Detection of bovine viral diarrhea virus (BVDV) in single or small groups of preimplantation bovine embryos (original) (raw)
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Quality controls for bovine viral diarrhea virus-free IVF embryos
Theriogenology, 1999
Introduction of bovine viral diarrhea virus (BVDV) with cumulus-oocyte-complexes (COCs) from the abattoir is a concern in the production of bovine embryos in vitro. Further, International Embryo Transfer Society (IETS) guidelines for washing and trypsin treatment of invivo-derived bovine embryos ensure freedom from a variety of pathogens, but these procedures appear to be less effective when applied to IVF embryos. In this study, COCs were exposed to virus prior to IVM, IVF and IVC. Then, virus isolations from cumulus cells and washed or trypsin-treated nonfertile and degenerated ova were evaluated as quality controls for IVF embryo production. The effect of BVDV on rates of cleavage and development was also examined. All media were analyzed prior to the study for anti-BVDV antibody. Two approximately equal groups of COCs from abattoir-origin ovaries were washed and incubated for 1 h in minimum essential medium (MEM) with 10% equine serum. One group was incubated in 10' cell culture infective doses (50% endpoint) of BVDV for 1 h, while the other was incubated without virus. Subsequently, the groups were processed separately with cumulus cells, which were present throughout IVM, IVF and IVC. Cleavage was evaluated at 4 d and development to morulae and blastocysts at 7 d of IVC. After IVC, groups of nonfertile and degenerated ova or morulae and blastocysts were washed or trypsin-treated, sonicated and assayed for virus. Cumulus cells collected at 4 and 7 d were also assayed for virus. Anti-BVDV antibody was found in serum used in IVM and IVC but not in other media. A total of 1,656 unexposed COCs was used to produce 1,284 cleaved embryos (78%), 960 embryos >5 cells (SS%), and 194 morulae and blastocysts (12%). A total of 1,820 virus-exposed COCs was used to produce 1,350 cleaved embryos (74%) 987 embryos >5 cells (54%), and 161 morulae and blastocysts (9%). Rates of cleavage (P=O.O21), cleavage to 15 cells (P=O.O26) and development to morula and blastocyst (P=O.OOS) were lower in the virus-exposed group (Chi-square test for heterogeneity). No virus was isolated from any samples from the unexposed group. For the exposed group, virus was always isolated from 4-and 7-d cumulus cells, from all washed nonfertile and degenerated ova (n=40) and morulae and blastocysts (n=57) and from all trypsin-treated nonfertile and degenerated ova (n=SO) and morulae and blastocysts (n=91). Thus, virus persisted in the system despite the presence of neutralizing antibody in IVM and IVC media, and both washing and trypsin treatment were ineffective for removal of the virus. Presence of virus in 4-and 7-d cumulus cells as well as in nonfertile and degenerated ova were good indicators of virus being associated with morulae and blastocysts.
Brazilian Journal of Microbiology, 2020
Detection of bovine viral diarrhea virus (BVDV) in aborted fetus samples is often difficult due to tissue autolysis and inappropriate sampling. Studies assessing different methods for BVDV identification in fetal specimens are scarce. The present study evaluated the agreement between different diagnostic techniques to detect BVDV infections in specimens from a large number of bovine aborted fetuses and neonatal deaths over a period of 22 years. Additionally, genetic, serological, and pathological analyses were conducted in order to characterize BVDV strains of fetal origin. Samples from 95 selected cases from 1997 to 2018 were analyzed by antigen-capture ELISA (AgELISA), nested RT-PCR (RT-nPCR), and real-time RT-PCR (RT-qPCR). In addition, amplification and sequencing of the 5′UTR region were performed for phylogenetic purposes. Virus neutralization tests against the BVDV-1a, BVDV-1b, and BVDV-2b subtypes were conducted on 60 fetal fluids of the selected cases. Furthermore, the frequency and severity of histopathological lesions were evaluated in BVDV-positive cases. This study demonstrated that RT-nPCR and RT-qPCR were more suitable than AgELISA for BVDV detection in fetal specimens. However, the agreement between the two RT-PCR methods was moderate. The BVDV-1b subtype was more frequently detected than the BVDV-1a and BVDV-2b subtypes. Neutralizing antibodies to any of the three subtypes evaluated were present in 94% of the fetal fluids. Microscopically, half of the BVDV-positive cases showed a mild non-suppurative inflammatory response. These results emphasize the need to consider different methods for a diagnostic approach of BVDV associated to reproductive losses.
