Interstate Equine Semen and Embryo Shipment Regulations in the United States and Their Implications on Control of Disease Transmission

Outbreaks of both equine viral arteritis and contagious equine metritis (CEM) in the United States have occurred in recent years. Shipped semen has been implicated in disease transmission and rapid spread. A survey was performed of state regulations regarding testing for equine arteritis virus (EAV) and CEM before interstate semen or embryo shipment. Results demonstrated lack of any requirements in 31 states. Four states had regulations regarding EAV; 17 required a Certificate of Veterinary Inspection (CVI) to accompany a semen or embryo shipment. Ten states required a negative equine infectious anemia test, primarily as a requirement of a CVI versus because of risk of dissemination of disease. No states required CEM testing. A comparison of state department of agriculture or veterinary medical association Web sites and direct communication with state veterinarians or their offices demonstrated contradictory information in six states. The lack of uniform regulations concerning CEM and EAV testing for shipped semen and embryos, and the threat they pose to the equine breeding industry and horse health, should be alarming to veterinarians and horse owners. Routine testing of animals before shipment of semen or embryos and veterinary involvement in collection and breeding activities are appropriate and necessary to help prevent future outbreaks and protect equine health.     

Hygienic Applications of Biotechnical Measures in Pigs*

A key factor in the hygienic application of embryo transfer (ET) and its associated technologies is effective risk management. This depends on knowing what the risks of infectious disease transmission are and how they can be reduced or removed. Risk assessment depends on a knowledge of the pathogenesis of the disease, the procedures used in ET and its associated technologies and the results from research into infectious disease transmission by embryos or through ET. Two approaches can be taken to ensure the safe health status of the embryo: 1) by determining that the donor animals (male and female) are free from specific diseases, or 2) by ensuring that the zona pellucida (ZP)-intact embryo is collected in a sanitary manner and handled (washed, treated, evaluated, etc.) according to recommended procedures. The former approach is applicable to diseases on which no or insufficient research has been done to determine the risk of their being transmitted by embryos or where the results of research done indicate a risk of the disease being transmitted. The latter approach is a preferred option with diseases where results of research done indicate that the risk of their being transmitted by embryos collected from infected or recovered donors is negligible. Infectious diseases for which research results indicate negligible risk of transmission by ZP-intact (ZP-I) embryos collected from infected or recovered donors include: pseudorabies (Aujesrky's disease), hog cholera (swine fever), foot and mouth disease, and swine vesicular disease. Infectious diseases on which insufficient research has been done include: African swine fever, vesicular stomatitis, enterovirus disease, parvouirus disease and leptospirosis. A problem associated with this approach is ensuring that procedures shown to be effective under experimental conditions are properly carried out under field conditions: the use of nationally accredited embryo collection teams may solve this problem. Healthy recipients and good record keeping are also critical factors in the hygienic application of ET. The health status of an embryo collected from a specific disease-free donor will not be adversely affected by damage to its ZP. Where research has shown that ZP-I embryos that have been exposed to particular pathogens are not infected or contaminated after proper washing or washing and treatment, it can be assumed that the risks of transmission of the diseases caused by these pathogens will not be increased by deliberate (micromanipulation) or accidental damage to the ZP after the washing/treatment procedures have been carried out. The risks of infectious disease transmission when embryos are produced by in vitro fertilization or blastomere transplantation to mature oocytes, followed by culture, have yet to be determined.     

Embryo transfer: a discussion on its potential for infectious disease control based on a review of studies on infection of gametes and early embryos by various agents

Studies on laboratory animals have shown that viruses vary as to whether or not they are transmissible by the gametes or are capable of passing through the zona pellucida and infecting the embryo.

Methods of studying early embryos for the presence of infectious agents include electron microscopy, immunocytochemistry and cell cultivation.

Determination that early bovine embryos do not become infected by certain agents might allow for easing of restrictions in the current import and export regulations for cattle embryos.

Embryo transfer could be used as a means of controlling or eliminating disease in a herd or flock if the causal agent does not infect the early embryo via the gametes or by penetrating the zona pellucida.

     

Import risk analysis in animal disease control

At the Swiss Federal Veterinary Office risk analyses are conducted according to international standards. A risk analysis contains the elements risk management, risk assessment and risk communication. A risk assessment is based on risk profile, hazard identification and a pathway model. All available information is gathered, documented and assessed and the risk estimated. The question. "What is the probability that unprocessed wild boar meat imported to Switzerland from the federal state Mecklenburg Western Pommerania is contaminated with classical swine fever virus?" was answered by a release assessment. The hazard identification recognized classical swine fever virus and attenuated live virus vaccine used for oral immunization as hazards. The probability of contamination was estimated to be small. The question: "What is the likelihood to introduce Aujeszky's disease to Switzerland and infect the indigenous pig population with the disease, by means of importing pork and meat products?" was answered by assessing the release, exposure and resulting consequences. The risk of an infection of the indigenous pig population was estimated to be very small, as 80% of the imported products derive from countries or zones free from Aujeszky's disease. Furthermore the majority of the imported products are processed. The strict implementation of the regulations governing feeding of food wastes to pigs reduces the probability of exposure. In all assessments the risk management decides on a strategy to deal with the risk, taking into consideration the results and recommendations derived from the risk assessment as well as other relevant factors.     

