Biologic, antigenic, and full-length genomic characterization of a bovine-like coronavirus isolated from a giraffe

Coronaviruses (CoVs) possess large RNA genomes and exist as quasispecies, which increases the possibility of adaptive mutations and interspecies transmission. Recently, CoVs were recognized as important pathogens in captive wild ruminants. This is the first report of the isolation and detailed genetic, biologic, and antigenic characterization of a bovine-like CoV from a giraffe (Giraffa camelopardalis) in a wild-animal park in the United States. CoV particles were detected by immune electron microscopy in fecal samples from three giraffes with mild-to-severe diarrhea. From one of the three giraffe samples, a CoV (GiCoV-OH3) was isolated and successfully adapted to serial passage in human rectal tumor 18 cell cultures. Hemagglutination assays, receptor-destroying enzyme activity, hemagglutination inhibition, and fluorescence focus neutralization tests revealed close biological and antigenic relationships between the GiCoV-OH3 isolate and selected respiratory and enteric bovine CoV (BCoV) strains. When orally inoculated into a BCoV-seronegative gnotobiotic calf, GiCoV-OH3 caused severe diarrhea and virus shedding within 2 to 3 days. Sequence comparisons and phylogenetic analyses were performed to assess its genetic relatedness to other CoVs. Molecular characterization confirmed that the new isolate belongs to group 2a of the mammalian CoVs and revealed closer genetic relatedness between GiCoV-OH3 and the enteric BCoVs BCoV-ENT and BCoV-DB2, whereas BCoV-Mebus was more distantly related. Detailed sequence analysis of the GiCoV-OH3 spike gene demonstrated the presence of a deletion in the variable region of the S1 subunit (from amino acid 543 to amino acid 547), which is a region associated with pathogenicity and tissue tropism for other CoVs. The point mutations identified in the structural proteins (by comparing GiCoV-OH3, BCoV-ENT, BCoV-DB2, and BCoV-Mebus) were most conserved among GiCoV-OH3, BCoV-ENT, and BCoV-DB2, whereas most of the point mutations in the nonstructural proteins were unique to GiCoV-OH3. Our results confirm the existence of a bovine-like CoV transmissible to cattle from wild ruminants, namely, giraffes, but with certain genetic properties different from those of BCoVs.     

Parasites, diseases and health status of sympatric populations of sambar deer and white-tailed deer in Florida

From December 1983 to December 1984 a study on parasites, diseases and health status was conducted on sympatric populations of sambar deer (Cervus unicolor) and white-tailed deer (Odocoileus virginianus) from St. Vincent Island, Franklin County, Florida. Ten sambar and six white-tailed deer were examined. White-tailed deer had antibodies to epizootic hemorrhagic disease virus and bluetongue virus. Serologic tests for antibodies to the etiologic agents of bovine virus diarrhea, infectious bovine rhinotracheitis, vesicular stomatitis, parainfluenza 3, brucellosis, and leptospirosis were negative in both species of deer. White-tailed deer harbored 19 species of parasites; all were typical of the parasite fauna of this species in coastal regions of the southeastern United States. Sambar deer harbored 13 species of parasites, which apparently were derived largely from white-tailed deer. The only exception was Dermacentor variabilis which occurs frequently on wild swine on the island. The general health status of sambar deer appeared to be better than that of white-tailed deer. This was hypothesized to result from the sambar deer's utilization of food resources unavailable or unacceptable to white-tailed deer and to the absence and/or lower frequency of certain pathogens in sambar deer.     

Viruses isolated from captive and free-ranging wild ruminants in Alberta.

Nasal secretions, leukocytes and preputial or vaginal swabs from a group of 15 captive wild ruminants, comprising six pronghorn antelope (Antilocapra americana), seven fallow deer (Dama dama) and two mule deer (Odocoileus hemionus), and from 50 free-ranging pronghorns in southern Alberta, were examined for viral agents. Captive animals were given injections of dexamethasone daily for 6 days in attempts to reactivate latent infections. Specimens were collected at 2-3 day intervals from days 0 to 18. Free-ranging pronghorns were sampled only once, at the time of capture. Fifteen viral isolates were obtained from the animals: six isolates of parainfluenza 3 (PI3) from nasal swabs from one fallow deer and one mule deer; five isolates of herpesvirus from leukocytes, vaginal, preputial and nasal swabs from three fallow deer; and four isolates of PI3 from nasal secretions of the 50 free-ranging pronghorns.     

