Neospora caninum antibodies detected in Midwestern white-tailed deer (Odocoileus virginianus) by Western blot and ELISA

White-tailed deer (Odocoileus virginianus) serve to maintain the Neospora caninum life cycle in the wild. Sera from white-tailed deer from south central Wisconsin and southeastern Missouri, USA were tested for antibodies to N. caninum by Western blot analyses and two indirect ELISAs. Seroreactivity against N. caninum surface antigens was observed in 30 of 147 (20%) of WI deer and 11 of 23 (48%) of MO deer using Western blot analysis. Compared to Western blot, the two indirect ELISAs were found to be uninformative due to degradation of the field-collected samples. The results indicate the existence of N. caninum antibodies in MO and WI deer, and that Western blot is superior to ELISA for serologic testing when using degraded blood samples collected from deer carcasses.     

Lesion development in white-tailed deer (Odocoileus virginianus) experimentally infected with Mycobacterium bovis

The recent discovery of tuberculosis in free-living white-tailed deer in northeastern Michigan underscores the need for increased understanding of the pathogenesis of tuberculosis in wildlife species. To investigate lesion development in white-tailed deer, 32 deer were experimentally infected by intratonsilar instillation of 300 colony-forming units of Mycobacterium bovis. Three deer each were euthanatized and examined at days 15, 28, 42, and 56 after inoculation, and five deer each were euthanatized and examined at days 89, 180, 262, and 328 after inoculation. Microscopic lesions first were seen in the medial retropharyngeal lymph node and lung 28 and 42 days after inoculation, respectively. Lung lesions were present in 12 (38%) of 32 deer, involving 23 lung lobes. Left caudal and right middle and caudal lobes were involved in 17 (74%) of the 23 affected lung lobes. Lesions in the medial retropharyngeal lymph node first appeared as granulomas composed of aggregates of macrophages and Langhans-type giant cells. Some early granulomas contained centrally located neutrophils. As granulomas developed, neutrophils were replaced with a central zone of caseous necrosis that first showed signs of mineralization 42 days after inoculation. Granulomas increased in size as the zone of caseous necrosis expanded. Peripheral fibrosis, first seen at 56 days after inoculation, progressed to only a thin fibrous capsule by 328 days after inoculation. By the termination of the study, the central necrotic core of the granuloma contained abundant liquefied necrotic material and grossly resembled an abscess. Although tuberculous lesions in white-tailed deer follow a developmental pattern similar to that in cattle, fibrosis is less pronounced and the advanced lesions may liquefy, a change seldom reported in cattle. An understanding of lesion development will aid in the identification of the spectrum of disease that may be seen in this important wildlife reservoir of tuberculosis.     

Hepatocellular adenocarcinoma in a white-tailed deer (Odocoileus virginianus).

A white-tailed deer (Odocoileus virginianus), shot during the 1978-79 New Jersey hunting season, was presented with an enlarged, multinodular liver and numerous skin growths. The skin lesions were found to be fibromas and the liver tumor was identified as a hepatocellular adenocarcinoma, a rare neoplasm, not only in deer but all wild animals.     

Tuberculin skin testing in white-tailed deer (Odocoileus virginianus).

The comparative cervical skin test for antemortem diagnosis of tuberculosis was done 169 times on 116 different white-tailed deer of known Mycobacterium bovis infection status. The sensitivity and specificity were 97 and 81%, respectively. The magnitude of change in skin thickness at test sites was not significantly influenced by dosage of inoculum, dissemination of the disease process, or repeated skin testing. However, the magnitude of change in skin thickness was significantly greater in deer infected for less than 109 days than in deer infected for more than 109 days. As used in the present study, the comparative cervical skin test is a sensitive method of antemortem diagnosis of M. bovis infection in white-tailed deer.     

Isolation and characterization of epizootic hemorrhagic disease virus from white-tailed deer (Odocoileus virginianus) in eastern Washington.