Biosecurity and the Various Types of Embryos Transferred

The aim of the present paper was to review some features related to the risk analysis of three types of embryos to be transferred, namely the in vivo derived, the in vitro produced and the cloned ones. For in vivo-collected embryos, a considerable number of experiments and scientific investigations have been performed and hundreds of thousands of embryos are transferred annually with no contamination of associated diseases. Provided that the code of practice such as that published by the International Embryo Transfer Society is strictly followed by the embryo transfer practitioners, the statement made some 17 years ago saying that the in vivo-derived embryo transfer was the safest way of exchanging genes remains entirely true, thanks to the professionalism of the embryo transfer industry. For the in vitro-produced embryos, some particular rules have to be followed because of specific risks for some pathogens to strongly adhere to the zona pellucida of such embryos. There are some means to monitor and control those effects, and the transfer of in vitro-produced embryos can also be a very safe way to exchange genes around the world. The third type of embryos, the cloned ones, is a quite different category and the risk analysis to be soundly made still needs a lot of investigations so as to characterize the potential risks if there are, in terms not only of disease transmission but also in terms of public health, zoonotic risks as well as those related to quality and safety of food. The problem in this regard, is more directly addressed for offspring of clones than to the cloned embryos themselves. Published data on this issue are increasing in numbers so that progress in that area is expected in the few years to come.     

The potential of embryo transfer for infectious disease control in livestock

This review of the general epidemiological aspects of embryo transfer indicates that the transfer of embryos provides the opportunity to introduce genetic material into populations of livestock while greatly reducing the risk for transmission of infectious diseases. Studies of specific diseases of livestock confirm that many pathogens are likely to be excluded when embryos are transferred. Twenty years of commercial embryo transfer under field conditions in a variety of species has not resulted in a single documented transmission of an infectious disease agent. Strategies for insuring that embryos are free of pathogens include the use of donors that are specific pathogen-free, washing of embryos, the trypsin treatment of embryos, or a combination of these methods. Although researchers continue to develop new procedures for the treatment of embryos to provide an even greater margin of safety, it is clear that existing techniques for the handling and transfer of embryos can be used to limit the spread of infectious diseases.     

Pathogens associated with mammalian embryo (a review)

The advantages of transporting mammalian preimplantation embryos rather than postnatal living animals include reduced costs of transportation and rapid dissemination of genetic material between countries. However, the risk of transmission of diseases through the embryos must be considered. The disease control potential of embryos will depend on proper handling and washing, and the integrity of zona pellucida. Researches on embryo-pathogen interactions have shown that some pathogens are carried through the gametes and others could not infect the gametes. Some pathogens were found to adhere to the zona pellucida and others could penetrate the zona pellucida. To date, data presented appear to suggest no concrete model guidelines for embryo-pathogen interaction. The interaction seems to depend on the species and the pathogen involved.     

Use of a Markov-chain Monte Carlo model to evaluate the time value of historical testing information in animal populations

Quantitative risk assessments are now required to support many regulatory decisions involving infectious diseases of animals. Current methods, however, do not consider the relative values of historical and recent data. A Markov-chain model can use specific disease characteristics to estimate the present value of disease information collected in the past. Uncertainty about the disease characteristics and variability among animals and herds can be accounted for with Monte Carlo simulation modeling. This results in a transparent method of valuing historical testing information for use in risk assessments. We constructed such a model to value historical testing information in a more-transparent and -reproducible manner. Applications for this method include trade, food safety, and domestic animal-health regulations.     

Understanding stakeholders’ opinions and preferences for non-native pet trade management in Florida

There is growing recognition of the link between the non-native pet trade and the introduction and establishment of invasive species due to the release and escape of non-native pets. However, it is unclear whether participants in the pet trade recognize the magnitude of this invasion risk. Successful mitigation of the pet trade invasion risk requires stakeholder support for, and participation in, regulations. We conducted 29 interviews in Florida to investigate key stakeholders’ opinions about the pet trade invasion risk and the effectiveness of potential regulations to mitigate this risk. Respondents framed the effectiveness of regulations in terms of their feasibility. Respondents also identified lack of trust and the existence of an adversarial relationship among stakeholder groups as major barriers to managing the pet trade invasion risk. Compliance with regulations may be improved if policy makers and managers utilize participatory decision-making to engage stakeholders in management of this risk.     