Prevalence and phylogeny of coronaviruses in wild birds from the Bering Strait area (Beringia)

Coronaviruses (CoVs) can cause mild to severe disease in humans and animals, their host range and environmental spread seem to have been largely underestimated, and they are currently being investigated for their potential medical relevance. Infectious bronchitis virus (IBV) belongs to gamma-coronaviruses and causes a costly respiratory viral disease in chickens. The role of wild birds in the epidemiology of IBV is poorly understood. In the present study, we examined 1,002 cloacal and faecal samples collected from 26 wild bird species in the Beringia area for the presence of CoVs, and then we performed statistical and phylogenetic analyses. We detected diverse CoVs by RT-PCR in wild birds in the Beringia area. Sequence analysis showed that the detected viruses are gamma-coronaviruses related to IBV. These findings suggest that wild birds are able to carry gamma-coronaviruses asymptomatically. We concluded that CoVs are widespread among wild birds in Beringia, and their geographic spread and frequency is higher than previously realised. Thus, Avian CoV can be efficiently disseminated over large distances and could be a genetic reservoir for future emerging pathogenic CoVs. Considering the great animal health and economic impact of IBV as well as the recent emergence of novel coronaviruses such as SARS-coronavirus, it is important to investigate the role of wildlife reservoirs in CoV infection biology and epidemiology.     

A serologic investigation of epizootic hemorrhagic disease virus in China between 2014 and 2019

Epizootic hemorrhagic disease virus (EHDV) is a member of the genus Orbivirus, family Sedoreoviridae. It was firstly recognized in 1955 to cause a highly fatal disease of wild white-tailed deer in America. So far, EHDV was detected and isolated in many wild or domestic ruminants, and widely distributed all over the world. Although the domestic cattle and sheep infected by EHDV were usually asymptomatic or subclinical, several outbreaks of epizootic hemorrhagic disease (EHD) in deer and cattle had been reported. Many EHDV strains were isolated and sequenced in last two decades in China, which promoted a general serologic investigation of EHDV in China. In this study, 18,122 sera were collected from asymptomatic or subclinical domestic ruminants (cattle, cow, yaks, sheep, goats, and deer) in 116 regions belonging to 15 provinces in China. All the sera were tested by EHDV C-ELISA, and the results were obtained by big data analysis. EHDV infections were detected in the 14 of 15 provinces, and only Tibet (average altitude ≥ 4000 m) which was the highest province in China was free of EHDV. The numbers of seropositive collections in both bovine and goat/sheep were in an inverse proportion to the latitude. However, the seropositive rates in bovine were ranged from 0% to 100%, while the seropositive rates in goat/sheep were no more than 50%. The results suggested that bovine was obviously more susceptive for EHDV infection than goat and sheep, therefore might be a major reservoir of EHDV in China. The prevalence of EHDV was consistent with the distribution of Culicoides which were known as the sole insect vectors of EHDV. In particular, the seropositive rates of EHDV were very high in the southern provinces, which required the enhanced surveillance in the future.     

Glycosylated hemoglobin as a stable alternative to serum glucose in white-tailed deer

We compared serum glucose concentration and percent glycosylated hemoglobin (GH) in captive and wild white-tailed deer (Odocoileus virginianus) to determine stability of glucose relative to GH. Temporal changes in levels of serum glucose and GH were ascertained from serial blood samples collected from three captive deer over a 2-week period. State of glycemia also was determined for 17 wild deer that were collected from three populations in southeastern Oklahoma and southwestern Arkansas (USA). Concentration of serum glucose of captive deer decreased (P = 0.04) from 190.4 to 155.8 mg/dl over the 2 weeks; percent GH did not differ temporally (P = 0.30). Percent GH of wild deer did not differ (P = 0.23) when deer were separated into 2 groups (high and low state of glycemia) based on the median serum glucose concentration. We found a significant difference (P = 0.04) in percent GH among wild deer populations; serum glucose concentration did not differ (P = 0.72) among populations. Our results indicate that percent GH is more stable than serum glucose concentration and may be useful in population comparisons of nutritional condition.     

