Experimental equine herpesvirus-1 infection in llamas (Lama glama).

Three llamas (Lama glama) were experimentally infected intranasally with an isolate of equine herpesvirus-1 (EHV-1) from the brain of an alpaca that had experienced severe neurologic signs. Two of the 3 llamas developed severe neurologic disorders following inoculation; 1 died, and 1 was euthanized in a moribund state. The third llama showed only mild neurologic signs. The euthanized llama had preexisting antibodies to EHV-1, and the remaining 2 llamas were seronegative (virus neutralization titer less than 6) at the time of inoculation. One of the seronegative llamas died acutely without production of detectable antibodies, and the other developed antibodies typical of a primary immune response. The EHV-1 virus was recovered only from a sample of the thalamus of the llama that died acutely. Histopathologic lesions were observed in the brain and retina of the dead and euthanized animals. This study verifies the pathogenic potential of EHV-1 for llamas.     

Longevity of protective immunity to experimental bovine herpesvirus-1 infection following inoculation with a combination modified-live virus vaccine in beef calves

OBJECTIVE: To determine whether a combination viral vaccine containing modified-live bovine herpesvirus-1 (BHV-1) would protect calves from infection with a recent field isolate of BHV-1. DESIGN: Randomized controlled trial. ANIMALS: Sixty 4- to 6-month-old beef calves. PROCEDURE: Calves were inoculated with a placebo 42 and 20 days prior to challenge (group 1; n = 10) or with the combination vaccine 42 and 20 days prior to challenge (group 2; 10), 146 and 126 days prior to challenge (group 3; 10), 117 and 96 days prior to challenge (group 4; 10), 86 and 65 days prior to challenge (group 5; 10), or 126 days prior to challenge (group 6; 10). All calves were challenged with BHV-1 via aerosol. Clinical signs, immune responses, and nasal shedding of virus were monitored for 14 days after challenge. RESULTS: Vaccination elicited increases in BHV-1-specific IgG antibody titers. Challenge with BHV-1 resulted in mild respiratory tract disease in all groups, but vaccinated calves had less severe signs of clinical disease. Extent and duration of nasal BHV-1 shedding following challenge was significantly lower in vaccinated calves than in control calves. In calves that received 2 doses of the vaccine, the degree of protection varied with the interval between the last vaccination and challenge, as evidenced by increases in risk of clinical signs and extent and duration of viral shedding. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that this combination vaccine provided protection from infection with virulent BHV-1 and significantly reduced nasal shedding of the virus for at least 126 days after vaccination.     

Antemortem detection of latent infection with neuropathogenic strains of equine herpesvirus-1 in horses

OBJECTIVE: To evaluate a technique for identifying horses latently infected with neuropathogenic strains of equine herpesvirus-1 (EHV-1). ANIMALS: 36 adult mares, 24 of which were experimentally infected as weanlings with neuropathogenic or nonneuropathogenic EHV-1. PROCEDURES: Mandibular lymph node (MLN) tissue was obtained from each horse via biopsy during general anesthesia. Purified DNA from MLNs was tested for EHV-1 DNA by use of a magnetic bead, sequencecapture, nested PCR assay. For MLNs that contained EHV-1 DNA, the 256-bp DNA fragments amplified via sequence-capture nested PCR were sequenced to determine the nucleotide at the polymorphic site that determines pathotype (ie, neuropathotype [G(2254)] or non-neuropathotype [A(2254)]). RESULTS: Latent viral DNA was detected in 26 of the 36 (72%) mares tested. Neuropathogenic and nonneuropathogenic EHV-1 genotypes were detected in the latently infected horses. In each mare previously infected with known EHV-1 pathotypes, the open reading frame 30 genotype of latent EHV-1 was identical to that of the strain that had been inoculated 4 to 5 years earlier. Latent viral DNA was detected in 10 of the 12 mares that were inoculated as weanlings with neuropathogenic strains of EHV-1. The detection rate of the sequence-capture PCR method for EHV-1 latency was double that of conventional nested or realtime PCR assays performed on the same MLN DNA preparations. CONCLUSIONS AND CLINICAL RELEVANCE: The magnetic bead, sequence-capture, nested PCR technique enabled low-threshold detection of DNA from latent neuropathogenic strains of EHV-1 in MLN specimens from live horses. The technique may be used to screen horses for latent neuropathogenic EHV-1 infection.     

