetected. If B. dendrobatidis was present at the time of our sampling, this survey supports evidence of low prevalence of B. dendrobatidis in North American headwater stream salamander populations.

Blastomycosis in man after kinkajou bite.

We report transmission of Blastomyces dermatitidis fungal infection from a pet kinkajou to a man. When treating a patient with a recalcitrant infection and a history of an animal bite, early and complete animal necropsy and consideration of nonbacterial etiologies are needed.

Development and use of an indirect enzyme-linked immunosorbent assay for detection of iridovirus exposure in gopher tortoises (Gopherus polyphemus) and eastern box turtles (Terrapene carolina carolina).

Iridoviruses, pathogens typically associated with fish and amphibians, have recently been shown to cause acute respiratory disease in chelonians including box turtles, red-eared sliders, gopher tortoises, and Burmese star tortoises. Case reports of natural infections in several chelonian species in the United States have been reported, however the prevalence remains unknown in susceptible populations of free-ranging chelonians. To determine the prevalence of iridovirus exposure in free-ranging gopher tortoises (Gopherus polyphemus) in the southeast United States, an indirect enzyme-linked immunosorbent assay (ELISA) was developed and used to evaluate plasma samples from wild gopher tortoises (G. polyphemus) from: Alabama (n=9); Florida (n=658); Georgia (n=225); Louisiana (n=12); Mississippi (n=28); and unknown locations (68) collected between 2001 and 2006. Eight (1.2%) seropositive tortoises were identified from Florida and seven (3.1%) from Georgia for an overall prevalence of 1.5%. Additionally, a population of eastern box turtles was sampled from a private nature sanctuary in Pennsylvania that experienced an outbreak of iridovirus the previous year, which killed 16 turtles. Only 1 turtle out of 55 survivors tested positive (1.8%). Results suggest a low exposure rate in chelonians to this pathogen; however, it is suspected that this is an underestimate of the true prevalence. Since experimental transmission studies and past outbreaks have shown a high rate of mortality in infected turtles, turtles may die before they develop an antibody response. Further, the duration of the antibody response is unknown and may also cause an underestimate of the true prevalence.

Consensus nested PCR amplification and sequencing of diverse reptilian, avian, and mammalian orthoreoviruses.

The orthoreoviruses are segmented RNA viruses that infect diverse vertebrate host species. While the most common human orthoreovirus, Mammalian Reovirus, is not typically associated with significant disease, the majority of Orthoreovirus species have been shown to cause significant and often fatal disease in reptiles, birds, and primates. There is significant potential for jumping species. A consensus nested-PCR method was designed for investigation of the RNA-dependent RNA polymerase gene of Orthoreovirus and Aquareovirus. This protocol was used to obtain sequencing template from reoviruses of three different vertebrate classes. Bayesian and maximum likelihood phylogenetic analysis found that all viruses analyzed clustered in the genus Orthoreovirus, that reptile reoviruses formed three distinct clusters, and that an African grey parrot reovirus clustered with Nelson Bay virus from bats. This PCR method may be useful for obtaining templates for initial sequencing of novel orthoreoviruses from diverse vertebrate hosts.

Ranavirus infection of free-ranging and captive box turtles and tortoises in the United States.

Iridoviruses of the genus Ranavirus are well known for causing mass mortality events of fish and amphibians with sporadic reports of infection in reptiles. This article describes five instances of Ranavirus infection in chelonians between 2003 and 2005 in Georgia, Florida, New York, and Pennsylvania, USA. Affected species included captive Burmese star tortoises (Geochelone platynota), a free-ranging gopher tortoise (Gopherus polyphemus), free-ranging eastern box turtles (Terrapene carolina carolina), and a Florida box turtle (Terrepene carolina bauri). Evidence for Ranavirus infection was also found in archived material from previously unexplained mass mortality events of eastern box turtles from Georgia in 1991 and from Texas in 1998. Consistent lesions in affected animals included necrotizing stomatitis and/or esophagitis, fibrinous and necrotizing splenitis, and multicentric fibrinoid vasculitis. Intracytoplasmic inclusion bodies were rarely observed in affected tissues. A portion of the major capsid protein (MCP) gene was sequenced from each case in 2003-2005 and found to be identical to each other and to Frog virus 3 (FV3) across 420 base pairs. Ranavirus infections were also documented in sympatric species of amphibians at two locations with infected chelonians. The fragment profiles of HindIII-digested whole genomic DNA of Ranavirus, isolated from a dead Burmese star tortoise and a southern leopard frog (Rana utricularia) found nearby, were similar. The box turtle isolate had a low molecular weight fragment that was not seen in the digestion profiles for the other isolates. These results suggest that certain amphibians and chelonians are infected with a similar virus and that different viruses exist among different chelonians. Amphibians may serve as a reservoir host for susceptible chelonians. This report also demonstrated that significant disease associated with Ranavirus infections are likely more widespread in chelonians than previously suspected.

