Biomedical subjects

D E Stallknecht

Publications and source records attributed to D E Stallknecht.

At least 19 recordsLinked to original sources

PubMed2006

White-tailed deer (Odocoileus virginianus) develop spirochetemia following experimental infection with Borrelia lonestari.

Borrelia lonestari is considered a putative agent of southern tick-associated rash illness (STARI) and is known to occur naturally only in lone star ticks (Amblyomma americanum) and white-tailed deer (Odocoileus virginianus). We used a low passage isolate of B. lonestari (LS-1) to inoculate white-tailed deer, C3H mice, Holstein cattle, and beagles. Animals were monitored via examination of Giemsa and acridine orange stained blood smears, polymerase chain reaction (PCR), indirect fluorescent antibody (IFA) test, and/or culture isolation. Spirochetes were visualized in blood smears of both deer on days post-inoculation (DPI) 6, 8, 12 and one deer on DPI 15. Whole blood collected from deer tested PCR positive starting on DPI 4 and remained positive as long as DPI 28. Both deer developed antibody titers of >64, with a maximum IFA titer of 1024. The organism was reisolated from the blood of both deer on DPI 6 and one deer on DPI 12. All isolation attempts from mice, calves, or dogs were negative, although one of seven mice was transiently PCR positive. Mice and dogs developed an IFA titer > or =64, while calves lacked a detectable antibody response. These preliminary experimental infection trials show that white-tailed deer are susceptible to infection with B. lonestari and develop a spirochetemia following needle-inoculation, while C3H mice, calves, and dogs do not. Results suggest that deer may serve as a vertebrate reservoir host. Tick transmission studies are needed to confirm that this organism can be maintained in a natural cycle involving deer and A. americanum.

Animals↗

PubMed2005

Proteomic profiling and neurodegeneration in West-Nile-virus-infected neurons.

West Nile virus, a mosquito-borne flavivirus, is a human, equine, and avian pathogen. High-resolution two-dimensional differential-gel electrophoresis (2D-DIGE) was used to characterize protein expression in primary rat neurons and to examine the proteomic profiling to understand the pathogenesis of West-Nile-associated meningoencephalitis. Three pH ranges, 3-10, 4-7, and 5-6, were used to analyze the protein spots. The proteins are labeled with fluorescent dyes Cy3 and Cy5 before being separated on the basis of charge and size respectively on a two-dimensional platform. About 55 proteins showed altered expression levels. These were then subsequently digested and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis using peptide mass fingerprinting and database searching. These cellular proteins could represent distinct roles during infection related to apoptosis. Our findings show that two-dimensional differential gel electrophoresis combined with mass spectrometry is a powerful approach that permits the identification of proteins whose expression was altered due to West Nile virus infection.

Journal Article↗

PubMed2005

Wintering of neurotropic velogenic Newcastle disease virus and West Nile virus in double-crested cormorants (Phalacrocorax auritus) from the Florida Keys.

During November 2002, six double-crested cormorants (DCCs; Phalacrocorax auritus) were found moribund in Big Pine Key, FL, exhibiting clinical signs indicative of neurologic disease. Postmortem diagnostic evaluations were performed on two adult birds. Virulent Newcastle disease virus (NDV) was isolated from a cloacal swab from cormorant 1. West Nile virus (WNV) was isolated from the brain and lung of cormorant 2. Nucleotide sequence analysis of a portion of the fusion (F) protein gene of the NDV cormorant isolate revealed it shared a 100% deduced amino acid identity with only two viruses: the 1992 epizootic cormorant isolate from Minnesota and the 1992 turkey isolate from North Dakota. The epidemiologic significance of the recognition of virulent NDV on cormorant wintering grounds during a nonepizootic period, in addition to the potential implications of the concurrent isolation of NDV and WNV from cormorants, is discussed.

Amino Acid Sequence↗

PubMed2003

Avian influenza viruses in Minnesota ducks during 1998-2000.

