Apparent propagation of the equine infectious anemia virus in a mosquito (Culex pipiens quinquefasciatus Say) ovarian cell line.

A tissue culture of Culex pipiens quinquefasciatus Say ovarian cells appeared to support the growth of equine infectious anemia (EIA) virus. Shetland ponies inoculated with 2nd, 7th, 9th, and 11th passages of mediums harvested from infected tissue culture had clinical signs of the disease and became EIA positive on 11, 19, 23, and 43 days after inoculation, respectively.     

Role of horse flies in transmission of wquine infectious anemia from carrier ponies

Equine infectious anemia virus was transmitted from an acutely ill and an inapparently infected pony to uninfected ponies by the interrupted feeding of horse flies (tabanids). Transmission from acutely ill ponies was not accomplished following: (1) the interrupted feeding of a single horse fly, (2) bites of horse flies that had fed on an acutely affected pony 24 hours earlier, (3) bites of horse flies that had oviposited after feeding on an acutely affected pony, or (4) the inoculation of larval material derived from horse flies that had fed to repletion. It was concluded that horse fly transmission of equine infectious anemia virus is mechanical only and that infected horses that are free of clinical signs can be a source of virus for insect transmission.     

Culicoides: biological vectors of Akabane virus

Akabane virus replicated in Culicoides nubeculosus and Culicoides variipennis after intrathoracic inoculation and was maintained in both species of midge for at least 9 days post-infection. The virus also replicated to high concentration in C. variipennis after oral infection and was transmitted through a membrane by this species of midge 7-10 days after infection. The experiments described in this paper provided the first definitive evidence that Culicoides spp. are able to act as fully competent vectors of Akabane virus.     

Virulence and in vitro growth of a cell-adapted strain of equine infectious anemia virus after serial passage in ponies

Five serial passages of a cell-adapted strain of equine infectious anemia (EIA) virus were conducted in Shetland ponies. The 13 recipient ponies became agar-gel immunodiffusion test-positive by 25 days after they were inoculated. The virulence of the cell-adapted strain of EIA virus markedly increased through 3 serial passages, although individual variation within passages was high. The 1st serial-passage recipient remained afebrile through 200 days, whereas a febrile episode occurred about every 185, 44, 35, and 33 days in the 2nd, 3rd, 4th, and 5th serial-passage recipients, respectively. Severe clinical signs of EIA were noted in the ponies at each serial passage, but the mean virulence rating of each passage, graded on frequency of febrile episodes and number of clinical signs evident within 200 days after ponies were inoculated, increased from 0 through 4, 21, 24, and 29 for the 1st through 5th serial passages, respectively. Isolates of EIA virus, made in fetal equine kidney cells, were obtained from plasma of 75% of the samples of blood collected during febrile episodes and from 45% of the samples collected during afebrile periods, indicating that the cell culture growth capacity of this strain of EIA virus may be relatively stable through 5 serial passages in Shetland ponies.     

Demonstration of equine infectious anemia virus in primary leukocyte cultures by electron microscopy.

Electron microscopy was used to demonstrate the presence of viral particles in primary cultures of leukocytes taken from a horse after SC inoculation with the Wyoming strain of equine infectious anemia virus. Unlike previous studies, the exposure virus was not passaged through cell culture prior to horse inoculation. Cultures were begun approximately 1 week before and 1 week after the 1st pyrexic period after inoculation. In both samples, viral particles and cytoplasmic alterations were observed resembling those previously reported in equine infectious anemia virus and other retravirus-infected cells.     

Pathogenicity of equine herpesvirus 1 subtype 2 for foals and adult pony mares

Three pony mares and 4 pony foals were inoculated with a subtype 2 strain of equine herpesvirus 1. Foals had periods of fever 12 h and 2.5 days after inoculation and leukopenia, involving both neutrophils and lymphocytes, followed by leukocytosis. Mares had transient fever and leukopenia 24 hours after inoculation that were less severe than in foals. An increase in circulating virus-neutralizing antibody was seen in 2 of 3 inoculated mares, but not in foals. Attempts to isolate virus from blood were unsuccessful. These studies show that equine herpesvirus 1 subtype 2 is a mild pathogen for ponies and infection may result in inapparent clinical disease.     

Indirect hemagglutination test in equine infectious anemia.

