 Equine infectious anemia, also known as EIA or swamp fever, is an infectious viral disease of members of the horse family, which poses a major threat to equid populations throughout the world. Control of this disease caused by equine infectious anemia virus, a lentivirus, was first possible in 1970 when an accurate diagnostic test was described. Each year in the United States, most new cases of this disease occur in the 18 contiguous states which we have termed the hot zone, the majority of which border the Gulf of Mexico or the Mississippi River. Currently, EIA research assumes new importance and urgency because the virus is closely related to the human immunodeficiency virus, the causative agent of AIDS. Those viruses infect our hosts for life, infect similar target cells, and mutate at a very high rate. Both are transmitted to susceptible individuals through the transfer of infective blood, and once infected, the individual may remain without clinical symptoms or signs for extended periods of time. It is hoped that the knowledge gained through research on either disease will ultimately aid in the control of both diseases. The purpose of this film, a collaboration between the US Department of Agriculture, the University of Kentucky, and Louisiana State University, is to update veterinarians and horse owners about progress in EIA. We will discuss the multiplication of the EIA virus, transmission and diagnosis of the infection, regulations which control the movement of EIA test positive horses, and research to better protect horses against this important disease. EIA played a pivotal role in helping French scientists describe the cause of AIDS. The virus that they had seen looked identical to published pictures of the EIA virus and cross-reacted in serologic tests against serum from EIA test positive horses. Incidentally, EIA was one of the first animal diseases proven to be caused by a virus by French scientists in 1904. Today EIA is recognized as an important animal model for AIDS and research in antiviral and vaccine strategies, diagnostics, and mechanisms of disease pathogenesis are high priority for both EIA and AIDS. This model of an EIA virus particle demonstrates its surface and core features. The virus is a member of the retro virus family, is similar to other retro viruses which cause leukemia, and is genetically related to the AIDS virus. EIA virus is inactivated by many chemical disinfectants, for example, household bleach. And even though the virus is not usually transmitted by contact, a thorough cleaning and disinfection of materials that have come in contact with infected or suspect animals, or untested ones, should be routine sanitary measures. When the EIA virus infects a horse, the virus has a marked tendency to mutate in regions of the virus genetic material which code for the surface unit protein. By using this mechanism, the virus can multiply, stimulate antibodies, mutate, and continue to multiply in the presence of relatively high levels of antibody against the previous virus strains. The antigenic variation of EIA, a feature shared with HIV and influenza virus, allows the virus to escape immunosurveillance and persist in the body of the horse for life. The net result of these mutations is the existence of multiple strains of the virus in nature, which makes developing an effective vaccine difficult if not impossible. Once the EIA virus enters a new host, it must multiply. That multiplication, which is shown schematically, is dependent on the virus attaching to receptors on the surface of a cell and then penetrating the cell. The enclosed genetic material is then released. The viral genome, which is RNA, uses a viral enzyme to make a copy of DNA from the viral RNA. The resultant double-stranded DNA then migrates to the nucleus of the infected cell and serves as a template for the formation of messenger RNAs and the new viral RNA strands. The messenger RNAs serve as a template for new virus protein synthesis. Some of these proteins play an important role in regulating the virus infection. Others will form the core structure, which will enclose the newly synthesized viral RNA strands. Other viral proteins will be inserted into the host cell membrane and serve as sites for viral budding and release from the cell membrane. Thousands of new virus particles may be released from one infected cell. What are the series of events following inoculation of EIA virus into a horse? First, the virus multiplies locally in phagocytic cells or histiocytes, which migrate through the skin. Then the virus is released to infect other cells locally or may be released immediately into tissue fluid or the bloodstream where it can be seeded throughout the body. There it tends to multiply primarily within those cells known as macrophages, monocytes or histiocytes, depending on their location. The outcome of virus multiplication in the horse is dependent on the virulence of the virus and the immune responses of the horse. Immune horses infected with field strains of EIA virus have increases in body temperature with the first virus multiplication cycle. The acute form of EIA occurs very rapidly following virus infection and is characterized by a marked increase in body temperature and by small hemorrhages, a response to the decrease in number and or function of platelets. This form of the disease is very difficult to diagnose because of the nonspecific signs and the fact that the horse has not had time to develop antibodies