A virus was isolated from the spleen of a white-tailed deer (Odocoileus virginianus) that had died during an epizootic in Washington state in 1967. Inoculation of a 10% spleen suspension from the deer caused hemorrhagic disease in normal white-tailed deer. Studies were conducted on the biological, physicochemical, and serologic properties of the Washington isolate. An in vitro assay system, utilizing a cultured primary of white-tailed deer fetal cells from an entire fetus, was employed for isolation and propagation of the virus. Cytopathic effect was characterized by focal development of rounded and clumped cells. Propagation was unsuccessful in suckling mice, BHK-21, and Vero cell cultures. The virus was resistant to treatment with ether, sodium deoxycholate, trypsin, oxytetracycline hydrochloride, and was sensitive to chloroform. Virus yield was not affected when infected cultures were treated with 5-iodo-2'-deoxyuridine, but dactinomycin (actinomycin D) treatment of infected cultures reduced virus yield. The virus was inactivated when heated at 70 C for 5 minutes or when exposed to pH 5 for 18 hours at 4 C. The virus was completely excluded from the filtrate by a 0.10- micronm (APD) membrane filter. Staining of infected cells with acridine orange indicated the presence of double-standard nucleic acid in the cytoplasm. Serum-neutralization tests with antiserums against the homologous virus and the New Jersey and Alberta strains of epizootic hemorrhagic disease virus resulted in neutralization of the Washington isolate. The Washington virus was not neutralized by bluetongue virus antiserum. Cells infected with the Washington isolate exhibited intracytoplasmic fluorescence by the indirect fluorescent antibody method with New Jersey and Alberta epizootic hemorrhagic disease antiserums but not with bluetongue antiserum.     

Congenital polycystic kidney in a white-tailed deer (Odocoileus virginianus).

Polycystic kidney and liver disease was seen in a stillborn white-tailed deer (Odocoileus virginianus) fawn. Bilaterally enlarged kidneys were characterized by severe dilatation of all renal tubules. Glomeruli were sparse, small, and located within a dilated Bowman's capsule. The liver was characterized by marked periportal fibrosis, biliary hyperplasia, and bile duct ectasia with dilated ducts containing inspissated bile. The presentation and morphology of this case are most similar to autosomal recessive polycystic disease in humans.     

Susceptibility of male white-tailed deer (Odocoileus virginianus) to Brucella ovis infection

Infection with Brucella ovis was established by conjunctival instillation in 8 male white-tailed deer (Odocoileus virginianus). Infection was transient in young bucks, but persisted in bucks that were mature when inoculated. The deer were euthanatized and necropsied at various intervals after inoculation. Brucella ovis was recovered from a mature buck at necropsy on postinoculation day 429. Four deer had gross lesions and histopathologic changes of the epididymides. A mature noninfected buck confined for 7 months with an infected buck acquired infection and developed epididymal lesions.     

Isolation and molecular detection of Neospora caninum from naturally infected sheep from Brazil

Neospora caninum was isolated from a naturally infected sheep from Brazil by bioassay in dogs. Approximately 70g of brain from each of two 4-month-old sheep with indirect fluorescent antibodies (>or=1:50) to N. caninum was offered to a different IFAT negative dog (Sheep n. 302, IFAT 1:400-Dog 1 and Sheep n. 342, IFAT 1:50-Dog 2). Parasite DNA was detected in both sheep brains using a PCR targeting the Nc-5 gene of N. caninum. Shedding of Neospora-like oocysts was noticed only in Dog 1, from 10 days post-inoculation (PI) to 25 days PI (a total of approximately 27,600 oocysts). Seventy days after infection, Dog 1 was euthanized and brain/cerebellum and medulla were collected and submitted to molecular methods, as were the oocysts, to confirm the identity of the isolate. Serum samples collected weekly from both dogs from the infection to the end of the experimental period had no antibodies anti-N. caninum by IFAT (<1:50). Oocysts, brain/cerebellum and medulla specimens of Dog 1 proved positive by a PCR assay targeting the Nc-5 gene of N. caninum. In addition, the oocysts have the DNA amplified by a PCR based on primers directed to the common toxoplasmatiid ITS1 sequence. The PCR products of ITS1 were sequenced, confirming again the isolate as N. caninum. Oocysts were also orally inoculated in two Swiss white mice two Mongolian gerbils (Meriones ungulatus) and two large vesper mice (Calomys callosus) (10(3)oocysts/animal). The rodents were sacrificed 2 months PI, and fresh preparations of brains showed Neospora thick-walled cysts in gerbil brains, but molecular detection using the Nc-5 PCR assay revealed DNA parasite in gerbil and also C. callosus brains. This is the first report of isolation and sequencing of N. caninum from a Brazilian sheep and the first report of molecular detection of N. caninum from C. callosus.     