Although wild ducks are known to be a major reservoir for avian influenza viruses (AIV), there are few recent published reports of surveillance directed at this group. Predominant AIV hemagglutinin (HA) subtypes reported in previous studies of ducks in North America include H3, H4, and H6, with the H5, H7, and H9 subtypes not well represented in these host populations. The objective of this study was to determine whether these subtype patterns have persisted. Each September from 1998 to 2000, cloacal swabs were collected from wild ducks banded in Roseau and Marshall counties, MN. Mallards (Anas platyrhynchos) were sampled all years, and northern pintails (A. acuta) were sampled only in 1999. Influenza viruses were isolated from 11%, 14%, and 8% of birds during 1998, 1999, and 2000, respectively. Prevalence, as expected, was highest in juveniles, ranging from 11% to 23% in mallards. Viruses representative of the HA subtypes 2, 3, 4, 5, 6, 7, 9, 10, 11, and 12 were isolated. Viruses in the H5, H7, and H9 subtypes, which are associated with high-pathogenicity influenza in poultry or recent infections in humans, were not uncommon, and each of these subtypes was isolated in 2 out of the 3 years of surveillance.

Animals↗

PubMed2001

Photosensitive chemical and laser light treatments decrease epizootic hemorrhagic disease virus associated with in vitro produced bovine embryos.

Photoinactivation was employed to eliminate EHDV-2 from in vitro produced bovine embryos experimentally exposed to this virus. Immature oocytes were matured, fertilized, and cultured in chemically defined conditions. All treatments were performed on zygotes. Developmental potential of zygotes and cell numbers of resulting hatched blastocysts were assessed after exposure to a 1 mW helium neon laser (633 nm, red) for 1, 5, 10, and 15 min; the photosensitive chemicals hematoporphyrin (15 microM) and hypericin (1 and 10 microM) for 15 min; a combination of 10 microM hypericin and laser light for 1, 3, or 5 min; and a combination of 15 microM hematoporphyrin and laser light for 1, 2, or 3 min. There were no significant differences among proportions of embryos developing or cell numbers after treatment with or without exposure to laser light alone for up to 10 min. No differences were observed after exposure of zygotes to photosensitive chemicals alone. Exposure to 10 microM hypericin and 5 min of laser light or 15 microM hematoporphyrin and 2 min of laser light compromised zygote developmental potential. After exposure to 10(6) TCID50/mL EHDV-2 for 90 min groups of 10 zygotes were exposed to 10 microM hypericin or 15 microM hematoporphyrin and laser light to inactivate the virus. Hematoporphyrin was effective with 3 min light exposure at reducing the percentage of EHDV-2 contaminated zygote pools (16.7%) as compared to EHDV-2 exposed pools without treatment (88.9%) but hematoporphyrin + 1 min light was ineffective. Hypericin + 3 min light provided an intermediate effect (55.6%).

Animals↗

PubMed2001

Reduction of infectious epizootic hemorrhagic disease virus associated with in vitro produced bovine embryos by non-specific protease.

Infectious viruses bind more tenaciously to the zonae pellucidae of in vitro produced bovine embryos than to zonae of in vivo derived embryos. Currently, the International Embryo Transfer Society recommends that all in vivo derived embryos be subjected to a rigorous washing procedure in combination with exposure to trypsin to remove viruses adherent to the zonae. In contrast to in vivo derived embryos, this method is not effective for disinfecting in vitro produced embryos. Our hypothesis was that a more potent, non-specific protease from Streptomyces griseus (S. griseus) would provide a more effective treatment for virus removal from in vitro produced bovine embryos. Bovine oocytes were matured, fertilized, and cultured in completely defined in vitro conditions. Zygotes were washed according to the procedure outlined by the International Embryo Transfer Society, replacing trypsin with the experimental protease. Experimental incubations were with 0.1% (4 units/ml) protease for 0, 30, 45, 60 and 75s intervals. Embryos were able to withstand exposure to this enzymatic treatment for only 45s before their developmental potential was significantly reduced; 60s exposure was detrimental (P<0.05). Oocytes were exposed to epizootic hemorrhagic disease virus serotype 2 (EHDV-2, 10(6) TCID(50)/ml) during in vitro maturation. Resulting zygotes were washed according to the International Embryo Transfer Society procedure and either exposed to trypsin or protease. Exposure to EHDV-2 prevented cumulus expansion and markedly reduced embryonic development (P<0.05). There were no differences in development among virus exposed groups receiving no treatment or treatment with trypsin or protease. However, proportions of infected embryos were reduced after protease treatment versus positive controls and trypsin treated embryos.