Effects of human alpha interferon on experimentally induced equine herpesvirus-1 infection in horses

The immunotherapeutic effect of low-dose human alpha interferon on viral shedding and clinical disease was evaluated in horses inoculated with equine herpesvirus-1 (EHV-1). Eighteen clinically healthy weanling horses, 5 to 7 months old, were allotted to 3 equal groups. Two groups were treated orally with human alpha-2a interferon (0.22 or 2.2 U/kg of body weight), on days 2 and 1 before inoculation with EHV-1, the day of inoculation, and again on postinoculation day 1. The horses of the remaining group were given a placebo orally on the same days. The horses were monitored daily for changes in body temperature and for clinical signs of respiratory tract disease. Blood and nasal swab specimens were collected daily for virus isolation. Blood was also collected at intervals throughout the monitoring period for evaluation of CBC, serum IgG and IgM concentrations, and antibody titers to EHV-1. Febrile responses, nasal discharge, viral shedding, changes in CBC, and an increase in antibody titers to EHV-1 were noticed in all horses after inoculation. There was no significant difference (P greater than 0.05) in mean values of the factors measured between treatment and control groups.     

Prevalence of equine herpesvirus-1 infection among Thoroughbreds residing on a farm on which the virus was endemic

OBJECTIVE: To determine the incidence of equine herpesvirus-1 (EHV-1) infection among Thoroughbreds residing on a farm on which the virus was known to be endemic. DESIGN: Prospective cohort study. ANIMALS: 10 nonpregnant mares, 8 stallions, 16 weanlings, 11 racehorses, and 30 pregnant mares and their foals born during the 2006 foaling season. PROCEDURES: Blood and nasopharygeal swab samples were collected every 3 to 5 weeks for 9 months, and placenta and colostrum samples were collected at foaling. All samples were submitted for testing for EHV-1 DNA with a PCR assay. A type-specific EHV-1 ELISA was used to determine antibody titers in mares and foals at birth, 12 to 24 hours after birth, and every 3 to 5 weeks thereafter. RESULTS: Results of the PCR assay were positive for only 4 of the 1,330 samples collected (590 blood samples, 590 nasopharyngeal swab samples, 30 placentas, and 30 colostrum samples), with EHV-1 DNA detected in nasal secretions from 3 horses (pregnant mare, stallion, and racehorse) and in the placenta from 1 mare. Seroconversion was detected in 3 of 27 foals during the first month of life. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that there was a low prevalence of EHV-1 infection among this population of Thoroughbreds even though the virus was known to be endemic on the farm and that pregnant mares could become infected without aborting. Analysis of nasopharyngeal swab samples appeared to be more sensitive than analysis of blood samples for detection of EHV-1 DNA.     

Nasal immunization with major epitope-containing ApxIIA toxin fragment induces protective immunity against challenge infection with Actinobacillus pleuropneumoniae in a murine model

Actinobacillus pleuropneumoniae is an infective agent that leads to porcine pleuropneumonia, a disease that causes severe economic losses in the swine industry. Based on the fact that the respiratory tract is the primary site for bacterial infection, it has been suggested that bacterial exclusion in the respiratory tract through mucosal immune induction is the most effective disease prevention strategy. ApxIIA is a vaccine candidate against A. pleuropneumoniae infection, and fragment #5 (aa. 439–801) of ApxIIA contains the major epitopes for effective vaccination. In this study, we used mice to verify the efficacy of intranasal immunization with fragment #5 in the induction of protective immunity against nasal challenge with A. pleuropneumoniae and compared its efficacy with that of subcutaneous immunization. Intranasal immunization of the fragment induced significantly higher systemic and mucosal immune responses measured at the levels of antigen-specific antibodies, cytokine-secreting cells after antigen exposure, and antigen-specific lymphocyte proliferation. Intranasal immunization not only efficiently inhibited the bacterial colonization in respiratory organs, but also prevented alveolar tissue damage in infectious condition similar to that of a contaminated pig. Moreover, intranasal immunization with fragment #5 provided acquired protective immunity against intranasal challenge with A. pleuropneumoniae serotype 2. In addition, it conferred cross-protection against serotype 5, a heterologous pathogen that causes severe disease by ApxI and ApxII secretion. Collectively, intranasal immunization with fragment #5 of ApxIIA can be considered an efficient protective immunization procedure against A. pleuropneumoniae infection.     

The equine immune response to equine herpesvirus-1: the virus and its vaccines

Equine herpesvirus-1 (EHV-1) is an alphaherpesvirus which infects horses, causing respiratory and neurological disease and abortion in pregnant mares. Latency is established in trigeminal ganglia and lymphocytes. Immunity to EHV-1 lasts between 3 and 6 months. Current vaccines, many of which contain inactivated virus, have reduced the incidence of abortion storms in pregnant mares but individual animals, which may be of high commercial value, remain susceptible to infection. The development of effective vaccines which stimulate both humoral and cellular immune responses remains a priority. Utilising data generated following experimental and field infections of the target species, this review describes the immunopathogenesis of EHV-1 and the interaction between the horse's immune system and this virus, both in vivo and in vitro, and identifies immune responses, highlighting those which have been associated with protective immunity. It then goes on to recount a brief history of vaccination, outlines factors likely to influence the outcome of vaccine administration and describes the immune response stimulated by a selection of commercial and experimental vaccines. Finally, based on the available data, a rational strategy designed to stimulate protective immune responses by vaccination is outlined.     