Acute sarcocystosis in a captive white-tailed deer in Virginia

A 19-mo-old female captive white-tailed deer in a public wild animal park in Richmond (Virginia, USA) was necropsied and evaluated histologically following spontaneous death after a 1 wk period of lethargy in a captive herd of 22 deer. An acute necrotizing pneumonia was associated with intraendothelial protozoal schizonts that were identified immunohistochemically as Sarcocystis spp. This is the first confirmed report of acute sarcocystosis in a wild ruminant.     

Maternal strategies in sable antelope,Hippotragus niger: Factors affecting variability in maternal retrieval of hiding calves

Maternal behavior of wild ungulates has long been considered relatively invariant, yet recent reports from both wild and captive populations indicate considerable variability. Maternal behavioral tactics associated with the “hider” developmental strategy were investigated in six mother-infant pairs of sable antelope, Hippotragus niger, at the National Zoological Park's Conservation and Research Center near Front Royal, Virginia. When a calf moved away from its mother to hide, the mother tended to be 10 or more body lengths away and facing away from the calf. The presence of maternal vocalizations and the mother's ability to orient to the calf's resting site prior to calf activity periods were dependent on the age of the calf and whether the calf was lying alone or with other calves. The presence of maternal vocalizations and the ability of a mother to orient toward her calf were negatively related, with the mother vocalizing only when her calf could not be located visually. Variability in maternal behavior when searching for hidden calves was best explained by differences in calf conspicuousness. A mother apparently remembered only the general area in which her calf was lying, and not the precise location. Comparison of this study with previous studies of captive sable antelope suggests that mother-infant interactions are strongly influenced by enclosure size and the availability of tall vegetation; therefore, behaviors of individuals observed in small captive enclosures lacking tall vegetation may not be representative of those in the wild. © 1996 Wiley-Liss, Inc.     

Evidence Supporting a Zoonotic Origin of Human Coronavirus Strain NL63

The relationship between bats and coronaviruses (CoVs) has received considerable attention since the severe acute respiratory syndrome (SARS)-like CoV was identified in the Chinese horseshoe bat (Rhinolophidae) in 2005. Since then, several bats throughout the world have been shown to shed CoV sequences, and presumably CoVs, in the feces; however, no bat CoVs have been isolated from nature. Moreover, there are very few bat cell lines or reagents available for investigating CoV replication in bat cells or for isolating bat CoVs adapted to specific bat species. Here, we show by molecular clock analysis that alphacoronavirus (α-CoV) sequences derived from the North American tricolored bat (Perimyotis subflavus) are predicted to share common ancestry with human CoV (HCoV)-NL63, with the most recent common ancestor between these viruses occurring approximately 563 to 822 years ago. Further, we developed immortalized bat cell lines from the lungs of this bat species to determine if these cells were capable of supporting infection with HCoVs. While SARS-CoV, mouse-adapted SARS-CoV (MA15), and chimeric SARS-CoVs bearing the spike genes of early human strains replicated inefficiently, HCoV-NL63 replicated for multiple passages in the immortalized lung cells from this bat species. These observations support the hypothesis that human CoVs are capable of establishing zoonotic-reverse zoonotic transmission cycles that may allow some CoVs to readily circulate and exchange genetic material between strains found in bats and other mammals, including humans.     