Animals↗

PubMed2001

Pathogenesis of experimental vesicular stomatitis virus (New Jersey serotype) infection in the deer mouse (Peromyscus maniculatus).

The pathogenesis of vesicular stomatitis virus (VSV) infection has not been investigated previously in native New World rodents that may have a role in the epidemiology of the disease. In the present study, 45 juvenile and 80 adult deer mice (Peromyscus maniculatus) were inoculated intranasally with VSV New Jersey serotype (VSV-NJ) and examined sequentially over a 7-day period. Virus was detected by means of immunohistochemistry and in situ hybridization in all tissues containing histologic lesions. Viral antigen and mRNA were observed initially in olfactory epithelium neurons, followed by olfactory bulbs and more caudal olfactory pathways in the brain. Virus also was detected throughout the ventricular system in the brain and central canal of the spinal cord. These results support both viral retrograde transneuronal transport and viral spread within the ventricular system. Other tissues containing viral antigen included airway epithelium and macrophages in the lungs, cardiac myocytes, and macrophages in cervical lymph nodes. In a second experiment, 15 adult, 20 juvenile, and 16 nestling deer mice were inoculated intradermally with VSV-NJ. Adults were refractory to infection by this route; however, nestlings and juveniles developed disseminated central nervous system infections. Viral antigen also was detected in cardiac myocytes and lymph node macrophages in these animals. Viremia was detected by virus isolation in 35/72 (49%) intranasally inoculated juvenile and adult mice and in 17/36 (47%) intradermally inoculated nestlings and juveniles from day 1 to day 3 postinoculation. The documentation of viremia in these animals suggests that they may have a role in the epidemiology of vector-borne vesicular stomatitis.

Animals↗

PubMed2001

Contact transmission of vesicular stomatitis virus New Jersey in pigs.

OBJECTIVE: To determine how viral shedding and development or lack of clinical disease relate to contact transmission of vesicular stomatitis virus New Jersey (VSV-NJ) in pigs and determine whether pigs infected by contact could infect other pigs by contact. ANIMALS: 63 pigs. PROCEDURE: Serologically naive pigs were housed in direct contact with pigs that were experimentally inoculated with VSV-NJ via ID inoculation of the apex of the snout, application to a scarified area of the oral mucosa, application to intact oral mucosa, or ID inoculation of the ear. In a second experiment, pigs infected with VSV-NJ by contact were moved and housed with additional naive pigs. Pigs were monitored and sampled daily for clinical disease and virus isolation and were serologically tested before and after infection or contact. RESULTS: Contact transmission developed only when vesicular lesions were evident. Transmission developed rapidly; contact pigs shed virus as early as 1 day after contact. In pens in which contact transmission was detected, 2 of 3 or 3 of 3 contact pigs were infected. CONCLUSIONS AND CLINICAL RELEVANCE: Transmission was lesion-dependent; however, vesicular lesions often were subtle with few or no clinical signs of infection. Contact transmission was efficient, with resulting infections ranging from subclinical (detected only by seroconversion) to clinical (development of vesicular lesions). Long-term maintenance of VSV-NJ via contact transmission alone appears unlikely. Pigs represent an efficient large-animal system for further study of VSV-NJ pathogenesis and transmission.

Animals↗

PubMed2001

Persistent Ehrlichia chaffeensis infection in white-tailed deer.

Four white-tailed deer (Odocoileus virginianus) were inoculated intravenously with a deer-origin isolate (15B-WTD-GA) of Ehrlichia chaffeensis. The course of infection was monitored using indirect fluorescent antibody (IFA), polymerase chain reaction (PCR), and culture over a 9 m period. All deer became rickettsemic within 24 days post inoculation (DPI), and all developed antibody titers >1:64 to E. chaffeensis by 17 DPI. Titers in all deer fell below 1:64 during 87 to 143 DPI. One deer exhibited a second period of seropositivity (peak titer of 1:256) from 207 to 271 DPI but was culture and PCR negative during this period. Rickettsemia was confirmed by reisolation of E. chaffeensis as late as 73 to 108 DPI in three deer. Positive PCR results were obtained from femur bone marrow of one deer and from rumenal lymph node of another (leer at 278 DPI. None of the deer developed clinical signs, hematologic abnormalities, or gross or microscopic lesions attributable to E. chaffeensis. Two uninoculated control deer were negative on all tests through 90 DPI at which time they were removed from the study. Herein we confirm that white-tailed deer become persistently infected with E. chaffeensis, have initial rickettsemias of several weeks duration and may experience recrudescence of rickettsemia, which reaffirm the importance of deer in the epidemiology of E. chaffeensis.