An indirect hemagglutination was developed for the diagnosis of equine infectious anemia using sheep red blood cells coated with group specific virus antigen which had been highly purified by affinity chromatography. The presence of indirect hemagglutination antibodies was demonstrated in horses with equine infectious anemia since the cells were specifically agglutinated by all the serum samples obtained from experimentally infected horses. Antibodies appeared within 35 days after inoculation, and development of which coincided well with that of precipitating and complement fixing antibodies. Titer of indirect hemagglutination antibodies were ten to 320 times greater than those of precipitating antibodies. Test results could be read more clearly by the indirect hemagglutination test especially in weakly positive cases. Ninety-six samples from suspected field cases collected from every region of Japan which were positive on the immunodiffusion test were also positive on indirect hemagglutination test. Serum samples from 420 horses in one race track were examined by both the indirect hemagglutination and immunodiffusion tests to determine the reliability of the indirect hemagglutination test for diagnosis of equine infectious anemia. The same result was obtained on both tests. Based on this evidence, the indirect hemagglutination test can be employed as a very sensitive serological test for the diagnosis of equine infectious anemia.     

Clinical and hematologic variables in ponies with experimentally induced equine ehrlichial colitis (Potomac horse fever)

The clinical and hematologic variables of 10 ponies with experimentally induced equine ehrlichial colitis (EEC; syn: Potomac horse fever) were studied for a 30-day period (6 ponies) or until death (4 ponies). The earliest clinical sign indicative of EEC was fever (rectal temperature exceeding 39 C). All ponies became depressed (CNS) at various times during the disease, and 90% of the ponies developed diarrhea between 9 and 15 days after infection was induced. The most significant hematologic change was an increase in plasma protein concentration after the onset of fever (P less than 0.05). The PCV in all ponies became increased above base line during the diarrheic phase of EEC. Forty percent of the ponies developed anemia (PCV less than or equal to 23%) during the study. White blood cell counts were highly variable, with 80% of the ponies developing leukopenia (WBC less than 5,000/microliters) during the illness and 60% of the ponies developing leukocytosis (WBC greater than 14,000/microliters) after leukopenia was observed. Differential WBC changes varied widely and included neutropenia with a left shift, lymphopenia, and eosinopenia. Serial thrombocyte counts, which were done for only 1 pony, identified the development of marked thrombocytopenia. Some hematologic changes in ponies with EEC were similar to those reported in canine monocytic and equine granulocytic ehrlichioses. These data are discussed in the context of the pathogenesis and differential diagnosis of EEC.     

Efficacy of a canarypox-vectored recombinant vaccine expressing the hemagglutinin gene of equine influenza H3N8 virus in the protection of ponies from viral challenge

OBJECTIVE: To determine onset and duration of immunity provided by a 2- or 3-dose series of a new canarypox-vectored recombinant vaccine for equine influenza virus (rCP-EIV vaccine) expressing the hemagglutinin genes of influenza H3N8 virus strains A/eq/Kentucky/94 and A/eq/Newmarket/2/93 in ponies. ANIMALS: Forty-nine 1- to 3-year-old male Welsh Mountain Ponies that were seronegative for equine influenza virus. PROCEDURES: Vaccinated and control ponies were challenged with aerosolized influenza virus A/eq/Sussex/89 (H3N8), representative of the Eurasian lineage of circulating influenza viruses. In trial 1, control ponies and ponies that received rCP-EIV vaccine were challenged 2 weeks after completion of the 2-dose primary vaccination program. In trial 2, ponies were challenged 5 months after 2 doses of rCP-EIV vaccine or 1 year after the first boosting dose of rCP-EIV vaccine, administered 5 months after completion of the primary vaccination program. After challenge, ponies were observed daily for clinical signs of influenza and nasal swab specimens were taken to monitor virus excretion. RESULTS: The challenge reliably produced severe clinical signs consistent with influenza infection in the control ponies, and virus was shed for up to 7 days. The vaccination protocol provided clinical and virologic protection to vaccinates at 2 weeks and 5 months after completion of the primary vaccination program and at 12 months after the first booster. CONCLUSION AND CLINICAL RELEVANCE: The rCP-EIV vaccine provided protection of ponies to viral challenge. Of particular importance was the protection at 5 months after the second dose, indicating that this vaccine closes an immunity gap between the second and third vaccination.     

