Effect of Rift Valley fever virus on pregnant and non-pregnant local Barki ewes after artificial infection

Studies were conducted into effects of Rift Valley fever virus on oestrous and vaginal cytology in non-pregnant ewes. 6 pregnant and 8 non-pregnant local Barki ewes were subcutaneously inoculated with different doses of virus isolated from Sharkia Province. Observed were irregularities in the oestrous cycle (20-28 days inoculation) and anoestrum. The anoestrum smears included large non-cornified epithelial cells with large well stains centrally located nuclei together with a large number of bacteria. Abortion and retention of placenta occurred in pregnant ewes with prolonged puerperal heat up to 70 days. Inoculation of virus was followed by thermal response. Re-isolation of virus in BHK cells was possible from aborted foetuses and placenta.     

Bluetongue disease in Swiss sheep breeds: clinical signs after experimental infection with bluetongue virus serotype 8

Clinical disease of bluetongue (BT) in sheep may differ depending on breed, age and immunity of infected sheep and may also vary between serotype and strain of BT virus (BTV). Since there are no data available on the susceptibility of Swiss sheep breeds for BT, we performed experimental infection of the 4 most common Swiss sheep breeds and the highly susceptible Poll Dorset sheep with the BTV serotype 8 (BTV-8) circulating in Northern Europe since 2006. Clinical signs were assessed regarding severity, localisation, progression and time point of their appearance. The results clearly show that the Swiss sheep breeds investigated were susceptible to BTV-8 infection. They developed moderate, BT-characteristic symptoms, which were similar to those observed in Poll Dorset sheep. Regardless of breed, the majority of infected animals showed fever, swelling of the head as well as erosions of the mouth and subcutaneous haemorrhages.     

IgE responses to bluetongue virus (BTV) serotype 11 after immunization with inactivated BTV and challenge infection

To test the hypothesis that development of a BTV-specific IgE response plays a role in clinical disease manifestation, the humoral immune response of cattle to inactivated and virulent BTV was studied. Three calves received three sensitizing immunizations of inactivated BTV, 3 weeks apart. The BTV-sensitized animals, two non-sensitized BTV-seropositive and 4 BTV-seronegative control cattle. were challenge-exposed with BTV-11, UC8 strain. All cattle inoculated with inactivated BTV developed group-specific non-neutralizing and serotype-specific neutralizing antibodies. The development of post-challenge-exposure neutralizing antibody titers was inversely correlated with protective immunity. None of the BTV-challenged animals showed clinical disease. The levels of IgE were greatest in the sensitized calves after virus challenge in comparison with control groups. The sequential development, specificity and intensity of virus protein-specific humoral responses were evaluated using immunostaining. After challenge exposure of BTV-sensitized and non-sensitized cattle, total and IgE antibodies reacted consistently within BTV structural proteins VP2, VP5 and VP7. Although no correlation was found between clinical disease and IgE, results add support to the hypothesis that IgE may be involved in the pathogenesis of clinical disease, since infection with BTV causes an increase in serum IgE levels. However, these results suggest that the levels of virus-specific reactivity may be an important factor in determining whether or not clinical disease manifestation occurs.     

Failure to establish congenital bluetongue virus infection by infecting cows in early pregnancy.

Two groups of 10 pregnant cows were inoculated with bluetongue virus type 11 at either 40 or 60 days of gestation. All the cows became infected as judged by the detection of viraemia and seroconversion but they showed no clinical signs. Seventeen of the cows produced live calves none of which showed any evidence of prenatal infection. After challenge with the same virus all the calves became viraemic and seroconverted. The response to challenge of the two groups did not differ from that of a control group challenged at the same time. It was concluded that the infection of pregnant cows in early gestation with this virus did not result in the transplacental infection of the fetuses and did not produce immunotolerant, latently infected calves.     