against the EIA virus proteins. Almost all develop antibodies to EIA virus by three to four weeks after infection. The most commonly diagnosed form of the disease EIA is the chronic form where the affected horse has recurring bouts of fever often accompanied by depression, weight loss, patechial hemorrhages, anemia and dependent edema. We think that the chronic form of EIA occurs because mutation of the virus results in changes in viral surface proteins which allows the virus to escape surveillance by the immune system. This results in a marked increase in virus multiplication and recurring clinical disease. In many cases the clinical signs of EIA are directly related to virus multiplication in and destruction of infected macrophages and the resultant inflammatory changes and immune responses to the viral antigens. For example, the anemia with EIA is probably caused by EIA virus attaching to red blood cells followed by anti-EIA virus antibodies attaching to the virus and their subsequent coating by a series of complement proteins which are attracted to the antigen antibody complexes. The red blood cells that are coated with complement are more fragile than normal and are removed from the circulation by phagocytic cells because they are viewed as foreign. Because these red blood cells are being destroyed so rapidly, the affected horse spends a great amount of energy trying to make more red blood cells in the face of active virus replication. Research on the mechanisms used by lentiviruses to cause disease are active subjects of investigation. This virus can be persistently demonstrated in the blood of test positive horses. The transfer of blood from one horse to another is an effective means of EIA virus transmission. This mechanical transmission can be done by man or blood-feeding insects. The transmission of EIA virus by man can be reduced markedly by adhering to the principles of universal precautions originally described to protect health care workers against blood-borne diseases. Veterinarians can reduce the potential of blood transmission between horses by using techniques to minimize blood contamination of people and objects such as the use of sterile evacuated glass tubes and needles designed to minimize the risk of blood leakage. The widespread use of and adherence to one needle, one horse has dramatically lowered the transmission of EIA virus by horse owners. Transmission of EIA virus by insects requires that the vector take part of its blood meal from an infected host, be interrupted in its feeding, and transferred to another host where it continues its feeding. The infected blood which contaminated the insect's mouth parts during the initial meal is deposited in the second host. Although the probability of insect transmission of EIA virus is dependent on the interaction among several variables, the most important are the amount of virus in the blood of the infected host and the amount of blood transferred from an infected to a susceptible host by the vector. When insect vectors such as mosquitoes and horse flies feed on horses, their mouth parts become contaminated with blood in quantities related to mouth part size and design. The finely structured mouth parts of mosquitoes allow for little blood contamination since they are designed to directly penetrate small blood vessels. The larger horse fly mouth parts have scissor-like blades and a sponge-like libellum that are designed for cutting, slashing, and imbibing the blood. The feeding creates a pool of blood which contaminates the mouth parts. When the horse fly is interrupted, this blood can be transferred to a second host. The concept of the hands in the candy jar will serve to estimate the probability of different vector species obtaining EIA virus from horses in different stages of the disease. These are used to represent red blood cells, white blood cells, EIA virus, and EIA virus-infected white blood cells. Cell-free virus as well as EIA virus-infected white blood cells are found circulating in the blood of donors showing clinical signs of illness. The level of cell-free virus reaches a million infective doses per milliliter during acute infection, and may exceed a thousand infective doses per milliliter during febrile episodes in horses with the chronic form of EIA. In contrast, little or no free EIA virus and few EIA virus-infected white blood cells are found in one milliliter of blood from the inapparently infected horse. The probability of virus transmission is greatest when large quantities of cell-free virus are present within the blood, fed on by relatively large blood-feeding vectors. In critical studies, mosquitoes have not been shown to transmit EIA virus even when horses with acute EIA are used as donors. The blood volume mechanically transmitted to the second host is exceptionally low, probably less than 1% of that transferred by a horsefly. When horseflies are allowed to feed on an acutely infected donor, transmission is easily affected. Our studies have shown that a single horsefly can transmit the virus to a susceptible host, and the virus can survive on the fly's mouth parts for at least 30 minutes. If the inapparently infected horse is used as a donor, the chance of virus transmission is significantly lower. We estimate that only one horsefly out of 6 million could transmit the virus after feeding on inapparently infected horses with low virus levels, because of the improbability that the horsefly will obtain and transfer the one infected