Nontransmission of bluetongue virus by embryos from bluetongue virus-infected sheep

Donor sheep were infected either by bites of bluetongue virus (BTV)-infected (serotype 11, "Texas Station strain") Culicoides variipennis or by inoculation with 100,000 median chicken embryo intravascular lethal doses of BTV (serotype 11) from a suspension made from infected C variipennis. Fourteen embryos from 4 BTV-infected ewes bred by rams not infected with BTV were transferred to 8 BTV-seronegative recipient ewes, and 35 embryos and 4 unfertilized eggs from 14 BTV-infected ewes bred by BTV-infected rams were transferred to 19 BTV-seronegative recipient ewes. Eleven pregnancies and 12 lambs resulted. None of the recipients or lambs seroconverted, and BTV was not isolated from the pregnant recipient ewes or their lambs at slaughter 30 days after parturition.     

Field performance of the equine parasiticide ivermectin in an oral paste

Ivermectin, a derivative of one of the avermectin compounds, was administered at 200 mcg per kg of body weight in an oral paste formulation to 80 mixed-breed ponies of both sexes and various ages. Twenty similar ponies received oral paste vehicle. Anthelmintic activity was determined by comparing fecal egg counts taken before and 14 days after ivermectin treatment to the counts of fecal samples from vehicle-treated controls. Commonly used equine vaccines were administered at the time of treatment. Sixteen of the 20 vehicle-treated ponies had positive counts prior to treatment and 17 were positive 14 days after treatment; 66 of the 80 ivermectin-treated ponies had positive counts prior to treatment; all 80 ponies had zero counts 14 days after treatment. The eggs were identified as strongylid in all the positive ponies while three ponies also hadOxyuris equi eggs prior to treatment.No adverse reactions were attributable to ivermectin oral paste treatment or concurrent vaccine administration.     

Immune response of ponies to experimental infection with Ehrlichia equi.

Four ponies experimentally infected with Ehrlichia equi developed substantial cell-mediated immune responses, as measured by the leukocyte migration-inhibition test. Serum anti-E equi antibodies up to 1:1,280 were demonstrated by the indirect fluorescent antibody test. Cell-mediated immune responses returned to a base-line value by day 200 after primary inoculation, but serum antibody titers persisted for at least 300 days after inoculation. Two additional susceptible ponies, which were inoculated with convalescent blood or organ homogenates from ponies recovered from acute equine ehrlichiosis, treated with tetracycline, and subsequently challenge exposed with E equi-infective blood, did not develop clinical disease. This study suggested that ponies are resistant to reinfection with E equi following clinical ehrlichiosis.     

Effect of equine ehrlichial colitis on the hemostatic system in ponies

Hemostatic function was determined in 10 ponies at various times after inoculation with Ehrlichia risticii to determine whether equine ehrlichial colitis (EEC) caused changes in the hemostatic system and to determine the prognostic value of hemostatic function tests during EEC. Mean platelet count; plasma fibrinogen, fibronectin, factor VIII: coagulant, alpha 2-antiplasmin, and plasminogen values; and serum concentrations of fibrin/fibrinogen degradation products changed significantly (P less than 0.05) from base line (day 0, before inoculation) during 18 days after inoculation with E risticii. Four ponies that died or were euthanatized because of severe clinical signs of EEC had significantly (P less than 0.05) greater mean plasma fibrinogen concentrations plasma factor VIII:coagulant values, and activated partial thromboplastin times immediately before death than did the 6 surviving ponies. Factor V concentrations were significantly (P less than 0.05) lower on postinoculation days 10 and 20 in nonsurvivors. Seemingly, changes in hemostasis took place during EEC. Ponies that did not survive EEC had greater laboratory evidence of coagulopathy.     

Infectious Center Assay of Intracellular Virus and Infective Virus Titer for Equine Mononuclear Cells Infected in vivo and in vitro with Equine Herpesviruses