PROCEEDINGS - Embryo Transfer in the Canadian Cattle Industry Status of Disease Transmission Studies and thier Relationship to the International Movement of Bovine Embryos

The current, generally accepted approach to formulating health requirements for the international movement of embryos is to base them on the health status of the male and female donor animals. The alternative approach of basing them on the health status of the embryos themselves has been blocked by the lack of scientific information about the potential of the early embryo to transmit agents of infectious disease. Consequently, most research into infectious disease transmission by embryos has had the objective of assessing the potential of the embryo to transmit infectious disease, at the stage of development at which it is transferred commercially, with the thought in mind that, for some diseases, it may be possible in the future to focus on the embryo rather than the donor when drawing up health requirements for import permits. Results from experiments involving the bovine leukemia virus, bluetongue virus, infectious bovine rhinotracheitis virus, foot and mouth disease virus and Brucella abortus are encouraging to the point where, with the exception of foot and mouth disease virus, they could and should be put to the test in field studies. Research on several other bovine pathogens is underway, but the studies are not sufficiently advanced for a judgement to be made on the potential of embryos to transmit them. There is evidence that the research done is starting to have a positive effect through the relaxation of some health requirements for the international movement of embryos.     

Prospects for the use of embryos in the control of disease and the transport of genotypes

Transfer and low temperature storage of embryos are now proven techniques for a number of mammalian species. These techniques are useful in control of disease and in saving genotypes from infected animals. The place of embryos in the epidemiology of disease depends upon whether the causative organism can gain entry to the oocyte before or at fertilisation and on whether the young embryo can be invaded by organisms in the uterine environment. There is little evidence that important live-stock diseases are transmitted via gametes. The zona pellucida surrounding the embryo is an effective barrier against a number of important disease organisms; in some cases the embryo is susceptible once it has hatched from the zona pellucida. It is important therefore in considering the use of embryos in disease control, to ensure that virus is not attached to the surface of the zona pellucida from where it can infect the recipient and/or the embryo after hatching. Washing procedures have been devised together with the use of enzymes and antisera to remove virus from the surface of embryos. Some viruses enter pores and sperm tracks in the zona and removal of these may present a problem. African swine fever virus has been shown to resist removal by treatment with enzymes. There are no guidelines as to the likely interaction between a certain virus and embryos. Therefore each virus of interest must be tested to determine whether it can be transmitted via washed embryos. Nevertheless there are numerous instances of the use of embryo transfer to eradicate a specific disease or to save valuable genetic material from infected animals without transmitting disease.     

新城疫病毒山东强毒株的分离鉴定

从山东流行烈性传染病的鸡群中分离出7株具有血凝活性的病毒，该7株病毒的血凝活性均能被新城疫La Sota株标准阳性血清所抑制，但病毒不能被中和，仍能致死鸡胚。经分离鉴定，分离毒均为新成疫病毒株。通过鸡胚半致死量（ELD50）、最小致死量致鸡胚的平均时间（MDT）、1日龄雏鸡脑内致死指数（ICPI）、6周龄雏鸡静脉致死指数（IVPI）、血凝解脱及血凝素稳定性等试验表明：7株分离病毒均为新城疫病毒强毒性，其毒力与标准强毒株F48E9株相似。     

The importance of the zona pellucida for disease control in livestock by embryo transfer

From the point of view of disease risk, the movement of livestock by embryo transfer is undoubtedly much safer for trading than the movement of live animals or semen. Nevertheless, strict governmental control by veterinary certification of health of embryos is still vital. In cattle, sheep and pigs, unlike laboratory species such as the mouse, infectious agents do not appear to pass through the zona pellucida (ZP) into the embryo proper. Some agents do, however, adhere firmly to the outer surface of the ZP, especially onto those of the pig. Disease risks associated with the inadvertent transmission of infectious agents when embryos are moved are, therefore, intimately connected with the nature and properties of the ZP. This article reviews current knowledge on the physical and adhesive properties of the ZP and discusses how risks associated with the possible presence of infection on its surface can be minimized. Further research is urgently needed so that realistic but safe veterinary certification of the health of embryos for international trade can be devised.     

Food safety surveillance through a risk based control programme: approach employed by the Belgian Federal Agency for the Safety of the Food Chain

The principal responsibility of the Belgian Federal Agency for the Safety of the Food Chain (FASFC) is to guarantee the safety along the food chain. In order to accomplish this responsibility, the FASFC has developed an integrated official control program to check compliance with various regulations. The original methodology developed and applied by FASFC is presented. This methodology is based on risk evaluation, statistical tools and current scientific knowledge.     