Use of melengestrol acetate in feed for contraception in herds of captive ungulates

Herds of blackbuck antelope (Antilopa cervicapra) and barasingha (Cervus duvauceli), axis (Cervus axis), sambar (Cervus unicolor), and Formosan sika (Cervus nippon taiwanaus) deer at the Wildlife Conservation Society/Bronx Zoo (WCS/BZ) were fed melengestrol acetate (MGA) at a concentration of 0.000154% in pelleted feed for various periods of times during 1991–2001. The target dose per animal of MGA was 1–2 mg per day. Contraceptive rates during treatment were 100% for blackbuck antelope and barasingha, sambar, and sika deer, and approximately 93% for axis deer. There were no observed adverse effects from MGA treatment on gestation. Post‐treatment reproductive rates were lower than pretreatment rates. Zoo Biol 22:455–463, 2003. © 2003 Wiley‐Liss, Inc.     

A Frightening Device for Deterring Deer Use of Cattle Feeders

Abstract: The presence of bovine tuberculosis (TB) in cattle can negatively impact a state's economy and cattle industry. In Michigan, USA, wild white-tailed deer (Odocoileus virginianus) are a reservoir for reinfecting cattle herds. Although direct TB transmission between deer and cattle is rare, infected deer may contaminate cattle feed. To mitigate this risk, we designed and evaluated a deer-resistant cattle feeder (DRCF) device for deterring deer from feeders. The device delivered negative stimuli to condition deer to avoid cattle feeders. We tested the device by conducting a comparative change experiment at a high-density captive white-tailed deer operation in northeastern lower Michigan using pretreatment and treatment periods and random allocation of DRCF protection to 3 of 6 feeders during the treatment period. We used animal-activated cameras to collect data on deer use of feeders. Deer use was similar at protected and unprotected feeders during the pretreatment period but was lower at protected feeders during the treatment period. Deer-resistant cattle feeders were 100% effective during the first 2 treatment weeks, 94% during the first 5 weeks, but effectiveness then dropped to 61% during the final week. Excluding problems associated with low battery power and infrared sensors, DRCFs were 99% effective at deterring deer. Our results suggest that DRCFs can effectively limit deer use of cattle feed, potentially with minimal impact on feeding behavior of cattle, thus reducing potential transmission of bovine TB through contaminated feed. By employing DRCFs in bovine TB endemic areas, especially at times that deer are food stressed, agencies and producers can practically and economically reduce the potential for bovine TB to be transmitted from deer to cattle.     

Review of the 2012 Epizootic Hemorrhagic Disease Outbreak in Domestic Ruminants in the United States

An unusually large number of cases of Epizootic hemorrhagic disease (EHD) were observed in United States cattle and white-tailed deer in the summer and fall of 2012. USDA APHIS Veterinary Services area offices were asked to report on foreign animal disease investigations and state diagnostic laboratory submissions which resulted in a diagnosis of EHD based on positive PCR results. EHD was reported in the following species: cattle (129 herds), captive white-tailed deer (65 herds), bison (8 herds), yak (6 herds), elk (1 herd), and sheep (1 flock). A majority of the cases in cattle and bison were found in Nebraska, South Dakota, and Iowa. The majority of cases in captive white-tailed deer were found in Ohio, Iowa, Michigan, and Missouri. The most common clinical sign observed in the cattle and bison herds was oral lesions. The major observation in captive white-tailed deer herds was death. Average within-herd morbidity was 7% in cattle and bison herds, and 46% in captive white-tailed deer herds. The average within-herd mortality in captive white-tailed deer herds was 42%.     

Bovine viral diarrhea virus multiorgan infection in two white-tailed deer in southeastern South Dakota

The susceptibility of wild ruminants, especially cervids, to bovine viral diarrhea virus (BVDV) has remained an enigma. Two white-tailed deer (Odocoileus virginianus) were submitted to the Animal Disease Research and Diagnostic Laboratory (ADRDL) in the fall of 2003 by the South Dakota Game Fish and Parks for chronic wasting disease (CWD) testing. Both animals were CWD negative. The animals were necropsied and histopathology, viral antigen detection, and virus isolation were performed. A noncytopathic (NCP) BVDV was isolated from the lungs and several other tissues of both animals. Formalin-fixed ear notches from both animals were positive for BVDV antigen by immunohistochemistry. The BVDV isolates were typed with the use of polymerase chain reaction in 5' untranslated region (UTR) and one isolate was typed a Type 2a and the other a Type 1b. Future field surveys to determine the incidence of BVDV along with experimental studies to determine if white-tailed deer fawns can be persistently infected with BVDV are needed.     