cell necessary for disease transmission. We conducted an 8-year field study on transmission of EIA virus using infected horses and negative sentinels in two ecologically divergent areas in Louisiana. Site 1 was a 40-acre pasture separated by 1,000 yards from any wooded areas. No EIA virus transmission was noted in this area, although mosquito populations were relatively high. Site 2 was a 20-acre pasture surrounded on three sides by a flooded hardwood forest. Mosquito populations were equal to those at Site 1. But horseflies were about 50 times as numerous. EIA virus transmission occurred at this site when peak horsefly populations were noted. At this site, over 1,000 horseflies landed on a given horse in a one-hour period. Dr. Lane Foyle of the Anthemology Department at Louisiana State University has conducted pioneering studies on the dynamics of feeding behavior of horseflies. The relative abundance of horseflies and deer flies can be estimated by the use of canopy traps. The traps are erected and made more attractive by dry ice, a carbon dioxide source which stimulates to banted host-seeking behavior. The trapping data are then correlated with field transmission, which is verified by sequential serologic tests. Over a three-year period, we have correlated transmission with the early emergence or increasing populations of different horsefly species. Feeding persistence or intensity by which different to banted species pursue the completion of the blood meal from an initial host is also an important factor which influences transmission. If feeding persistence is high and the flies normally complete the meal on one horse, then the chances of this to banted species being a potential mechanical vector are small. A great deal has been learned about the feeding behavior of different to banted species by marking flies feeding on horses with non-toxic water-based paints. Four horses are positioned in a square formation, each accompanied by a researcher with a different colored paint. The flies are marked on the thorax with a dot of paint. Any to banted which is repelled by a horse and which flies to another horse is captured. By comparing the number of flies which transfer between horses to the number completing the meal on the original horse, considerable differences have been observed in the feeding persistence of different to banted species. In general, the larger horse flies, like to bannis atratus and to bannis americanus, are more easily repelled by the horses and pursue a different host, while the smaller species, like to bannis lineola and deer flies of the genus chrysops, complete the blood meal on the initial host. A similar experimental design was used to test the efficacy of spatial barriers on reducing the probability of mechanical transmission of pathogens by horse flies. By marking flies with paint, capturing and releasing them one foot from the feeding site, we artificially create the event of repelling of the fly by the horse. A linear correlation was found between the distance separating the horses and the percentage of flies refeeding on the original horse. That is, the farther the animals are separated, the higher the probability of the to banted completing the blood meal on the original animal. Therefore, spatial barriers can be used to reduce the potential of equine infectious anemia virus transmission by horse flies. If foals from positive dams are weaned at an early age and are moved from contact with positive horses, the majority of the foals become test negative. Horse fly feeding behavior may at least partially account for this apparent protection of foals from transmission. The most commonly used diagnostic procedure to detect antibodies to EIA in the serum of horses that are infected with EIA virus is the immunodefusion or Kagan's test. The results of this test can be reported in 24 hours. The immunodefusion test works on the principle that antigen and antibody react visibly, forming a line of precipitation where they're found in optimal proportions. If we sample the blood serum of a horse every 10 days following infection, we can see that the first sample contains no antibody. There is no line of precipitation between the test sample and the antigen that is common with the reference line of reaction from a known positive horse. But by 30 days following infection, most horses have produced detectable antibody against the viral antigen and remain positive for the rest of their lives. In contrast, the uninfected foal of the positive mare at the time of birth has no detectable antibody to EIA virus. 24 hours after suckling colostrum, the foal has absorbed antibody against EIA virus and tests positive for up to and occasionally beyond six months of age. At that time, the colostral immunity wanes to an undetectable level, assuming the foal has not been infected. The immunodefusion test is an excellent diagnostic tool to determine which horses are infected with EIA virus, assuming the samples were collected at least 30 days after infection to give them sufficient time to produce antibody. Two additional tests are approved for diagnosis of EIA and use enzyme-based formats, the so-called ELISA tests. The SELISA and SA ELISA test results are available from 30 minutes to two hours after the test is set up. Both of these tests give results with color changes, which in general are easier to interpret than those in the immunodefusion test, especially on samples with low levels of antibody. Both of these tests