M.S.D. Marley

Infectious causes of embryonic and fetal mortality

Abstract The purpose of this review is to summarize bacterial, fungal, protozoan, and viral causes of reproductive dysgenesis in cattle, sheep, goats, pigs, horses, dogs, and cats. The clinical presentations of disease due to reproductive pathogens are emphasized, with a focus on assisting development of complete lists of causes that result in abortion and infertility in these species. Clinicians are encouraged to assess clinical presentation, create complete lists of differential diagnoses, obtain appropriate diagnostic samples, maximize diagnostic laboratory support, and avoid zoonotic infections resulting from reproductive pathogens of animals. The foundation of an accurate diagnosis of reproductive loss due to infectious pathogens facilitates the prudent use of immunization and biosecurity to minimize reproductive losses.

Pathogens that cause infertility of bulls or transmission via semen

The purpose of this paper is to review scientific evidence regarding pathogens that cause infertility of bulls or that could be transmitted via bovine semen. Although several pathogens can cause male infertility and potentially be transmitted via semen, adhering to disease control recommendations provided by Certified Semen Services (CSS) and the World Organization for Animal Health (OIE) can prevent infectious male infertility and ensure that the risk of pathogen transmission via semen is negligible. Regarding bulls to be used for natural breeding, quarantine prior to herd introduction and appropriate diagnostic testing during quarantine will commonly prevent introduction of pathogens that adversely affect reproduction.

Bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced preimplantation bovine embryos following artificial exposure

The objective was to determine the average amount of bovine viral diarrhea virus (BVDV) associated with single in vivo-derived and in vitro-produced bovine embryos following recommended processing procedures for embryos. In vivo-derived and in vitro-produced bovine embryos at 7d post-fertilization were exposed (for 2h) to 2 x 10(5-7) cell culture infective dose (CCID(50))/mL of SD-1 (a noncytopathic, Type 1a strain of BVDV), and then washed according to International Embryo Transfer Society (IETS) guidelines prior to testing. Of the 87 in vivo-derived embryos tested, 27% were positive for virus by quantitative polymerase chain reaction (qPCR). The range in amount of virus associated with 99% of the contaminated embryos was <or=6.62+/-1.57 copies/5 microL; 90% of the contaminated embryos had <or=4.64+/-1.57 viral copies/5 microL of embryo-associated virus, using tolerance intervals (P<0.05). The SEM was 0.33 and the mean of averages was 1.12/5 microL. Of the 87 in vitro-produced embryos, 42% were positive for virus. The range in amount of virus associated with 99% of the contaminated embryos was <or=3.44+/-0.89 copies/5 microL; 90% of the contaminated embryos had <or=2.40+/-0.89 viral copies/5 microL of embryo-associated virus using tolerance intervals (P<0.05; S.E.M. was 0.14 and the mean of averages was 0.55/5 microL). Therefore, although many embryos were positive for virus, there were limited numbers of copies, thereby posing doubt regarding their potential for contamination following embryo transfer.

Development of a duplex quantitative polymerase chain reaction assay for detection of bovine herpesvirus 1 and bovine viral diarrhea virus in bovine follicular fluid

The objective of this study was to develop a duplex quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of bovine herpesvirus 1 (BoHV-1) and bovine viral diarrhea virus (BVDV) type I and type II. Follicular fluid was collected from a BoHV-1 acutely infected heifer, a BVDV I persistently infected heifer, and from 10 ovaries recovered from an abattoir. Both the BoHV-1 and BVDV contaminated follicular fluid were diluted 1:5 to 1:10(7) using the pooled, abattoir-origin follicular fluid. Each dilution sample was analyzed using the duplex qPCR, virus isolation, reverse transcription-nested PCR (RT-nPCR), and BoHV-1 qPCR. The duplex qPCR was able to simultaneously detect BoHV-1 and BVDV I in the fluid diluted to 1:100 and 1:1000, respectively. These results corresponded with the reverse transcription-nested PCR and BoHV-1 qPCR. Therefore, the duplex qPCR might be used for quality assurance testing to identify these two viruses in cells, fluids and tissues collected from donor animals and used in reproductive technologies.