Columbid herpesvirus-1 in two Cooper's hawks (Accipiter cooperii) with fatal inclusion body disease.

We report two separate naturally occurring cases of fatal herpesviral disease in Cooper's Hawks (Accipiter cooperii). Gross lesions included splenomegaly and hepatomegaly, with diffuse pale mottling or scattered small white foci. Histologic lesions included splenic and hepatic necrosis associated with eosinophilic intranuclear inclusion bodies characteristic of herpesvirus. In one case, necrosis and inclusions were also noted in bone marrow, thymus, bursa of Fabricius, thyroid gland, parathyroid gland, ceca, and the enteric system. Transmission electron microscopy demonstrated viral particles typical of herpesvirus within hepatocyte nuclei and budding from the nuclear membrane. Herpesviral DNA was amplified via polymerase chain reaction (PCR) of paraffin-embedded liver and spleen, and sequence data were consistent with columbid herpesvirus-1, an alphaherpesvirus of Rock Pigeons (Columba livia). PCR results provide evidence that this disease is transmitted to raptors via Rock Pigeons, most likely through ingestion of Rock Pigeons as prey.

Household responses to school closure resulting from outbreak of influenza B, North Carolina.

School closure is a proposed strategy for reducing influenza transmission during a pandemic. Few studies have assessed how families respond to closures, or whether other interactions during closure could reduce this strategy's effect. Questionnaires were administered to 220 households (438 adults and 355 children) with school-age children in a North Carolina county during an influenza B virus outbreak that resulted in school closure. Closure was considered appropriate by 201 (91%) households. No adults missed work to solely provide childcare, and only 22 (10%) households required special childcare arrangements; 2 households incurred additional costs. Eighty-nine percent of children visited at least 1 public location during the closure despite county recommendations to avoid large gatherings. Although behavior and attitudes might differ during a pandemic, these results suggest short-term closure did not cause substantial hardship for parents. Pandemic planning guidance should address the potential for transmission in public areas during school closure.

Six novel gammaherpesviruses of Afrotheria provide insight into the early divergence of the Gammaherpesvirinae.

The Afrotheria represent an early branching of placental mammals. Only two herpesviruses from Afrotheria have been previously identified, and the genus Proboscivirus in the subfamily Betaherpesvirinae has been proposed for them. Six novel gammaherpesviruses were identified in four species in the superorder Afrotheria by detection and analysis of their DNA polymerase genes. Elephantid herpesvirus 3 (ElHV3) and Elephantid herpesvirus 4 (ElHV4) were identified from conjunctival swabs from Asian elephants (Elephas maximus). ElHV3 was also found in a vaginal swab from one elephant with vaginitis. Elephantid herpesvirus 5 (ElHV5) was identified from vaginal swabs of two Asian elephants with vaginal plaques. Elephantid herpesvirus 6 was discovered in a conjunctival swab from an African elephant (Loxodonta africana). Procavid herpesvirus 1 (PrHV1) was found in spleen and conjunctival swabs of rock hyrax (Procavia capensis). Trichechid herpesvirus 1 (TrHV1) was identified from skin and buffy coats of Florida manatees (Trichechus manatus latirostris). ElHV3 and ElHV4 form a distinct cluster, and ElHV5, ElHV6, TrHV1, and PrHV1 form a second cluster. These viruses may have codiverged with their host species. Phylogenetic analysis of these novel herpesviruses suggests that two separate groups of gammaherpesviruses may have codiverged with the Afrotheria.

Antemortem diagnosis and characterization of nasal intranuclear coccidiosis in Sulawesi tortoises (Indotestudo forsteni).

Intranuclear coccidia and Mycoplasma spp. were identified from the nasal cavity of 5 Sulawesi tortoises (Indotestudo forsteni) affected by chronic rhinosinusitis and oronasal fistulae. This study provides the first antemortem diagnosis of intranuclear coccidiosis in tortoises and the first cytomorphologic descriptions of this disease. Histopathologic and ultrastructural morphology of the intranuclear coccidia were identical to those previously described in tortoises. Nucleic acid sequence data of a 1715 base-pair fragment of the 18S small subunit rRNA gene identify this coccidian as a novel species.