Theriogenology, 2017
As production of in vitro (IVP) bovine embryos steadily increases, the sanitary risk associated with IVP embryos remains a concern. One of the greatest concerns is how BVDV may be transmitted through IVP embryos. The objective of this study was to evaluate the effects caused by BVDV-1, BVDV-2 and Hobi-like virus exposure during in vitro maturation on embryo development and viral infection. Abittior-derived oocytes were randomly assigned for in vitro maturation with serial concentrations of BVDV-1 (3.12 Â 10 2-2.50 Â 10 3 TCID 50 /100 mL), BVDV-2 (6.25 Â 10 1-5.20 Â 10 2 TCID 50 /100 mL) or Hobi-like virus (1.90 Â 10 2-1.58 Â 10 3 TCID 50 /100 mL) for 22e24 h. After maturation, oocytes were fertilized and embryo cultured following standard in vitro procedures. Embryo development was evaluated and percentage of respective, positive BVDV degenerated and viable embryos were evaluated by RT-qPCR. No concentration of BVDV-1 altered embryo development as measured by cleavage and blastocyst rates, compared to negative control group. However 100% of degenerated embryos and 50e100% of viable embryos tested positive for BVDV-1, depending on the viral concentration. BVDV-2 exposed oocytes had higher cleavage rates than the negative control group (60.2e64.1% vs 49.8%; P ¼ 0.003e0.032). However, no difference was detected for blastocyst rates. In aadition, 100% of degenerated embryos and 20e50% of viable embryos tested positive for BVDV-2. Hobi-like virus treated oocytes had reduced cleavage rates for the three highest viral concentrations (33.3e38.0% vs 49.8% for negative controls; P 0.001e0.014). Blastocyst rates were only reduced in the 7.9 Â 10 2 Hobi-like virus concentration (6.9 ± 0.9% vs 15.1 ± 1.6%; P ¼ 0.009), when calculated by oocyte number. 50e80% of degenerated embryos tested positive for Hobilike virus. No viable embryos from the Hobi-like virus treated oocytes tested positive. These results suggest that IVP embryos from BVDV-1 and-2 infected oocytes develop normally, but carry the virus. However, Hobi-like virus infected oocytes had reduced cleavage and cause pre-implantation embryo loss, but viable embryos did not carry the virus.
The Southeast Asian journal of tropical medicine and public health, 2006
The effect of infection with teratogenic viruses at early stages of pregnancy is not fully understood. This study aimed to look at the effect of infection with teratogenic viruses such as bovine viral diarrhea virus (BVDV) and border disease virus (BDV), on early stage embryos at the hatched blastocyst stage. BVDV and BDV are known to cross the placenta of infected mothers and lead to congenital defects and death of developing fetuses. This study can be a good model for better understanding the effects of other teratogenic viruses such as Rubella virus in humans.
Reproduction, 1995
Large-scale in vitro bovine embryo production systems commonly use genital tracts obtained from an abattoir as a source of both cumulus\p=n-\oocytecomplexes and co-culture feeder cells. Tissues derived from this source may be contaminated with non-cytopathogenic bovine viral diarrhoea virus (BVDV) since, in several countries surveyed, approximately 1% of animals tested are persistently infected with this pathogen. Therefore, the use of such material in in vitro fertilization systems presents a potential risk for the transmission of BVDV to bovine embryos and via embryo transfer. This potential was investigated by obtaining oviduct epithelial cells and granulosa cells, which are commonly used as feeder cells, from cattle persistently infected with BVDV and examining them for the presence of BVD viral antigen (p80 non-structural protein and gp53 envelope glycoprotein) by indirect immunofluorescent histochemistry, and also viral RNA (encoding the p80 region) by in situ hybridization. In addition, titres of virus present in oviduct, ovary and blood were assayed by immunodetection on calf testis cell cultures. Luminal epithelial cells from the oviduct and primary cultures of granulosa cells and oviduct epithelial cells from such cattle were shown to contain both viral antigen and RNA. The susceptibility of both cell types to BVDV infection was further established by inoculating primary cell cultures of cells derived from cattle not infected with BVDV with a cloned isolate of non-cytopathogenic BVDV (Pe515). RNA encoding BVDV and the antigen were detected 12 h after inoculation. Viral titres present in oviduct, ovary and blood were between \m=ge\102.2 and 107; \m=ge\102.2 and 106.75; and 103.5 and 104.25 tissue culture infective doses, (TCID)50 g \ m=-\ 1, respectively. Control tissues from cattle not infected with BVDV, tested in each of the preceding techniques, were negative. These data establish that ovary and oviduct of persistently infected animals harbour non-cytopathogenic BVDV and that granulosa cells and oviduct epithelial cells, which are used as co-culture cells during bovine embryonic development in vitro and which, in the case of granulosa cells, constitute the cumulus investment surrounding the oocyte, are a vehicle for the potential transmission of BVDV to developing embryos.