Efficacy of a recombinant trypsin product against bovine herpesvirus 1 associated with in vivo- and in vitro-derived bovine embryos

Although porcine-origin trypsin will effectively remove bovine herpesvirus 1 (BHV-1) associated with in vivo-derived embryos, TrypLE, a recombinant trypsin-like protease, has not been evaluated. In Experiment 1, 17 groups of 10 in vivo-derived embryos were exposed to BHV-1, treated with TrypLE Express or TrypLE Select (10x concentration) for varying intervals, and assayed as 2 groups of 5 embryos. TrypLE Select treatment for 5 and 10 min (two and seven groups of five embryos, respectively) effectively inactivated BHV-1. In Experiment 2, 22 groups of 10 IVF embryos were treated and assayed. Treatment with TrypLE Select for 7 and 10 min (six groups of five embryos each) and with TrypLE Select diluted 1:2 for 10 min (seven groups of five embryos) was also effective. In Experiment 3, 17 groups of 10 IVF embryos were further evaluated with TrypLE Select undiluted and diluted 1:2 for 10 min. Treatment with the diluted product was effective (18 groups of five embryos), whereas the undiluted product was not completely effective (virus isolated from 2 of 16 groups). In Experiment 4, IVF embryos were treated as described in Experiment 3 and then cultured individually or as groups of five on uterine tubal cells (UTCs) for 48 h; 60% of UTC samples associated with groups of embryos and 35% of UTC associated with individual embryo samples were positive for BHV-1. Therefore, although TrypLE Select appeared to have promise for the treatment of in vivo-derived embryos, it cannot be recommended for treatment of in vitro-derived embryos.

Risk and prevention of bovine viral diarrhea virus (BVDV) transmission through embryo production via somatic cell nuclear transfer (SCNT) using oocytes from persistently infected donors

The objective was to assess the risk of transmission of bovine viral diarrhea virus (BVDV) through embryo production via somatic cell nuclear transfer (SCNT), with oocytes obtained from persistently infected (PI) donors. Using ultrasound-guided follicular aspiration following superstimulation, oocytes were obtained from five female beef cattle, including three that were PI and two that were negative for BVDV. In the three PI cattle, seven aspirations yielded 32 oocytes (PI-1: three aspirations yielding six oocytes; PI-2: two aspirations yielding 14 oocytes; and PI-3: two aspirations yielding 12 oocytes). The oocyte recovery rate was better in negative control cattle, with 32 oocytes obtained from the two cattle in a single superstimulation and aspiration session. Oocytes were processed individually for SCNT, evaluated, and tested for BVDV. Nearly all (31/32) oocytes from the three PI donors were positive for BVDV by PCR, with detected viral RNA copy number ranging from 1 to 1.1 x 10(5). The proportion of oocytes acceptable for SCNT embryo production (based on oocyte quality and maturation status) was only 16 to 35% from PI donors, but was 81% from control donors. Therefore, routine testing of unacceptable (discarded) oocytes could be an effective approach to identify batches that might contain infected oocytes from PI donors. Identification and removal of high-risk batches of oocytes would minimize the risk of BVDV transmission through SCNT embryo production.

Intrauterine inoculation of seronegative heifers with bovine viral diarrhea virus concurrent with transfer of in vivo―derived bovine embryos

Bovine viral diarrhea virus (BVDV) has been shown to be associated with single transferable in vivo-derived bovine embryos despite washing and trypsin treatment. Hence, the primary objective was to evaluate the potential of BVDV to be transmitted via the intrauterine route at the time of embryo transfer. In vivo-derived bovine embryos (n=10) were nonsurgically collected from a single Bos tarus donor cow negative for BVDV. After collection and washing, embryos were placed into transfer media containing BVDV (SD-1; Type 1a). Each of the 10 embryos was individually loaded into an 0.25-mL straw, which was then nonsurgically transferred into the uterus of 1 of the 10 seronegative recipients on Day 0. The total quantity of virus transferred into the uterus of each of the 10 Bos tarus recipients was 878 cell culture infective doses to the 50% end point (CCID(50))/mL. Additionally, control heifers received 1.5 x 10(6) CCID(50) BVDV/.5 mL without an embryo (positive) or heat-inactivated BVDV (negative). The positive control heifer and all 10 recipients of virus-exposed embryos exhibited viremia by Day 6 and seroconverted by Day 15 after transfer. The negative control heifer did not exhibit a viremia or seroconvert. At 30 d after embryo transfer, 6 of 10 heifers in the treatment group were pregnant; however, 30 d later, only one was still pregnant. This fetus was nonviable and was positive for BVDV. In conclusion, the quantity of BVDV associated with bovine embryos after in vitro exposure can result in viremia and seroconversion of seronegative recipients after transfer into the uterus during diestrus.