Animals↗

PubMed2000

VSV-NJ on Ossabaw Island, Georgia. The truth is out there.

Ossabaw Island, Georgia, is the only recognized enzootic focus of vesicular stomatitis virus New Jersey (VSV-NJ) in the United States and has been the subject of VSV-NJ research since 1981. To date, VSV-NJ antibodies have been detected only from feral swine, cattle, equines, deer, and raccoons. VSV-NJ transmission occurs annually, is seasonal, and is associated with the maritime forest. Despite high transmission rates the clinical disease is rarely detected. A sand fly (Lutzomyia shannoni) occurs on the Island, and experimental and field data suggest that it is a biological vector of VSV-NJ at this site. Many questions relating to the epidemiology of VSV-NJ on Ossabaw remain. What is the maintenance cycle of VSV-NJ? Is a vertebrate amplifying host(s) needed? Are other insect vectors involved in mechanical or biological transmission? Why do vesicular lesions develop on some but not all infected animals? Do native and domestic animals play the same role in the maintenance cycle? These questions challenge researchers in all areas where VSV-NJ occurs. It is our hope that Ossabaw Island will provide a much needed model system for gaining insight into the epidemiology of this virus.

Animals↗

PubMed2000

Natural infection of domestic goats with Ehrlichia chaffeensis.

Thirty-eight domestic goats from an area of Ehrlichia chaffeensis endemicity were tested for antibodies reactive to E. chaffeensis and for E. chaffeensis-specific 16S rRNA gene fragments by an indirect fluorescent antibody test and a nested PCR assay, respectively. Twenty-eight of 38 (73.7%) goats had antibodies reactive to E. chaffeensis (>/=1:128), and 6 of 38 (15.8%) goats were positive by diagnostic PCR; E. chaffeensis was isolated in cell culture from one goat. Our data indicate that goats in areas of endemicity are naturally exposed to and infected with E. chaffeensis.

Animals↗

PubMed2000

An epizootic of hemorrhagic disease in white-tailed deer in Missouri.

As part of a white-tailed deer (Odocoileus virginianus) survival study in Missouri (USA) we were actively monitoring 97 radio-collared deer when 8 (8%) died. This mortality, which occurred from 20 August to 23 September 1996, consisted of five adult females, two yearling females and one yearling male. Based on the seasonality of this mortality and the isolation of epizootic hemorrhagic disease virus (EHDV) serotype 2 from one of these animals, we believe that these losses resulted from an epizootic of hemorrhagic disease. The remains of five unmarked deer that may have died from HD also were found on the study area during this same period. During the fall following this mortality, we tested serum from 96 deer taken by hunters in the immediate area. Fifteen (16%) were positive for EHDV or bluetongue virus (BTV) antibodies as determined by agar gel immunodiffusion tests. Serum neutralization test results indicated that previous infections were caused by EHDV virus serotype 2. Based on these data, and assuming that there was no prior exposure to EHDV serotype 2 in this population, the exposure rate for this epizootic was 24% of which 8% died. We noted hoof interruptions in only two of the 96 deer sampled. During this mortality event, the Missouri Department of Conservation received no reports of dead deer, and without the radio-monitored animals the event would have been undetected.

Animals↗

PubMed1999

Novel Ehrlichia organism (Rickettsiales: Ehrlichieae) in white-tailed deer associated with lone star tick (Acari: Ixodidae) parasitism.