Parelaphostrongylus tenuis in captive pronghorn antelope (Antilocapra americana) in Nebraska

Lesions in four captive pronghorn antelope (Antilocapra americana) naturally infected with Parelaphostrongylus tenuis in eastern Nebraska (USA) are described in this report. Animals were bright and alert with hind limb ataxia that progressed to sternal or lateral recumbency between July 28 and October 17, 1998. Animals were euthanized due to disease progression despite therapy. Multifocal decubital ulcers over bony prominences occurred in two animals and chronic unilateral otitis media was present in one animal. Histopathologic examination revealed severe Wallerian degeneration randomly scattered throughout the spinal cords of all four animals. Spinal cord sections from two animals contained adult nematode parasites consistent with P. tenuis. This is the first report of naturally occurring P. tenuis infection in pronghorn antelope. Pronghorn antelope should be considered susceptible to P. tenuis infection and contact with infected white-tailed deer as well as intermediate gastropod hosts of P. tenuis should be prevented in endemic areas.     

Epidemiology of chronic wasting disease in captive white-tailed and mule deer

The natural occurrence of chronic wasting disease (CWD) in a 1993 cohort of captive white-tailed deer (Odocoileus virginianus) afforded the opportunity to describe epidemic dynamics in this species and to compare dynamics with those seen in contemporary cohorts of captive mule deer (O. hemionus) also infected with CWD. The overall incidence of clinical CWD in white-tailed deer was 82% (nine of 11) among individuals that survived >15 mo. Affected white-tailed deer died or were killed because of terminal CWD at age 49-76 mo (x = 59.6 mo, SE = 3.9 mo). Epidemic dynamics of CWD in captive white-tailed deer were similar to dynamics in mule deer cohorts. Incidence of clinical CWD was 57% (4/7) among hand-raised (HR) and 67% (4/6) among dam-raised (DR) mule deer; affected HR mule deer succumbed at 64-86 mo of age (x = 72 mo; SE = 5 mo), and affected DR mule deer died at age 31-58 mo (x = 41.3 mo; SE = 6.1 mo). Sustained horizontal transmission of CWD most plausibly explained epidemic dynamics, but the original source of exposures could not be determined. Apparent differences in mean age at CWD-caused death among these cohorts may be attributable to differences in the timing or intensity of exposure to CWD, and these factors appear to be more likely to influence epidemic dynamics than species differences. It follows that CWD epidemic dynamics in sympatric, free-ranging white-tailed and mule deer sharing habitats in western North American ranges also may be similar.     

Paratuberculosis in saiga antelope (Saiga tatarica) and experimental transmission to domestic sheep.

Mycobacterium paratuberculosis was isolated in low numbers from the small intestine and associated mesenteric lymph nodes of a saiga antelope (Saiga tatarica) using routine culture techniques in spite of histologic evidence of high numbers of acid-fast bacteria in these tissues. Two newborn domestic sheep were fed the ground intestinal tissue containing acid-fast bacteria and the progression of the experimental disease was followed by fecal culture, immunodiffusion (AGID) and lymphocyte stimulation (LST) tests. One experimentally infected sheep developed progressive clinical illness 1 yr postinoculation. Few M. paratuberculosis were isolated from feces or tissues although an extensive granulomatous mycobacterial enteritis, lymphadenitis and lymphangitis were observed containing large numbers of typical acid-fast organisms. No clinical illness was observed in the second inoculated sheep after 18 mo of observation, although infection was demonstrated at necropsy. Both sheep developed AGID and LST reactions indicative of paratuberculosis. This study demonstrated that a difficult to culture isolate of M. paratuberculosis was responsible for paratuberculosis in captive wild ruminants and was transmissible to domestic sheep. Diagnosis of paratuberculosis in four of eight of the imported saiga antelope and in eleven of their 18 offspring indicates the importance of this disease in management of captive wild ruminants and the ease with which this organism can be transmitted.     