A novel, simple method of infectious center assay was developed to detect and quantitate the intracellular existence of equine herpesvirus 1 and equine herpesvirus 2 in peripheral blood mononuclear cells infected in vivo and in vitro with the viruses by cocultivation of these cells with a permissive equine cell culture. The infectious center titers were correlated with the infectious virus titers. In vivo equine herpesvirus 1-infected mononuclear cells obtained from ponies experimentally infected with the virus and equine herpesvirus 2-infected mononuclear cells obtained from selected naturally infected ponies with the virus gave by infectious center assay a mean value of 67 infectious center/2 x 10⁶ cells as a peak titer on day 4 postinfection and 26 infectious center/2 x 10⁶ cells for equine herpesvirus 1 and equine herpesvirus 2 respectively. The mononuclear cells, in both cases, did not contain detectable infectious virus, but the infectious virus was detected from the respective cells when they were cultured in the presence of mitogen. The equine herpesvirus 1 infected mononuclear cells in culture gave a mean count of 8.05 x 10² infectious center/2 x 10⁶ cells/mL and contained 1.08 x 10⁴ plague assay/mL of infectious virus. Similarly the equine herpesvirus 2 infected mononuclear cells in culture gave a mean count of 7.1 x 10¹ infectious center/2 x 10⁶ cells/mL and contained <10¹ tissue culture infective dose₅₀/mL of infectious virus. Mononuclear cells infected in vitro with equine herpesvirus 1 gave a mean count of 9.3 x 10⁴ infectious center/2 x 10⁶ cells/mL and contained 5.75 x 10³ plaque assay/mL of infectius virus. Culturing these cells in the presence of mitogen gave a mean count of 5.5 x 10³ infectious center/2 x 10⁶ cells/mL and contained 9 x 10³ plague assay/mL of infectious virus. A correlation between infectious center assay and infectious virus assay is discussed.     

Detection of equine infectious anemia virus in a horse with an equivocal agar gel immunodiffusion test reaction

A horse whose serum reacted equivocally in the agar gel immunodiffusion (AGID) test for equine infectious anemia was studied over a 3-year period. The horse remained afebrile and virus was detected in only 1 of 6 horse inoculation tests. The intensity of AGID test reactions increased temporarily following this evidence for virus. Although the AGID test reaction was equivocal and 5 of the 6 transmission attempts failed, the 1 successful transmission proved the horse was infected.     

Equine infectious anemia in mules: virus isolation and pathogenicity studies

There appears to be a lack of information concerning responses of mules to natural infection or experimental inoculation with equine infectious anemia virus (EIAV). In the present study EIAV was isolated from mules, for the first time, and its pathogenicity in naturally infected and experimentally inoculated animals was investigated. Two naturally infected (A and B) and three EIAV free mules (C, D and E) were used for this purpose. Mule A developed clinical signs, whereas mule B remained asymptomatic until the end of the study. Mules C and D were each inoculated with 10ml of blood from mule A and developed signs of the disease; they were euthanatized or died at day 22 and 25 post-inoculation, respectively. Mule E served as a negative control. The virus was isolated from the plasma samples of mules with clinical signs of the disease (A, C and D), but not from the asymptomatic mule B. Both proviral DNA and viral RNA were amplified from blood and tissues of the infected animals by nested polymerase chain reaction (nPCR). Antibodies were not detected in the two experimentally infected mules until their natural death or euthanasia. Clinicopathological and laboratory findings showed that, in mules, EIAV produced clinical signs similar to those observed in horses and ponies. Nested PCR proved to be a rapid, sensitive and specific diagnostic method for the detection of EIAV, regardless of the disease stage.     

Duration of circulating antibody and immunity following infection with equine influenza virus

The duration of immunity as measured by virological, serological and clinical responses following infection with influenza A/equine/Newmarket/79 (H3N8) was assessed in repeated challenge experiments in which ponies were infected by exposure to aerosols of infectious virus. Previous infection stimulated complete clinical protection which persisted for at least 32 weeks as demonstrated by the absence of febrile responses and coughing in two groups of ponies infected 16 weeks or 32 weeks after the first infection. Partial clinical protection persisted for over a year as demonstrated by the absence of coughing and a reduction in the number of febrile responses in a group of ponies infected 62 weeks after their first infection. These results contrasted with those observed in immunologically naive control ponies which developed pyrexia, dyspnoea and nasal discharge and coughing. The kinetics of virus specific antibody production in primary and secondary infections with equine influenza were studied by the single radial haemolysis test and a radioisotopic antiglobulin binding assay which measured virus specific IgGab antibody isotype. Antibody to the haemagglutinin, as measured by the single radial haemolysis test, declined rapidly after primary infection whereas the IgGab responses to whole virus antigens persisted for longer. The single radial haemolysis test was therefore particularly useful for the detection of antibody responses in multiple infections or exposures to influenza antigens. The radioisotopic antiglobulin binding assay was more sensitive for identifying infections which had occurred more than six months previously, as evidenced by anamnestic IgGab responses in ponies with low levels of antibody before rechallenge.     

Inactivation of equine infectious anemia virus by chemical disinfectants.

