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.     

牛病毒性腹泻病毒BVDV-JL株的分离与鉴定

本研究从吉林某牛场表现严重腹泻症状濒死牛的胸腺病料样品中分离一株病毒,该病毒在MDBK细胞中盲传4代无细胞病变产生,而通过RT-PCR和间接免疫荧光试验、微量血清中和试验检测表明该分离病毒株为牛病毒性腹泻病毒(BVDV),并命名为BVDV-JL.将BVDV-JL株F4代细胞培养液(10<'7.13>TCID<,50>/...     

Effects of incubation temperatures and bovine viral diarrhea virus immune serum on bovine turbinate cells persistently infected with a noncytopathogenic isolate of bovine viral diarrhea virus

Noncytopathogenic bovine viral diarrhea virus (BVDV) on initial inoculation in receptive cells induced a persistence in vitro at incubation temperatures of 33, 37, and 39 C. The mechanisms of this persistence were not the result of the induction of defective interfering particles or the selection of temperature-sensitive mutants, but were the result of a naturally occurring noncytopathogenic isolate of BVDV. Persistently infected cells were not freed of the infection by continuous passage in media containing homologous antibody. Persistently infected cells appeared to undergo an earlier senescence than did noninoculated cells of the same passage level and age. This phenomenon was reversed by renewal of the culture media.     

Clinical response and immunomodulation following experimental challenge of calves with type 2 noncytopathogenic bovine viral diarrhea virus

Eight calves between 16 and 18 weeks of age that were seronegative to bovine viral diarrhea virus (BVDV), bovine leucosis virus and bovine immunodeficiency-like virus were infected (day 0) intranasally with the type 2 noncytopathogenic Canadian 24515 field isolate of BVDV in order to evaluate the effect of BVDV infection on certain clinical, hematological and immunological parameters. All virus-exposed animals developed fever and showed a significant (P < 0.05, 0.01 or 0.001) drop in the number of circulating leucocytes (neutrophils, lymphocytes and monocytes) by day 3 or 5 post-exposure (PE), which continued to the end of the experiment at day 12 PE. BVDV was consistently isolated from the peripheral blood buffy coat cells from day 5 PE, and also from selected tissues (spleen, thymus, mesenteric and submaxillary lymph nodes, small intestine, lungs and thyroid gland) that were collected at the time of euthanasia of the animals at day 12 PE. Diminished significant (P < 0.05) percentages of peripheral blood mononuclear cells (PBMCs) expressing at their surface either B7 and MHC II molecules were observed in virus-exposed calves at days 7, 10 and/or 12 PE, when compared to virus-nonexposed control calves (n = 5). However, no changes in the percentages of PBMCs expressing either B4 or MHC I molecules were observed throughout the experiment. Finally, a significant (P < 0.05 or 0.01) enhanced phagocytic capability of the PBMCs, as analyzed by flow cytometry, was observed in virus-exposed animals at days 3, 5, 7, 10 and 12 PE, when compared to control calves. These results demonstrated the virulence of the 24515 isolate of BVDV in 4 to 4.5 month-old calves, and suggest that type 2 BVDV infection in calves is associated with dysregulation of certain immunological functions.     

Adherence of bovine viral diarrhea virus to bovine oocytes and embryos with a hardened zona pellucida cultured in vitro

The purpose of this study was to investigate the adherence of bovine viral diarrhea virus (BVDV) to bovine mature, or immature, cumulus-free oocytes and to in vitro fertilized embryos, maintained in vitro in a ligated bovine oviduct to allow for the hardening of the zona pellucida. Incubation of the oocytes and embryos in the oviduct for 5 h caused hardening of the zona pellucida as measured by resistance to pronase digestion (which increased from approximately 3 min to 7 h; P >0.001). However, there was no difference between the number of infected oocytes and embryos (n = 965 in 193 samples) following experimental exposure to BVDV regardless of whether or not they were previously incubated in the oviduct (P > 0.05). It was concluded that the modification of the proteolytic resistance properties of the zona pellucida during in vitro oviductal incubation did not influence the adherence of BVDV to zona pellucida of oocytes or in vitro fertilized embryos.     

Reproductive consequences of infection with bovine viral diarrhea virus.