Experimentally induced bluetongue virus infection in white-tailed deer: coagulation, clinical pathologic, and gross pathologic changes

Ten yearling white-tailed deer (Odocoileus virginianus) were inoculated with bluetongue virus serotype 17. Two yearling white-tailed deer were inoculated with sonicated heparinized noninfected blood and served as controls. Clinical signs of bluetongue virus infection included increased rectal temperature, erythema, facial edema, coronitis, and stomatitis. By postinoculation day (PID) 8, excessive bleeding and hematoma formation at venipuncture sites, dehydration, and diarrhea developed. At necropsy, the most consistent findings were oral lesions and widespread hemorrhage, which ranged from petechia to massive hematoma formation. Bluetongue virus caused progressive prolongation of activated partial thromboplastin time and prothrombin time, and progressive reduction of Factors VIII and XII plasma activities beginning on PID 6. A progressive decrease in platelet numbers also developed on PID 6. Changes in platelet size were not detected. Mean thrombin time was shortened, but prolongation developed in 1 deer. Mean fibrinogen concentration and Factor V plasma activity initially increased and then decreased, but remained above preinoculation values. Factor V activity was low in a few deer. Results of screening tests for inhibitors of the intrinsic coagulation system were positive in 2 deer. High concentrations of fibrin(ogen) degradation products were first detected between PID 3 and 6. Hematologic changes included leukopenia, lymphopenia, neutrophilia, and low total plasma protein concentration. Differences in PCV, hemoglobin concentration, or RBC counts were not detected between infected and control deer. Serum total bilirubin concentration increased by PID 6, primarily because of increased unconjugated bilirubin concentration. Mild to severe increases in serum aspartate transaminase activity were accompanied by more marked increases in creatine kinase activity. Indirect Coombs test results were negative in all deer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Semen changes in boars after experimental infection with pseudorabies virus

Two groups of adult boars were inoculated with a field strain of pseudorabies virus (PRV) by intranasal droplet; one group was given 5 x 10(5) median tissue culture infective doses (TCID50), and the other, 5 x 10(6) TCID50. (A third group was maintained as controls.) Ejaculates were examined twice a week for volume, sperm numbers, sperm morphology, and presence of PRV. Severe clinical disease with fever followed administration of the larger virus dose. Death (one boar), testicular degeneration, and transient elevation in spermatozoa with proximal cytoplasmic droplets were seen in different members of this group. The smaller dose resulted in seroconversion, but did not produce signs of disease. In this group, volume, sperm numbers, and sperm morphology did not decline when compared with base-line values or data of control animals. The virus was not isolated from semen. Effects of PRV infection on semen quality in boars seem to be related to the associated clinical signs of systemic disease.     

Transplacental and oral transmission of wild-type bluetongue virus serotype 8 in cattle after experimental infection

Potential vertical transmission of wild-type bluetongue virus serotype 8 (BTV-8) in cattle was explored in this experiment. We demonstrated transplacental transmission of wild-type BTV-8 in one calf and oral infection with BTV-8 in another calf. Following the experimental BTV-8 infection of seven out of fifteen multi-parous cows eight months in gestation, each newborn calf was tested prior to colostrum intake for transplacental transmission of BTV by RRT-PCR. If transplacental transmission was not established the calves were fed colostrum from infected dams or colostrum from non-infected dams spiked with BTV-8 containing blood. One calf from an infected dam was born RRT-PCR positive and BTV-specific antibody (Abs) negative, BTV was isolated from its blood. It was born with clinical signs resembling bluetongue and lived for two days. Its post-mortem tissue suspensions were RRT-PCR positive. Of the seven calves fed colostrum from infected dams, none became infected. Of the six calves fed colostrum from non-infected dams spiked with infected blood, one calf became PCR-positive at day 8 post-partum (dpp), seroconverted 27 days later, and remained RRT-PCR and Abs positive for the duration of the experiment (i.e., 70 dpp). This work demonstrates that transplacental transmission in late gestation and oral infection of the neonate with wild-type BTV-8 is possible in cattle under experimental conditions.     