Twelve chemicals and commercial disinfectants were tested for inactivation of equine infectious anemia virus. In the presence of 10% bovine serum, all chemicals inactivated 4 log10 (based on 0.1 ml) of the virus within 5 minutes at 23 C. A reduction of at least 4 log10 was observed when the virus was exposed for 1 minute to substituted phenolic disinfectants (3 commercial preparations and sodium orthophenylphenate), halogen derivatives (iodophor and sodium hypochlorite), chlorhexidine, and 70% ethanol. Sodium hydroxide (5%), 2% formalin, and 2% glutaraldehyde were slower to inactivate the virus, but achieved 4 log10 reduction in titer by 5 minutes' contact time. The susceptibility of the equine infectious anemia virus to chemical disinfectants is similar to that of other enveloped viruses.     

Equine cell-mediated immune response to Rhodococcus (Corynebacterium) equi

A lymphocyte blastogenic assay was developed to serve as an in vitro correlate of cell-mediated immunity to Rhodococcus (Corynebacterium) equi (R equi) in the equine species. Lymphocytes obtained from a group of experimental ponies showed no response in cell culture to R equi heat extract or lysozyme extract antigens. Ponies were assigned to groups for experimental inoculation. Three ponies were inoculated subcutaneously with live R equi, 3 were given live R equi by intranasal and intratracheal routes, and 4 ponies were left untreated. Lymphocytes from all inoculated ponies had a mitogenic response to R equi antigens in lymphocyte blastogenic assays performed between the 7th and 40th days after inoculation. Lymphocytes from noninoculated control ponies remained unresponsive to R equi antigens. Delayed-type hypersensitivity reactions developed in all experimentally exposed ponies after intradermal administration of the R equi antigen preparations. In a 2nd phase of experimentation, blastogenesis assays were performed on lymphocytes from horses in herds with endemic R equi infections. Results indicated that many of the animals had significant (stimulation index greater than 2) cell-mediated responses to the bacterium, but there was no distinct correlation between the immune response and clinical history. These data indicated that cell-mediated immunity is involved in the interaction of the equine immune system with R equi.     

2. Persistence of Equine Herpesviruses in Experimentally Infected Horses and the Experimental Induction of Abortion

An 8-month-old filly (No. 2) developed an acute vulvo-vaginitis and respiratory disease following inoculation of equine herpesvirus (EH virus) type 1 (EH 39 virus; equine rhinopneumonitis virus) into the vestibule of the vagina. The same virus produced acute respiratory disease but not balanoposthitis following intranasal, intravenous and intrapreputial inoculation of a 12-month-old colt (No. 3). A second 8-month-old filly (No. 1) developed a mild respiratory disease but not vulvo-vaginitis following intravestibular inoculation of EH 39 virus. EH viruses that were slowly cytopathic for equine foetal kidney cell cultures and serologically unrelated to the inoculated EH 39 virus were isolated from the buffy coat cells at 3 days and from the nasal cavity at 6 days after inoculation of horse No. 1. EH virus that was slowly cytopathic and serologically unrelated to EH 39 virus was isolated at 16 days from the vagina of the filly (No. 2) that developed acute vulvovaginitis and was frequently isolated from the nasal cavities of 2 of the 3 horses for 83 days and from the nasal cavity of the third horse for 57 days under conditions that precluded reinfection from other equidae except from each other. EH viruses were recovered from the 3 horses for a further 58 days under conditions where contact with other equidae may, although was not known to, have occurred between 83 and 141 days postinoculation. It was concluded that these viruses represented a single virus type that was present in the nasal cavity (designated EH 1–6 virus) perhaps also the blood stream of filly No. 1 at the time the 3 horses were purchased and that this virus was subsequently transmitted to the vagina of 1 and the nasal cavities of the other 2 horses. Accordingly a carrier state for EH 39 virus was not shown to occur. These findings are discussed in relation to the natural history of EH virus infections. Attempts to reactivate the EH viruses to cause clinical respiratory disease, by a series of injections of adrenalin and cortisone, were inconclusive. The 3 horses showed no clinical evidence of respiratory disease when they were reinfected intranasally with EH 39 virus 360 days (1 horse) and 412 days (2 horses) after the initial infection with this virus. Abortion was produced when EH 39 virus was inoculated directly into the allantoic or amniotic cavity of a pregnant mare although naturally occurring EH virus abortion remains unrecognised in Australia.