Intracytoplasmic inclusions in circulating leukocytes from an eastern box turtle (Terrapene carolina carolina) with iridoviral infection.

A free-ranging adult female eastern box turtle (Terrapene carolina carolina) was presented to the University of Tennessee in October 2003 because of suspected trauma and blindness. Physical examination revealed lethargy, clear ocular and nasal discharges, and white oral and laryngeal plaques. Intracytoplasmic inclusions within heterophils and large mononuclear leukocytes were observed on routine blood smear examination. Postmortem findings included necrosis of epithelial and parenchymal cells with intracytoplasmic inclusions. Ultrastructurally, the leukocyte inclusions consisted of variably electron-dense granular material and viral particles consistent with the Iridoviridae family of viruses. The virus shared 100% sequence identity to a 420-base pair sequence of frog virus 3 (family Iridoviridae, genus Ranavirus) as determined by polymerase chain reaction and gene sequencing targeting a portion of the Ranavirus major capsid protein gene.

A novel mycoplasma detected in association with upper respiratory disease syndrome in free-ranging eastern box turtles (Terrapene carolina carolina) in Virginia.

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.

Lymphoid follicular cloacal inflammation associated with a novel herpesvirus in juvenile alligators (Alligator mississippiensis).

Multifocal hyperemic nodules and plaques associated with the cloacal mucosa of juvenile alligators (Alligator mississippiensis) at a public aquarium were investigated. Grossly, pale pink to dark red multifocal, circular lesions of varying degrees of severity were identified on the cloacal and, in males, phallus mucosa. Cloacal mucosa biopsies were obtained from 2 of the alligators. These samples were examined histologically and by polymerase chain reaction (PCR) using consensus primers targeting a conserved region of the herpesvirus polymerase gene. Microscopically, the lesions were characterized as submucosal lymphoid follicles with hyperemia and hemorrhage. No inclusion bodies were observed. Minimal to no anisokaryosis was present, and no etiologic agents were identified. Through PCR, a band consistent in size with herpesvirus was observed. Tissues showing similar clinical, histopathologic, and PCR findings were collected from animals at an alligator farm several months later. Sequencing of the PCR amplicon resulted in a 180-base pair sequence that shared 85% sequence identity with tortoise herpesvirus-1.

Identification of a novel herpesvirus from a California desert tortoise (Gopherus agassizii).

Herpesviruses are significant pathogens of tortoises, causing upper respiratory tract disease and necrotizing stomatitis, with infections often associated with high mortality rates. Herpesvirus infection in a captive California desert tortoise (Gopherus agassizii) was detected by light microscopic observation of intranuclear inclusion bodies in various tissues followed by transmission electron microscopic observation of herpesvirus-like particles, and amplification of herpesvirus nucleic acid sequences using polymerase chain reaction. Using an indirect enzyme linked immunosorbent assay, anti-tortoise herpesvirus antibodies were detected one month after initial onset of clinical signs. This novel herpesvirus is distinct from the previously described tortoise herpesvirus (tortoise herpesvirus-1, THV-1) sharing 83% sequence identity of 60 amino acids of a portion of the DNA polymerase gene and 79% sequence identity across 120 amino acids of a portion of the ribonucleotide reductase gene. Similar to THV-1, this novel herpesvirus, tortoise herpesvirus-2 (THV-2), also clusters with the alphaherpesviruses.

Fatal human herpesvirus type 1 infection in a white-handed gibbon (Hylobates lar).

This report documents a case of spontaneous, fatal, and likely recrudescent human herpesvirus type 1 (HHV-1) infection in a captive white-handed gibbon (Hylobates lar) confirmed by polymerase chain reaction (PCR). An approximately 44-year-old, captive, female, white-handed gibbon with a history of recurrent conjunctivitis and occasional seizures became acutely weak, disoriented, and ataxic. A postictal state was suspected by caretakers and veterinary staff, and euthanasia was ultimately elected because of lack of clinical improvement with supportive care. No significant abnormalities were detected at necropsy. Histologically, sections of cerebrum and midbrain contained minimal to mild, multifocal lymphoplasmacytic meningoencephalitis with numerous intranuclear viral inclusions within astrocytes and some neurons. The presumptive diagnosis of HHV-1-induced encephalitis was strengthened by nested PCR amplification of a segment of the herpesvirus DNA polymerase gene. Sequences from this region have been found to be unique to each herpesvirus species, thus identifying HHV-1 as the likely etiologic agent in this case. Positive HHV-1 serology from several years before the terminal episode suggested that the disease was most likely due to recrudescence of latent HHV-1 infection.