Journal of Veterinary Medicine Series B, 2003
Nineteen pregnant cows were experimentally infected with bovine viral diarrhoea virus (BVDV) between day 74 and 81 of pregnancy. All cows became infected and developed serum antibodies. Sixteen of the cows delivered persistently infected (PI) offspring, whereas the remaining three gave birth to calves with detectable serum antibodies and free from BVDV. The 16 cows with PI foetuses developed higher levels of antibodies in serum during pregnancy than did their three peers carrying non-PI calves. Multivariate analysis showed that the antibody levels in these two groups of cows were significantly different from day 135 of pregnancy. Foetal fluid was successfully collected from 18 of the 19 infected cows and from five uninfected control cows between 10 and 24 days before delivery by use of a percutaneous, blind puncture technique. No negative effects were observed in the cows or their offspring. BVDV was isolated and detected with an immunoperoxidase test in foetal fluid from 13 of the 16 cows carrying PI foetuses, and from 15 of the cows when a quantitative fluorescent polymerase chain reaction (PCR) technique was used. The negative sample in the PCR assay was positive for BVDV antibodies. The number of viral copies per microlitre in foetal fluids varied between 103 and 1080 in the positive samples. All samples taken from the cows carrying non-PI foetuses were negative for BVDV in both assays. In this experiment, examination of either serum or foetal fluids could identify the cows carrying a PI foetus. Examination of serum for BVDV antibodies was a reliable indicator of a PI foetus if the serum was collected during the last 2 months of pregnancy. For examination of foetal fluids, both viral and serological analyses should be performed. For viral analysis, PCR should be the test of choice. High levels of BVDV antibodies in conjunction with a negative result in the PCR may be indicative of a false-negative virus result. Further experience with the method of collection of foetal fluids is necessary for evaluation of its safety. Investigation of pregnant cows in order to discover a PI offspring before it is born could be a useful tool in control and eradication of BVDV.
Veterinary microbiology, 2018
Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus and can cause reproductive problems in cattle. However, there is still a lack of research to clarify its pathogenicity in different gestational periods of sows and its effects in neonates. In this study, 12 gilts divided into groups (G) were experimentally inoculated with the strain BVDV-2 (SV-253) oronasally at a dose of 10· TCID50; one group was inoculated 30 days before insemination (G0; n = 2), three groups were inoculated during gestation (first (G1; n = 2), second (G2; n = 3), third (G3; n = 3)), and a fourth was the control group (G4; n = 2). Samples of blood and nasal swabs from the gilts were collected every three days until delivery for a virus neutralization (VN) test, qRT-PCR, and blood count. On the day of delivery, 40% of the neonates were euthanized to obtain tissue and blood samples at necropsy for histopathology and qRT-PCR. The sows were seroconverted between 12 and 33 days after inoculation, and th...
Journal of Veterinary Diagnostic Investigation, 1995
The objective of this study was to examine a monoclonal antibody-based immunohistochemical staining method for its efficacy in diagnosis of bovine virus diarrhea virus (BVDV)-induced abortion and neonatal calf death. This method was compared to viral isolation and immunofluorescence staining of frozen tissue sections. Tissues from 105 cases, 53 fetuses and 52 neonates, were tested by the 3 methods. There were significant numbers of both false negatives and false positives with the immunofluorescence method and significant numbers of false negatives with the viral isolation method. Of the methods tested, immunohistochemical staining using monoclonal antibody 15C5 performed best, differentiating 97% of positive and negative cases. These results indicated that immunohistochemical staining can be applied to improve the accuracy of BVDV diagnosis in cases of abortion and perinatal death.