Polymerase chain reaction (PCR) evidence of a novel Ehrlichia organism was found recently in wild white-tailed deer, Odocoileus virginianus Zimmermann, and lone star ticks, Amblyomma americanum L., from the southeastern United States. To evaluate whether lone star tick parasitism was associated with the presence of this novel Ehrlichia organism in deer, 2 retrospective studies were conducted using specific nested PCR to test archived deer serum samples. The 1st study of 150 serum samples collected from a single deer population over a 15-yr period examined the temporal association between the presence of the Ehrlichia organism in deer and parasitism by lone star ticks. The deer Ehrlichia was not detected in serum samples collected before 1986, when lone star ticks were absent or rare, but was detected in samples collected in 1986 and every year thereafter, when lone star ticks became increasingly abundant. In the 2nd study, serum samples from 120 deer from 24 sites in 14 southeastern states were tested to evaluate if a site-specific, spatial association existed between the presence of the deer Ehrlichia and lone star ticks. All 60 serum samples from the 12 deer populations without evidence of lone star tick infestation were negative for the deer Ehrlichia, whereas 83% of the 12 populations infested by lone star ticks had PCR evidence of infection. These data suggest that lone star ticks may be a vector of the deer Ehrlichia; however, they do not preclude the involvement of other arthropods in maintaining infection with this organism in deer populations.

Animals↗

PubMed1999

Susceptibility of red and gray foxes to infection by Ehrlichia chaffeensis.

Red foxes (Vulpes vulpes) and gray foxes (Urocyon cinereoargenteus) were evaluated for their susceptibility to experimental infection with Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis. Two red foxes and three gray foxes were inoculated intravenously with E. chaffeensis (15B-WTD-GA strain) and were monitored at 7, 14, 21, and 28 days post inoculation (DPI) for evidence of infection using an indirect fluorescent antibody (IFA) assay, light microscopy, polymerase chain reaction (PCR), and cell culture methods. One red fox and one gray fox served as negative controls. Red foxes were susceptible to infection based on reisolation of E. chaffeensis from blood at 7 and 14 DPI, seroconversion by 7 DPI, and positive PCR assays on spleen and lymph nodes at 28 DPI. Morulae were not found in circulating leukocytes and clinical signs or lesions of ehrlichiosis were not observed. In contrast, gray foxes were refractory to infection based on negative results on all culture, PCR, serologic, and microscopic examinations. These findings imply that red foxes, but not gray foxes, are potential vertebrate reservoirs for E. chaffeensis. These findings also illustrate the need to verify serologic evidence of E. chaffeensis infection among wild animals.

Animals↗

PubMed1999

Experimental infection of calves with epizootic hemorrhagic disease virus.

OBJECTIVE: To determine whether experimental inoculation with a field strain of epizootic hemorrhagic disease virus serotype-2 (EHDV-2) suspected of causing clinical disease in naturally infected cattle would cause clinical disease in calves. ANIMALS: 8 calves. PROCEDURE: A strain of EHDV-2 isolated from a white-tailed deer that died of hemorrhagic disease was passaged twice in deer and used to inoculate 6 calves SC and ID; the other 2 calves were used as controls. Physical examinations, CBC, lymphocyte blastogenesis assays, and coagulation assays were performed; rectal temperature, interferon production, and serum neutralizing antibody responses were measured; and virus isolation was attempted every other day for 21 days after inoculation and then every fourth day for another 30 days. Calves were euthanatized on postinoculation day 51, and necropsy was performed. RESULTS: Calves inoculated with EHDV-2 became infected, as evidenced by development of viremia and seroconversion. However, the virus did not cause detectable clinical disease, clinicopathologic abnormalities, or gross lesions. Viremia was prolonged despite development of a serum neutralizing antibody response. A white-tailed deer inoculated with the same EHDV-2 strain developed clinical signs of epizootic hemorrhagic disease, demonstrating that the inoculum was virulent. CONCLUSION: Calves experimentally infected with EHDV-2 developed viremia and seroconverted but did not develop detectable clinical disease.

Animals↗

PubMed1999

Potential for contact and mechanical vector transmission of vesicular stomatitis virus New Jersey in pigs.