Experimentally induced bluetongue virus infection in white-tailed deer: ultrastructural findings

White-tailed deer (Odocoileus virginianus) were inoculated with bluetongue virus serotype 17 and sequentially euthanatized during infection. Ultrastructural changes in the microvasculature of tongue, buccal mucosa, heart, and pulmonary artery, platelets, and bone marrow were evaluated. Bluetongue virus was found in endothelial cells of the microvasculature by postinoculation day 4. Viral replication was associated with the development of viral matrices, viral-associated macrotubules, and aggregates of mature viral particles in the cytoplasm of infected cells. Viral infection of pericytes and vascular smooth muscle cells developed subsequent to endothelial cell infection. Viral infection was associated with striking changes in the endothelial lining of the microvasculature by postinoculation day 4. Endothelial cell degeneration and necrosis, which resulted in denudation of the endothelial lining, and endothelial cell hypertrophy frequently were observed. Thrombosis, hemorrhage, and vessel rupture developed subsequent to endothelial damage. Bluetongue virus neither infected nor directly damaged platelets or bone marrow cells. It was concluded that viral-induced endothelial damage is the primary triggering mechanism for disseminated intravascular coagulation in bluetongue virus infection. Vascular damage coupled with the development of disseminated intravascular coagulation is responsible for the hemorrhagic diathesis, which is characteristic of bluetongue virus infection in white-tailed deer.     

Susceptibility of in vitro produced hatched bovine blastocysts to infection with bluetongue virus serotype 8

ABSTRACT: Bluetongue virus serotype 8 (BTV-8), which caused an epidemic in ruminants in central Western Europe in 2006 and 2007, seems to differ from other bluetongue serotypes in that it can spread transplacentally and has been associated with an increased incidence of abortion and other reproductive problems. For these reasons, and also because BTV-8 is threatening to spread to other parts of the world, there is a need for more information on the consequences of infection during pregnancy. The aim of the present study was to investigate whether hatched (i.e. zona pellucida-free) in vitro produced bovine blastocysts at 8-9 days post insemination are susceptible to BTV-8 and whether such infection induces cell death as indicated by apoptosis. Exposure of hatched in vitro produced bovine blastocysts for 1 h to a medium containing 103.8 or 104.9 TCID50 of the virus resulted in active viral replication in between 25 and 100% of the cells at 72 h post exposure. The infected blastocysts also showed growth arrest as evidenced by lower total cell numbers and a significant level of cellular apoptosis. We conclude from this in vitro study that some of the reproductive problems that are reported when cattle herds are infected with BTV-8 may be attributed to direct infection of blastocysts and other early-stage embryos in utero.     

The effect of experimental infection with Corynebacterium pseudotuberculosis on reproduction in adult ewes

Fifteen ewes were inoculated subcutaneously with 2 x 10(6) cells of Corynebacterium pseudotuberculosis seven days before mating, during the embryonic stage of gestation and during the fetal stage of gestation. The clinical signs, antibody response and the consequences of the infection on reproduction were studied. None of the ewes showed any change in general condition during pregnancy. The effects of experimental infection during gestation were evident after incubation periods that ranged from 25 to 140 days after inoculation and were dependent on the time of the inoculation in relation to the period of gestation. Ewes inoculated before gestation can resolve infection. Ewes inoculated during the embryonic stage were severely affected and some aborted. In others lambs were stillborn or, if born alive, infected. Ewes inoculated during the fetal stage of gestation did not show reproductive disorders although some remained chronically infected.     

Changes in the concentration of mRNAs for the inhibin subunits in ovarian follicles after administration of gonadotropins to progestin treated ewes.

RNA was extracted from single or small groups of ovine ovarian follicles after treatment of ewes with FSH and/or LH. The content of mRNA for the alpha-inhibin and beta A-inhibin subunits was analyzed by hybridization with specific cDNA probes. All ewes were treated with progestin vaginal pessaries to suppress spontaneous preovulatory follicle maturation and ewes were given three intramuscular injections of gonadotropins at 8-hr intervals starting 24 hr prior to collection of ovaries. In experiment I, both Schering-FSH and NIDDK-oFSH-17 (oFSH) significantly increased alpha- and beta A-inhibin mRNA per ewe in 2-5 mm follicles and tended to increase alpha- and beta A-inhibin mRNA in large (greater than 5 mm) follicles. In experiment II, oFSH and NIDDK-oLH-25 (oLH) were administered in a 2X2 factorial arrangement. Separate administration of oFSH or oLH increased (P less than .05) the alpha-inhibin mRNA concentration in large follicles. alpha-inhibin mRNA concentration in 4-5 mm follicles was also increased by oFSH but was decreased by oLH. Concomitant treatment with oFSH and oLH did not change alpha-inhibin mRNA concentrations from those measured in oFSH treated ewes. In experiment II, beta A mRNA concentrations followed a pattern similar to that of alpha A mRNA, but the differences were not statistically significant. We conclude that, in the ewe, exogenous FSH increases the concentration of inhibin mRNA in the whole follicle. The ability of exogenous oLH to alter expression of the inhibin subunit genes may depend upon the stage of follicle maturation.     