Reproductive efficiency is imperative for the maintenance of profitability in both dairy and cow-calf enterprises. Bovine viral diarrhea virus is an important infectious disease agent of cattle that can potentially have a negative effect on all phases of reproduction. Reduced conception rates,early embryonic deaths, abortions, congenital defects, and weak calves have all been associated BVDV infection of susceptible females. In addition, the birth of calves PI with BVDV as a result of in utero fetal exposure is extremely important in the perpetuation of the virus in an infected herd or spread to other susceptible herds. Bulls acutely or PI with BVDV may bea source of viral spread through either natural service or semen used in artificial insemination. Management practices including elimination of PI cattle, biosecurity measures and strategic use of vaccination can be implemented to reduce the risk of BVDV related reproductive losses.Development of vaccines and vaccine strategies capable of providing better protection against fetal infection would be of benefit.     

Characteristics of the polyriboinosinic acid:polyribocytidylic acid assay for noncytopathogenic bovine viral diarrhea virus

The use of polyriboinosinic acid:polyribocytidylic acid (poly I:C) for noncytopathogenic bovine viral diarrhea virus (NC-BVDV) assay (PINBA) was studied. Several viruses were tested for their suitability as test challenge viruses. In addition to vesicular stomatitis virus, which previously was shown to be a suitable challenge virus, bovine enteroviruses also were found to be suitable, whereas infectious bovine rhinotracheitis virus and parainfluenza type 3 virus were only marginally suitable. Bovine embryonic skin (BES) cultures developed resistance to PINBA within a few in vitro passages, whereas bovine embryonic lung (BEL) cultures did not. At certain passages, BES cultures were 500,000 times more resistant than BEL cultures. To determine whether the difference in viral titers on BEL and BES cultures was due to NC-BVDV replication, PINBA and fluorescent antibody assays were compared on the cultures. Resistance of BES cultures to PINBA was not due to an inability of the virus to replicate in the cultures, but was due to an inability of PINBA to detect the virus. Viral titers were comparable by fluorescent antibody assay titers on BES and BEL cultures, but were considerably higher than viral titers on BES cultures with PINBA. Variations in viral titers, using PINBA on BEL cultures, were observed and were considered to be due to cultural conditions, such as the presence of low levels of BVDV antibodies in bovine fetal serum used in the medium. Treatment of BEL cultures with poly I:C or interferon showed that NC-BVDV was sensitive to interferon as determined by virus-yield reduction.     

Effect in vitro of bovine viral diarrhea virus on bovine embryos with the zona pellucida intact,damaged and removed

The in vitro effect of bovine viral diarrhea virus (BVDV) on the survival of day 7 to day 7.5 bovine embryos collected from superovulated donors was studied. Fifty-four experimental embryos with the zona pellucida (ZP) intact, damaged or removed were exposed to 1×10⁴ TCD₅₀/ml of the NADL cytopathic strain of BVDC at 37°C for 24 hrs and compared to 36 control embryos that were cultured for 24 hr. Seven embryos with the ZP-removed were similarly exposed for 48 hrs and compared to five control embryos. The overall survival rate was 68% for embryos exposed to BVDV for 24 hrs and 77% for embryos not exposed (P>0.05). Extended exposure of the embryos with the ZP removed to virus for 48 hrs did not affect their survival rate compared to controls. Damage to the ZP by cracking or total removal of the ZP by micromanipulation or acidic Tyrode's solution had no effect on subsequent embryonic survival in the presence of BVDV. It was concluded that exposure to BVDV in vitro is not cytopathic for morula and blastocyst stage bovine embryos over a 48 hr period, even when they are not protected by the ZP.     

Evaluation of bovine viral diarrhea virus uptake by preimplantation embryos

Bovine embryos were exposed to bovine viral diarrhea (BVD) virus in vitro. An uptake of BVD virus by the embryos could not be detected by several assay systems. A significant decrease in the titer of BVD virus was found to occur when the virus was incubated in saline solution + 5% goat serum or minimal essential medium + 5% goat serum for 24 hours at 37 C. Since there was significant inactivation of the BVD virus during the incubation period, lack of viral infectivity of the embryos may have been due to adverse effects of the experimental environmental conditions on the virus or the embryos or upon viral-embryo interaction.     

Response of cattle persistently infected with noncytopathic bovine viral diarrhea virus to vaccination for bovine viral diarrhea and to subsequent challenge exposure with cytopathic bovine viral diarrhea virus

Nine steers persistently infected with noncytopathic bovine viral diarrhea (BVD) virus were allotted into 3 groups (3 cattle/group). Cattle in group A were vaccinated with a modified-live BVD virus vaccine of porcine cell origin, cattle in group B with a modified-live BVD virus vaccine of bovine cell origin, and cattle in group C with a killed BVD virus vaccine of bovine cell origin. Detrimental effects due to vaccination were not seen. Six weeks after vaccination, the steers were challenge exposed with a cytopathic BVD virus. All steers developed mucosal disease after challenge exposure, produced antibodies that neutralized various isolates of BVD virus, and remained persistently infected until death. Steers given killed virus vaccine had a minimal neutralizing-antibody response and developed mucosal disease as quickly as reported for challenge-exposed, nonvaccinated, persistently infected cattle. Steers given modified-live virus vaccines had higher neutralizing-antibody response and longer intervals from challenge exposure to development of mucosal disease. The specificity of the neutralizing-antibody response differed between groups of vaccinated cattle.     