Choroid plexitis in white-tailed deer (Odocoileus virginianus) in southern New York State

Brains, spinal cords, nerve roots, nerves and muscle tissues were removed from deer in southern New York State and examined for histologic evidence of infection by the causative agent of Lyme disease, Borrelia burgdorferi. There was no histologic evidence of this infection and only four of 26 deer had serologic evidence of past infection despite the fact that all were parasitized by the tick vector, Ixodes dammini. Of these ticks, 21% were carrying B. burgdorferi. In contrast, most of the deer had choroid plexitis. All but one of 48 deer tested were infected with Trypanosoma cervi, 20 of 24 deer had sarcocystis in skeletal muscles and two had dural lesions probably due to the nematode Pneumostrongylus tenuis. The causal relationship between choroid plexitis and trypanosomiasis is discussed.     

Surgical technique for tubal ligation in white-tailed deer (Odocoileus virginianus).

Surgical tubal ligation was used to sterilize urban free-ranging white-tailed deer (Odocoileus virginianus) as a methodology of a larger study investigating the influences of intact, sterile females on population dynamics and behavior. Deer were either trapped in clover traps (n = 55) and induced with an i.m. injection of xylazine and tiletamine/zolazepam or induced by a similar protocol by dart (n = 12), then intubated and maintained on isoflurane in oxygen. Over 3 yr, individual female deer (n = 103) were captured in Highland Park, Illinois, with a subset of females sterilized using tubal ligation by ventral laparotomy (n = 63). Other sterilization procedures included tubal transection by ventral (n = 1) or right lateral (n = 2) laparoscopy and ovariohysterectomy by ventral laparotomy (n = 1). One mortality (1/ 67, 1.5%) of a doe with an advanced pregnancy was attributed to a lengthy right lateral laparoscopic surgery that was converted to a right lateral laparotomy. The initial surgical modality of laparoscopy was altered in favor of a ventral laparotomy for simplification of the project and improved surgical access in late-term gravid does. Laparotomy techniques included oviductal ligation and transection (n = 14), application of an oviductal mechanical clip (n = 9), ligation and partial salpingectomy (n = 40), and ovariohysterectomy (n = 1). As of 2 yr poststerilization, no surgical does were observed with fawns, indicating that these procedures provide sterilization with low mortality in urban white-tailed deer.     

Estradiol and progesterone metabolite concentration in white-tailed deer (Odocoileus virginianus) feces.

Assays of reproductive hormone metabolites require validation in each animal species. For validation of methodology in white-tailed deer (Odocoileus virginianus), fecal samples were collected from females that had been injected by blowgun with estradiol, progesterone, or a control substance. Analysis by radioimmunoassay revealed that estradiol and pregnanediol were more abundant fecal metabolites of estrogens and progestins than were estrone or progesterone. Peak excretion rates of estradiol and pregnanediol occurred within 12 and 24 hr of injection, respectively. Ovulation time was estimated by measuring the frequency of occurrence of eight behavior patterns, including copulation. Profiles were compiled for three deer over the course of estrus and early pregnancy for estradiol, estrone, progesterone, and pregnanediol using radioimmunoassay. Pregnanediol was excreted at concentrations about 1,000 times higher than those of the other three fecal steroid metabolites, and pregnanediol differed in concentration during estrus, the luteal phase, and early pregnancy. Consequently, a simpler enzyme immunoassay was adapted and used to measure pregnanediol levels over the course of estrus and early pregnancy for seven deer. Measurement of fecal pregnanediol is useful for monitoring reproductive events in female white-tailed deer.     