日粮不同能量和蛋白水平对多浪羊繁殖性能及血液生化指标的影响

试验于2009年3月至2010年3月在喀什地区麦盖提县多浪羊繁育基地进行,研究日粮不同能量和蛋白水平对多浪羊繁殖性能及血液生化指标的影响。90只母羊分为第Ⅰ组、第Ⅱ组和第Ⅲ组共3个组进行试验,3组均饲喂不同能量和蛋白水平的日粮,分别计算春季和秋季3个组的繁殖性能指标值,并检测血清中TP、ALB、GLB、ALP、BUN、GLU、TG和LDH的含量。结果发现,提高日粮能量水平对多浪羊春季发情率的提高有明显效果,但日粮高能量水平对受胎率和双羔率的提高均无效果;高蛋白水平日粮对多浪羊春季与秋季的发情率、受胎率和双羔率的提高没有明显效果;多浪羊春季血清中BUN含量高于秋季,秋季血清中ALP含量高于春季。从血液生化指标分析可以看出,秋季的繁殖性能比春季好。     

Dot immunobinding assay for the detection of bluetongue virus antibodies in sheep experimentally inoculated with bluetongue virus type 1.

Dot immunobinding assay (DIA) was evaluated for the detection of bluetongue virus (BTV) antibodies in sheep experimentally inoculated with BTV 1. Serum samples collected on 14, 21, 28, 43 and 60 day post infection (dpi) were positive for precipitating antibodies by the agar gel precipitation test (AGPT) while antibodies could be detected as early as 7 dpi by DIA and ELISA. Virus neutralizing antibodies were detected first at 14 dpi. The sensitivity of the four tests was compared on the same serum samples collected at different intervals. The results indicated that DIA was more sensitive than AGPT and the serum neutralization test and as sensitive as ELISA. Thus due to sensitivity simplicity and economy, DIA could replace AGPT for diagnosis and serological survey for BTV infection in animals.     

Detection of bluetongue virus RNA by in situ hybridization: comparison with virus isolation and antigen detection.

An in situ nucleic acid hybridization (ISH) technique was developed to detect bluetongue virus (BTV) RNA in cell culture. The sensitivity of the ISH technique was compared with virus isolation (VI) and antigen detection, using an indirect fluorescent-antibody (IFA) or an enzyme immunocytoassay (EICA) technique, for detection of 5 BTV serotypes indigenous to the United States. The VI was the most sensitive technique, detecting BTV early after infection of the cells. The IFA and EICA were of similar sensitivity; BTV antigen could be detected shortly after demonstration of virus by isolation. The sensitivity of ISH for detection of BTV-17 was equivalent to that of antigen detection. The ISH was not as sensitive as VI or antigen detection when assaying for the other BTV serotypes.     

Serologic and virologic evidence of bluetongue virus infection in cattle and sheep in Mexico

Three independent 1-year studies were conducted during 3 consecutive years to better define the prevalence of bluetongue virus (BTV) infection in Mexico. Serologic data were obtained by use of agar-gel immunodiffusion for identification of BTV group-reactive antibodies, and virologic data were obtained by virus isolation. Samples were obtained from sheep in 6 states over a 1-year period, with 9% seropositive; samples were obtained from cattle in 11 states during the same 1-year period, with 35% seropositive. Two years later, samples were obtained from cattle in 4 additional states, with 69% seropositive. Virus isolation was conducted on pooled blood samples obtained from cattle in 7 states. Six virus isolates were recovered and included 2 isolates each of BTV serotypes 11 and 13 and 1 isolate each of serotypes 10 and 17. All virus isolates were partially characterized by electrophoretic analysis of genomic RNA migration profiles (electropherotypes) in polyacrylamide gels. All Mexican isolates of BTV differed considerably in electropherotype profile, as compared with their respective US prototype strain of the same serotype. Such differences appeared to be much more extensive than those described to exist between numerous California isolates of the same serotype.