Bovine viral diarrhea virus is inactivated when whole milk from persistently infected cows is heated to prepare semen extender

Bovine viral diarrhea virus (BVDV) can be present in cryopreserved bovine semen and be transmitted through artificial insemination. Because BVDV can be shed in milk, the virus might also be introduced as a contaminant of milk-based semen extenders. Thus, the purpose of this study was to evaluate the epidemiologic risk of using heated, BVDV-contaminated milk to prepare semen extender. Milk was obtained from cows free of and persistently infected (PI) with BVDV. Six replicates of milk samples were processed by heating (85-92.2 degrees C, 10min). Samples of milk collected before and after heating were assayed for BVDV. Additionally, milk was injected intravenously into eight BVDV seronegative calves to monitor for seroconversion and viral infection. Virus was not detected in any milk samples from negative animals. Virus was consistently isolated from unheated milk samples from PI cows by passage of somatic cells, ultracentrifugation, and animal inoculation. Virus was usually detected in these samples by RT-nPCR (reverse transcription nested polymerase chain reaction). In heated milk samples from PI cows, no infectious BVDV was detected using any technique, but viral RNA was detected using RT-nPCR in four of six replicates. Bovine viral diarrhea virus in milk from PI cows was inactivated by heating. Therefore, properly heated milk used in semen extenders will not result in transmission of infectious BVDV. Although RT-nPCR detected the presence of viral RNA in milk samples after heating, the virus was not infectious as demonstrated by lack of replication despite using multiple sensitive techniques.

Inactivation at various temperatures of bovine viral diarrhea virus in beef derived from persistently infected cattle.

Bovine viral diarrhea virus (BVDV) is a pestivirus that is enzootic in most cattle populations throughout the world. This virus is present throughout the body of persistently infected (PI) cattle. Previous research has not assessed the cooking temperature at which BVDV in meat from PI cattle can be inactivated. Therefore, muscle tissue from 6 PI cattle was harvested, refrigerated, frozen, and heated to various internal temperatures. The concentration of virus present was determined by virus isolation. Average cell culture infective doses (50% endpoint; CCID(50)) of BVDV per gram of frozen, uncooked meat from PI cattle were 10(5.85) CCID(50)/g of whole cuts and 10(6.02) CCID(50)/g of ground meat. The virus in whole and ground meat was consistently inactivated when cooked to temperatures greater than or equal to 75°C. A second objective of this research was to thoroughly reassess if Vero cells were permissive to BVDV infection in our laboratory to provide further indication of whether primates, including humans, might be susceptible to BVDV. Vero cells were not permissive to infection with any of 43 different strains of BVDV that readily replicated in Madin Darby bovine kidney cells. In conclusion, this bovine pathogen, which is not considered to be a human pathogen, can be inactivated by cooking ground or whole cuts of meat to 75°C or higher. Care should be taken to ensure that susceptible hosts such as pigs are not fed improperly cooked meat, meat by-products, or waste food originating from PI cattle.

Lactoferrin from bovine milk inhibits bovine herpesvirus 1 in cell culture but suppresses development of in vitro-produced bovine embryos

Bovine herpesvirus 1 (BoHV-1) is widely distributed among cattle populations and has been associated with cells, fluids, and tissues collected from donor animals for use in reproductive technologies. The purpose of this study was to determine if lactoferrin would inhibit BoHV-1 in cell culture and to evaluate if embryos could develop normally when cultured in vitro with lactoferrin. In Experiment 1, lactoferrin (10 mg/mL) inhibited up to 25,000 plaque forming units (PFU)/mL of BoHV-1 in Madin Darby bovine kidney (MDBK) cell culture. In Experiment 2, lactoferrin (10 mg/mL) combined with cidofovir (62.5 microg/mL) inhibited up to 100,200 PFU/mL of virus in cell culture. In Experiment 3, following fertilization, presumptive zygotes were cultured in media containing lactoferrin (10, 5, and 2.5 mg/mL). Embryonic development and quality were assessed, and embryonic viability was determined by counting the nucleated cells of developed blastocysts. While lactoferrin did not affect the nucleated cell count of the treated embryos, it did significantly decrease blastocyst development. In conclusion, lactoferrin from bovine milk can inhibit BoHV-1 in cell culture. However, supplementation of in vitro culture medium with lactoferrin inhibits blastocyst development of in vitro-produced embryos.