have the advantage of speed and potentially increased sensitivity compared with the Coggins test as a result of their ELISA-based formats. Both procedures are also inherently less specific and a nonspecific positive reaction will give the same result as a true positive, that is, a color change. By contrast, nonspecific reactions in the Coggins test can be distinguished from those which are specific because a separate line of reaction occurs. In cases where results in the approved test do not agree, more sensitive and specific procedures, such as the immunoblot test, have been used. In this test, purified EIA virus is chemically disrupted and its proteins are separated in a gel-like material by applying an electric current. The proteins which are stained in this gel are transferred to a membrane, and serum is then applied onto the membrane. If the horse is infected, antibodies against EIA virus will specifically bind to the proteins against which they were produced. The power of the immunoblot test resides in its ability to detect antibodies against all of the proteins of VIA found in the virus particle. Generally, infected horses produce antibodies against all three major EIA virus structural proteins, the major core, P26, and the envelope proteins, GP90 and GP45, even though the envelope proteins are present in the virus in low quantities and cannot be seen in the gel. Serum samples from horses with very low levels of antibody to the major core antigen and with very weak positive reactions in the Kagan's test, which may be interpreted falsely as negative, are generally positive in the immunoblot test. In dealing with AIDS, the immunoblot test has been accepted as the confirmatory test for antibodies against the human immunodeficiency virus because it is sensitive and can detect antibodies against multiple viral antigen. All of the tests described above detect antibodies to EIA virus, none detect the virus itself. Additional, more sensitive tests for EIA virus, for example, a PCR-based test to amplify the genetic material of EIA virus, are being developed to maintain and improve the accuracy of our testing programs. Since horses are the only known hosts for EIA virus, the best method of controlling its transmission is to control the movement of infected individuals. Routine testing of all horses should be done if controlled EIA is to be effective. When positive horses are found, their separation from other horses is very important in breaking the transmission of EIA virus in nature. The goodwill and honesty of horse owners is essential in the control of EIA. Control of the movement of infected horses is more easily accomplished than control of the blood-feeding vectors. Although pyrethrin sprays may effectively reduce vectors in enclosed facilities, the large-scale application of insecticide treatments that would effectively reduce vector pressures on horses in nature is very limited. The ultimate control of EIA is the responsibility of horse owners. One of the challenges of the next decade will be the development of electronic identification of horses that will provide both permanent Hain application and access to an expansional database. An electronic microchip inserted into the ligament and mucchi of the horse could serve as a license plate or code to access a computer database which could be included in the microcomputer of a smart card, replacing the traditional equine health certificate or passport. Such databases could store full registry or racing, vaccination, movement, medication records and photographs for identification. In our opinion, all horses that are positive on EIA tests should be visibly marked to identify them as EIA virus carriers. All horses should be tested, especially those in herds where EIA has been diagnosed. By controlling the movement of positive horses and by segregating and isolating them from negative horses, we feel that transmission can be blocked very effectively since horses are the only known reservoir for this virus in nature. Our current testing regulations do not require all horse owners to have their horses tested for EIA. We estimate that over 80% of the American horse population remains untested, a potential and unquantified reservoir for the EIA virus. The highest proportion of these reservoirs appears to be in the hot zone. It is our intention to improve the quality of testing and intensify control efforts in this zone where horses are at the greatest risk of acquiring infection. Federal regulations control only the quarantine and movement of reactors between states. While each state has its own regulations, the similarities outnumber the differences and should facilitate the creation of a sound and uniform policy program for all states. A frequent request of the industry, this would allow for ease of movement of horses between states without compromising biosecurity. The United States Department of Agriculture and the Gluck Equine Research Center at the University of Kentucky are dedicated to improving the knowledge of equine infectious anemia through educational means such as this film. And by stimulating and continuing discussion on EIA among veterinarians, horse owners, researchers, and regulatory authorities. Our surveillance of this disease will improve through more accurate laboratory testing and through field studies to determine the location of reservoirs. For more information concerning EIA virus infection of horses and the disease EIA, contact your local veterinarian.