Experimental persistent infection with bovine viral diarrhea virus in white-tailed deer

Bovine viral diarrhea virus (BVDV) infections cause substantial economic losses to the cattle industries. Persistently infected (PI) cattle are the most important reservoir for BVDV. White-tailed deer (Odocoileus virginianus) are the most abundant species of wild ruminants in the United States and contact between cattle and deer is common. If the outcome of fetal infection of white-tailed deer is similar to cattle, PI white-tailed deer may pose a threat to BVDV control programs. The objective of this study was to determine if experimental infection of pregnant white-tailed deer with BVDV would result in the birth of PI offspring. Nine female and one male white-tailed deer were captured and housed at a captive deer isolation facility. After natural mating had occurred, all does were inoculated intranasally at approximately 50 days of pregnancy with 10(6) CCID(50) each of a BVDV 1 (BJ) and BVDV 2 (PA131) strain. Although no clinical signs of BVDV infection were observed or abortions detected, only one pregnancy advanced to term. On day 167 post-inoculation, one doe delivered a live fawn and a mummified fetus. The fawn was translocated to an isolation facility to be hand-raised. The fawn was determined to be PI with BVDV 2 by serial virus isolation from serum and white blood cells, immunohistochemistry on skin biopsy, and RT-PCR. This is the first report of persistent infection of white-tailed deer with BVDV. Further research is needed to assess the impact of PI white-tailed deer on BVDV control programs in cattle.     

Experimental fetal infection with bovine viral diarrhea virus. II. Morphological reactions and distribution of viral antigen.

The effect of an infection with bovine viral diarrhea virus on fetal bovine tissues as well as the tissue-localization of viral antigen are described. Four bovine fetuses, 120-165 days of gestation, were inoculated in utero with a second passage virus strain. Lymphoid tissues were studied by light and electron microscopy. The infection induced precocious development of the secondary lymphoid organs. Characteristic changes were seen in postcapillary venules, cells of the mononuclear phagocyte system and lymphoid follicles. In all four fetuses the thymus was hypoplastic. In three fetuses this implicated a morphological immaturity, but no actual pathological alterations. In the fourth fetus, the hypoplasia was caused by necrosis and depletion of lymphocytes, attended by infiltration of macrophages. Histopathological changes were also noted in the cerebellum of three fetuses, consisting of necrosis in and depletion of the external germ layer and in the skin and mucous membranes of all four fetuses. The viral antigen was present in cells of the mononuclear phagocyte system, primarily in the lymphoid tissues. The infection was further demonstrated in the affected cerebelli.     

Experimental fetal infection with bovine viral diarrhea virus. I. Virological and serological studies.

The serological and virological results of an experimental infection of bovine fetuses with bovine viral diarrhea virus are presented. Four fetuses, 120-165 days gestational age, were inoculated in utero with a second passage virus strain. Two fetuses received a sham-inoculum. A humoral immune response in the virus-inoculated fetuses, was demonstrated three weeks later. In three fetuses only IgM and IgG1 were detectable. The serum from the fourth fetus also contained IgG2 and IgA. Bovine viral diarrhea virus-neutralizing antibodies were detected in two fetuses. These two fetuses inoculated at 135-150 days gestational age, represent the youngest reported bovids, giving a specific response in three weeks following an experimental infection with bovine viral diarrhea virus. The fetal sera did not contain heat-labile factors, which could mediate the neutralization. The virus was not reisolated from any of the fetuses, but viral antigen was nevertheless demonstrated by immunocytochemical methods in sections of several of the fetal organs, primarily lymphoid tissues.     