Two-way antigenic cross-reactivity between severe acute respiratory syndrome coronavirus (SARS-CoV) and group 1 animal CoVs is mediated through an antigenic site in the N-terminal region of the SARS-CoV nucleoprotein

In 2002, severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged in humans, causing a global epidemic. By phylogenetic analysis, SARS-CoV is distinct from known CoVs and most closely related to group 2 CoVs. However, no antigenic cross-reactivity between SARS-CoV and known CoVs was conclusively and consistently demonstrated except for group 1 animal CoVs. We analyzed this cross-reactivity by an enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using specific antisera to animal CoVs and SARS-CoV and SARS patient convalescent-phase or negative sera. Moderate two-way cross-reactivity between SARS-CoV and porcine CoVs (transmissible gastroenteritis CoV [TGEV] and porcine respiratory CoV [PRCV]) was mediated through the N but not the spike protein, whereas weaker cross-reactivity occurred with feline (feline infectious peritonitis virus) and canine CoVs. Using Escherichia coli-expressed recombinant SARS-CoV N protein and fragments, the cross-reactive region was localized between amino acids (aa) 120 to 208. The N-protein fragments comprising aa 360 to 412 and aa 1 to 213 reacted specifically with SARS convalescent-phase sera but not with negative human sera in ELISA; the fragment comprising aa 1 to 213 cross-reacted with antisera to animal CoVs, whereas the fragment comprising aa 360 to 412 did not cross-react and could be a potential candidate for SARS diagnosis. Particularly noteworthy, a single substitution at aa 120 of PRCV N protein diminished the cross-reactivity. We also demonstrated that the cross-reactivity is not universal for all group 1 CoVs, because HCoV-NL63 did not cross-react with SARS-CoV. One-way cross-reactivity of HCoV-NL63 with group 1 CoVs was localized to aa 1 to 39 and at least one other antigenic site in the N-protein C terminus, differing from the cross-reactive region identified in SARS-CoV N protein. The observed cross-reactivity is not a consequence of a higher level of amino acid identity between SARS-CoV and porcine CoV nucleoproteins, because sequence comparisons indicated that SARS-CoV N protein has amino acid identity similar to that of infectious bronchitis virus N protein and shares a higher level of identity with bovine CoV N protein within the cross-reactive region. The TGEV and SARS-CoV N proteins are RNA chaperons with long disordered regions. We speculate that during natural infection, antibodies target similar short antigenic sites within the N proteins of SARS-CoV and porcine group 1 CoVs that are exposed to an immune response. Identification of the cross-reactive and non-cross-reactive N-protein regions allows development of SARS-CoV-specific antibody assays for screening animal and human sera.     

Immunologic and molecular identification of Babesia bovis and Babesia bigemina in free-ranging white-tailed deer in northern Mexico

The suitability of white-tailed deer (Odocoileus virginianus) as hosts for the cattle ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus (Boophilus) annulatus, has been well documented. These ticks have a wide host range, and both transmit Babesia bovis and Babesia bigemina, the agents responsible for bovine babesiosis. Although this disease and its vectors have been eradicated from the United States and some states in northern Mexico, it still is a problem in other Mexican states. It is not known if wild cervids like white-tailed deer can act as reservoirs for bovine babesiosis. The purpose of this study was to determine if B. bovis and B. bigemina or antibodies against them occur in white-tailed deer in the states of Nuevo Leon and Tamaulipas, Mexico. Twenty blood samples from white-tailed deer from two ranches were collected and tested with a nested polymerase chain reaction (nested PCR) and indirect immunofluorescence antibody test (IFAT) for B. bovis and B. bigemina. Eleven samples were positive for B. bigemina and four for B. bovis by nested PCR; amplicon sequences were identical to those reported in GenBank for B. bovis (Rap 1) and B. bigemina. Results of the IFA test showed the presence of specific antibodies in serum samples. This is the first report of the presence of B. bovis and B. bigemina in white-tailed deer using these techniques and underscores the importance of cervids as possible reservoirs for bovine babesiosis.