OBJECTIVE: To determine whether swine could be infected with vesicular stomatitis virus New Jersey (VSV-NJ) by routes simulating mechanical vector and contact transmission, and to document clinical response and measure source, duration, and extent of associated viral shedding. ANIMALS: 48 approximately 10-kg male pigs. PROCEDURE: 7 inoculation routes were tested at 3 virus concentrations (10(6), 10(4), and 10(2) median tissue culture-infective doses [TCID50]). Inoculation routes included intradermal (snout), topical application of virus to a scarified area (ear or mucous membrane of the lip), mucosal (oral or conjunctiva) application, and nasal instillation (drop or aerosol). Swine were monitored daily for clinical disease, seroconversion, and virus isolation. RESULTS: Virus was isolated from swab specimens of nasal planum, nasal cavity, saliva, tonsil, and feces from 15 pigs infected by intradermal snout inoculation (10(6) and 10(4) TCID50), scarification of the lip (10(6) and 10(4) TCID50), and oral route (10(6) TCID50). Virus was not isolated from plasma. Vesicular lesions were restricted to pigs inoculated via intradermal snout and lip scarification routes. Seroconversion was detected in 23 pigs inoculated by all routes except intranasal aerosol and conjunctival. Frequency of virus isolation from these pigs was dependent on serologic status and presence of vesicular lesions. CONCLUSIONS: Viral titers obtained from swab specimens and infective doses indicated that sufficient virus is shed from infected swine for contact or mechanical transmission to occur. Amount of virus is highest when lesions are present and before seroconversion. Pigs may represent an effective large-animal model for studying pathogenesis and transmission of VSV-NJ.

Animals↗

PubMed1998

Natural Mycoplasma gallisepticum infection in a captive flock of house finches.

Naturally-occurring mycoplasmal conjunctivitis is described among 104 wild-caught, and initially seronegative, house finches (Carpodacus mexicanus) maintained in captivity for 12 wk during November 1995 through January 1996. Finches housed in three pens were monitored for clinical signs, and > or = 10 birds were euthanatized for necropsy and mycoplasma testing every 2 wk. Within 2 to 4 wk following initial detection of lesions, > 50% of the birds in each of three pens developed a debilitating disease characterized by mild to severe ocular swelling, conjunctivitis, and ocular and nasal discharge. Microscopic lesions in affected finches consisted of mild to severe lymphoplasmacytic inflammation with epithelial and lymphoid hyperplasia in conjunctivae, nasal turbinates, and trachea. Mycoplasma gallisepticum infection was confirmed by culture or polymerase chain reaction (PCR) in all birds with conjunctival lesions and in 43% of birds without lesions. An arbitrary primer PCR was used to confirm M. gallisepticum isolates as identical to a field strain previously associated with house finch conjunctivitis. Most birds (89%) with conjunctivitis developed a concurrent antibody response detectable by serum plate agglutination (SPA) within 2 wk of lesion development. Hemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA) tests were less sensitive than the SPA test. The clinical severity of this disease and high proportion of affected birds suggests that M. gallisepticum may have a negative impact on free-flying house finch populations.

Agglutination Tests↗

PubMed1998

Evaluation of two oral baiting systems for wild rodents.

Tetracycline hydrochloride (TC)-treated peanut butter or rodent chow baits were distributed during March 1990, on separate 0.53 ha sites in Oglethorpe County, Georgia (USA). Rodents were trapped on a control site prior to bait distribution and on two baited sites 6 days post-distribution. Cleaned skulls from euthanized mammals were grossly examined for TC fluorescence using an ultraviolet (UV) light. Mandibles were sectioned and examined for TC fluorescence using an ultraviolet light microscope. All 21 cotton rats (Sigmodon hispidus), four eastern harvest mice (Rithrodontomys humulis), and two golden mice (Ochrotomys nuttalli) captured on the control site were negative for TC fluorescence. On the peanut butter bait site, mandible sections from 29 of 32 (91%) cotton rats, three of three (100%) eastern harvest mice, two of three (66%) golden mice, zero of five (0%) white-footed mice (Peromyscus leucopus), one of three (33%) short-tailed shrews (Blarina brevicauda), and zero of two (0%) least shrews (Cryptotis parva) were positive for TC. Results from the rodent chow bait site indicated that 18 of 25 (72%) cotton rats, zero of three (0%) eastern harvest mice, two of seven (29%) golden mice, zero of four (0%) white-footed mice, and zero of four (0%) least shrews were positive for TC fluorescence in mandible sections. These results suggest that a large portion of a free-ranging small rodent population can be administered biological markers or vaccines using baits.

Animal Feed↗