Pharmacokinetics of imidocarb dipropionate in white-tailed deer (Odocoileus virginianus) after single intramuscular administration

This study was designed to investigate the pharmacokinetics of imidocarb, a carbanilide derivative, in white-tailed deer (Odocoileus virginianus). The pharmacokinetic properties of a single intramuscular (IM) dose of imidocarb were determined in 10 deer. A single IM injection of 3.0 mg/kg imidocarb dipropionate was administered, and blood samples were collected prior to, and up to 48 hr after imidocarb administration. Plasma imidocarb concentrations were determined by high-performance liquid chromatography with ultraviolet detection. The disposition of plasma imidocarb was best characterized by a two-compartment open model. The mean ± SE maximal imidocarb concentration in deer was 880.78 ± 81.12 ng/ml at 38.63 ± 5.30 min postinjection. The distribution phase had a half-life (t_1/2α) of 25.90 ± 10.21 min, and plasma imidocarb concentration declined with a terminal elimination half-life (t_1/2β) of 464.06 ± 104.08 min (7.73 ± 1.73 hr). Apparent volume of distribution based on the terminal phase (V_Z/F) was 9.20 ± 2.70 L/kg, and apparent total body clearance (Cl/F) was 15.97 ± 1.28 ml min⁻¹ kg⁻¹.     

Fatal pulmonary edema in white-tailed deer (Odocoileus virginianus) associated with adenovirus infection.

Sporadic sudden deaths in adult white-tailed deer occurred from November 1997 through August 1998 on an Iowa game farm. Three of the 4 deer necropsied had severe pulmonary edema, widespread mild lymphocytic vasculitis, and amphophilic intranuclear inclusion bodies in scattered endothelial cells in blood vessels in the lung and abdominal viscera. Immunohistochemistry with bovine adenovirus 5 antisera and transmission electron microscopy demonstrated adenoviral antigen and nucleocapsids, respectively, within endothelial cells. Adenovirus was isolated in cell culture from 1 of the affected deer. The isolate was neutralized by California black-tailed deer adenovirus antiserum. These findings indicate that adenovirus should be considered in the differential diagnosis of both black-tailed and white-tailed deer with pulmonary edema and/or hemorrhagic enteropathy.     

Seroprevalence of Neospora caninum and Toxoplasma gondii in black-tailed deer (Odocoileus hemionus columbianus) and mule deer (Odocoileus hemionus hemionus)

Deer are considered important intermediate hosts for the coccidian parasites, Toxoplasma gondii and Neospora caninum. Antibodies to N. caninum and T. gondii were determined in sera of 42 mule deer (Odocoileus hemionus hemionus) and 43 black-tailed deer (Odocoileus hemionus columbianus) from Washington state, USA, using direct agglutination test with specific antigens. A titer of 1:25 was considered diagnostic for both parasites. N. caninum antibodies were found in 7 of 42 mule deer and 8 of 43 black-tailed deer. T. gondii antibodies were found in 14 black-tailed deer but not in any of the mule deer. This is probably the first report of seroprevalence of N. caninum in these hosts.     

Isolation of Neospora caninum from the brain of a naturally infected adult dairy cow

Neospora caninum was isolated from the brain of a 2-year-old dairy cow that had aborted confirmed N. caninum-infected fetuses on two occasions. The cow had an indirect fluorescent antibody titer of 1:1600 to N. caninum. The cow was killed 24 days after its second abortion and the brain was bioassayed for N. caninum in nude mice. Multifocal areas of perivascular cuffing and glial nodules were observed in the cerebrum and mesencephalon of the cow, but N. caninum was not identified in histological sections of the brain. All three nude mice inoculated with brain homogenate of the cow, developed emaciation and paralysis. Microscopical examination of the nude mice revealed systemic N. caninum infection with demonstrable tachyzoites in various organs. The parasites isolated from fresh mouse brain were transferred successfully into Vero cell cultures. PCR procedure on the purified tachyzoites obtained from the Vero cell cultures amplified the specific DNA sequence for N. caninum.