病死孔雀中新城疫病毒的分离和鉴定

从疑似患新城疫的病死孔雀中分离出1株病毒,该病毒具有血凝活性,在血凝试验和血凝抑制试验中,能凝集鸡的红细胞,且血凝活性能被新城疫阳性血清所抑制.通过试验测定鸡胚最小致死量（EMLD）,鸡胚平均致死时间（MDT）,血凝素热稳定性,脑内接种致病指数（ICPI）及静脉接种致病指数（IVPI）,结果表明,该分离病毒为新城疫嗜内脏速发型毒株.     

Studies of embryo transfer from cattle clinically affected by bovine spongiform encephalopathy (BSE)

Semen from 13 bulls, eight with clinical bovine spongiform encephalopathy (BSE), was used to artificially inseminate (AI) 167 cows with clinical BSE, and their resultant embryos were collected non-surgically seven days after AI. The viable and non-viable embryos with intact zonae pellucidae were washed 10 times (as recommended by the International Embryo Transfer Society) then frozen. Later, 587 of the viable embryos were transferred singly into 347 recipient heifers imported from New Zealand, and 266 live offspring were born of which 54.1 per cent had a BSE-positive sire and a BSE-positive dam. The recipients were monitored for clinical signs of BSE for seven years after the transfer, and the offspring were monitored for seven years after birth. Twenty-seven of the recipients and 20 offspring died while being monitored but none showed signs of BSE. Their brains, and the brains of the recipients and offspring killed after seven years, were examined for BSE by histopathology, PrP immunohistochemistry, and by electron microscopy for scrapie-associated fibrils. They were all negative. In addition, 1020 non-viable embryos were sonicated and injected intracerebrally into susceptible mice (20 embryos per mouse) which were monitored for up to 700 days, after which their brains were examined for spongiform lesions. They were all negative. It is concluded that embryos are unlikely to carry BSE infectivity even if they have been collected at the end-stage of the disease, when the risk of maternal transmission is believed to be highest.     

Use of an immunoenzyme histochemical method for the improvement of the etiologic diagnosis of Aujeszky's disease in swine]

With regard to the legal regulations for the diagnosis of Aujeszky's disease in pigs, a retrospective immunoenzymatic study was performed on brains of 20 pigs, in which Aujeszky's disease had been diagnosed based only on clinical, macroscopical and histological findings, although the etiological agent could not be demonstrated neither by immunofluorescent technique nor by virological cultivation. Applying the peroxidase-antiperoxidase-(PAP) method, in 6 of 20 animals (30%) viral antigen of porcine herpesvirus type 1 was demonstrated in situ in at least two of five different locations of the brain. Reasons for negative results in immunohistological and virological investigations are discussed and the significance of the performed immunoenzymatic technique is pointed out.     

Delivery after transfer of frozen-thawed embryos from in vitro-matured oocytes in a woman at risk for ovarian hyperstimulation syndrome

The present report describes the birth of a healthy infant after cryopreservation of embryos produced from in vitro-matured oocytes retrieved from a woman at risk of developing ovarian hyperstimulation syndrome (OHSS) during conventional in vitro fertilization (IVF) cycles. A conventional long protocol including gonadotropin-releasing hormone agonist (GnRHa) and gonadotropins induced a risk of OHSS. Oocyte retrieval was performed on day 11 of the cycle, and 27 immature oocytes were obtained. Following incubation for 24 h in maturation medium, 74.1% (20/27) of the oocytes were at the metaphase II stage. Fourteen oocytes (14/20, 70.0%) were fertilized after intracytoplasmic sperm injection (ICSI) with her husband's spermatozoa and cultured for 3 days. On day 4 following oocyte retrieval, three embryos at the 8-16 cell stage were transferred into the woman's uterus, and five spare embryos were frozen. Since the fresh embryo transfer failed to result in pregnancy, three post-thaw embryos were transferred into the woman three months later. Transfer of the frozen embryos resulted in pregnancy with delivery of a healthy infant girl.     

Exploratory spatial relative risk mapping

The many faces of disease mapping include maps of disease case locations, regional counts of cases, and disease risk. Another approach is that of mapping the relative risk. Previous methods to map the relative risk were based on regression models of relative risk, given information about geographical locations and established risk factors. However, spatial epidemiological investigations are often exploratory with limited knowledge about the putative risk factors. Indeed, often the primary motivation for the analysis is to identify unknown geographically varying risk factors.

An exploratory approach to mapping the spatial relative risk is to scale the risk map using the background risk in the unexposed (or less-exposed) population. Exposure to unknown spatial risk factors is defined via specific cluster analysis. Identification of spatial disease clusters separates the population into those inside and those outside high risk areas (the exposed and unexposed populations). This exploratory approach to relative risk mapping gives the investigator an impression about the importance and geographical distribution of the unknown spatial risk factors.

Two examples illustrate the exploratory relative risk mapping approach using a spatial point data set on pseudorabies in pig-herds and a regional count data set on small fox tapeworm infections in red foxes.