Diagnosis of bovine viral diarrhea virus infection using monoclonal antibodies

The monoclonal antibody (MAb) D89 against bovine viral diarrhea virus (BVDV) was used in conjunction with fluorescein-conjugated anti-mouse immunoglobulin in an indirect fluorescent antibody (IFA) procedure on frozen tissue sections and cell culture. During the 2-year study, BVDV was isolated from specimens submitted in 460 cases. The D89 Mab detected all but 2 BVDV isolates, both cytopathic. In 316 of the cases in which BVD virus was detected by IFA, specimens were inoculated on bovine turbinate cells and examined for BVDV antigens at 3-5, 10, and 20 days postinoculation. The BVDV was detected in 238/316 cases (75%) after 3-5 days incubation. The remainder were not detected until 10 or 20 days postinoculation. Virus isolation was enhanced in the early test if plates were centrifuged at the time of inoculation. Results suggest that D89 monoclonal antibody is a suitable diagnostic reagent for the detection of BVDV isolated from diagnostic specimens. The D89 MAb can be used for the detection of BVDV in both cell culture and tissues. Combination of D89 with another BVDV MAb (C17) did not improve the ability to detect BVDV in tissues compared to using D89 only, and the combined Mab's resulted in an increase in nonspecific fluorescence when used on tissues. Although pooling of different BVDV monoclonal antibodies may be necessary to detect all strains of BVDV in cell culture, pooling should be used with caution on tissues. Early detection of BVDV in cell culture by this IFA procedure permits faster confirmation of BVDV diagnosis when compared to the usual routine testing for noncytopathic BVDV at termination of first passage in cell culture.     

Production of cattle immunotolerant to bovine viral diarrhea virus.

Inoculation of bovine virus diarrhea virus into 58 to 125 day old fetuses of bovine virus diarrhea virus seropositive pregnant cows, or inoculation of bovine virus diarrhea virus into seronegative cows 42 to 114 days pregnant, may produce clinically normal calves which are persistently infected with the specific isolate of bovine virus diarrhea virus yet seronegative to the homologous and heterologous isolates. Reinoculation of these persistently infected cattle with their homologous isolate produced no neutralizing antibody response to bovine virus diarrhea virus. These persistently infected cattle were immunocompetent as they developed neutralizing serotiters to infectious bovine rhinotracheitis, parainfluenza-3 viruses and agglutinating serotiters to Pasteurella hemolytica .     

Uterine tubal cells remain uninfected after culture with in vitro-produced embryos exposed to bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) has been isolated from washed and sonicated, in vitro-produced embryos, but the infectivity of BVDV associated with intact, developing, embryos has not been demonstrated. The objective of this study was to determine if a dose of BVDV infective for co-culture cells was associated with individual, developing embryos, following artificial exposure to the virus and washing. In 5 replicates, zona pellucida-intact, in vitro-produced embryos were assigned to a negative control embryo group, or were incubated in 10(5)-10(6) cell culture infective doses (50%, CCID50) per milliliter of a type I, noncytopathic (strain SD-1) BVDV for 2 h. Unexposed negative control embryos and exposed positive control embryos were washed, sonicated and assayed for BVDV using virus isolation with immunoperoxidase monolayer assay. Immediately or following cryopreservation, remaining virally-exposed, washed embryos were co-cultured individually with BVDV-negative cultures of bovine uterine tubal cells in a medium free of BVDV-neutralizing activity. After two days in culture, uterine tubal cells and embryos (including the zona pellucida) were separated and washed. The culture medium, uterine tubal cells and embryos were then assayed for BVDV. Bovine viral diarrhea virus was not isolated from any negative control embryo group, but was isolated from all positive control embryo groups. Although all uterine tubal cell populations were confirmed to be susceptible to BVDV, virus was never isolated from uterine tubal cells or embryos from post-exposure culture. In conclusion, although BVDV remains associated with washed in vitro-produced embryos, the virus associated with unsonicated embryos was not infective for uterine tubal cells in vitro.     

Pathogenetic studies of infection of the bovine fetus with bovine viral diarrhea virus. II. Ocular lesions.

Twenty-three susceptible pregnant heifers were inoculated with bovine viral diarrhea virus at 150 +/- 1 days of gestation. Seven additional heifers were inoculated between 65 and 115 days of gestation. Acute ocular lesions were seen in fetuses taken 17-21 days after inoculation of the dams at 150 days. By the fourth week, the acute lesions were beginning to resolve, and in newborn animals focal to total retinal atrophy was seen. The acute lesions were characterized by a mild to moderate retinitis that resulted in various degrees of destruction of the different layers, mononuclear cuffing of inner retinal vessels, proliferation of pigment epithelium, and choroiditis. Residually there was an absence of cellular elements in the atrophied areas of the retina, frequently a loss of layering and various numbers of pigment-containing cells. Moderately severe acute inflammation was seen in the retina of the fetus taken at 22 days after inoculation of its dam at 95 days. Ocular lesions did not occur in the other fetuses taken from heifers inoculated at earlier stages of gestation.