Duration of protective immunity and antibody responses in cattle immunised against alcelaphine herpesvirus-1-induced malignant catarrhal fever

ABSTRACT: Protection of cattle from alcelaphine herpesvirus-1 (AlHV-1)-induced malignant catarrhal fever (MCF) has been described previously, using an attenuated virus vaccine in an unlicensed adjuvant. The vaccine was hypothesised to induce a protective barrier of virus-neutralising antibody in the oro-nasal region, supported by the observation of high titre neutralising antibodies in nasal secretions of protected animals. Here we describe further analysis of this vaccine strategy, studying the effectiveness of the vaccine formulated with a licensed adjuvant; the duration of immunity induced; and the virus-specific antibody responses in plasma and nasal secretions. The results presented here show that the attenuated AlHV-1 vaccine in a licensed adjuvant protected cattle from fatal intranasal challenge with pathogenic AlHV-1 at three or six months. In addition, animals protected from MCF had significantly higher initial anti-viral antibody titres than animals that succumbed to disease; and these antibody titres remained relatively stable after challenge, while titres in vaccinated animals with MCF increased significantly prior to the onset of clinical disease. These data support the view that a mucosal barrier of neutralising antibody blocks infection of vaccinated animals and suggests that the magnitude of the initial response may correlate with long-term protection. Interestingly, the high titre virus-neutralising antibody responses seen in animals that succumbed to MCF after vaccination were not protective.     

Prevalence of equine herpesvirus-1 and equine herpesvirus-4 infections in equidae species in Turkey as determined by ELISA and multiplex nested PCR

In this report we examined the presence of specific antibodies against equine herpesvirus type 1 (EHV-1), and equine herpesvirus type 4 (EHV-4) in several equidae, including mules, donkeys, horses. The presence of EHV-1 and EHV-4 in respiratory diseases of equids, and ability of multiplex nested polymerase chain reaction (PCR) screening in simultaneous diagnosis of horses acutely infected by EHV-1 and EHV-4 were also investigated. Sera from 504 horses, mules and donkeys sampled were tested for the presence of EHV-1 and EHV-4 specific antibodies. Blood samples taken from 21 symptomatic horses and nasal swabs taken from 40 symptomatic horses were tested for the presence of EHV-1 and EHV-4 by a multiplex nested PCR. A total of 14.3% (3/21) of buffy coat samples and 32.5% (13/40) nasal swab samples were found to contain EHV-1 DNA, while 19% (4/21) buffy coat samples and 22.5% (9/40) nasal swab samples were found to be positive for EHV-4 DNA. By species, 14.5% of horses, 37.2% of mules and 24.2% of donkeys tested were EHV-1 seropositive. EHV-4 specific antibodies were detected in 237 (81.7%) of 290 horse sera tested. Results from this investigation demonstrate that EHV-1 and EHV-4 are prevalent throughout the equid population, and that donkeys and mules might also represent an important source of infection for other equids. We also showed that the multiplex nested PCR assay might be useful for diagnosis of mixed respiratory infections in horses due to EHV-1 and EHV-4.     

Characterization of a murine model of intranasal infection suitable for testing vaccines against C. abortus

Mouse models have been widely used to test candidate vaccines against Chlamydophila abortus infection in mice. Although the induction of a systemic infection by endogenous or intraperitoneal inoculation is a useful tool for understanding the immune mechanism involved in the protection conferred by the vaccination, a different approach is necessary to understand other factors of the infection, such as mucosal immunity or the colonization of target organs. To test whether C. abortus intranasal model of infection in mice is a useful tool for testing vaccines in a first group of experiments mice, were infected intranasally with C. abortus to characterize the model of infection. When this model was used to test vaccines, two inactivated experimental vaccines, one of them adjuvated with QS-21 and another with aluminium hydroxide, and a live attenuated vaccine (strain 1B) were used. Non-vaccinated control mice died within the first 8 days, after displaying substantial loss of weight. Histologically, the mice showed lobar fibrinopurulent bronchointerstitial pneumonia. Prior immunization with QS-21 adjuvated vaccine or 1B vaccine presented mortality and the recipients showed a greater number of T cells in the lesions, especially CD8(+) T cells, than the control mice and mice immunized with vaccine adjuvated with aluminium hydroxide. The results confirm that the C. abortus intranasal model of infection in mice is a useful tool for testing vaccines.     

Bovine herpesvirus-1 infection of cattle: kinetics of antibody formation after intranasal exposure and abortion induced by the virus

The kinetics of antibody formation in Holstein heifers after primary and secondary intranasal inoculation of bovine herpesvirus-1 (BHV-1) and after BHV-1-induced abortion was determined. Sera were fractionated by gel filtration and ion-exchange chromatography. The antibody activity within serum immunoglobulin (Ig) isotypes was assessed, using a plaque-reduction neutralization assay. The primary immune response to BHV-1 infection was characterized by the appearance of IgM and IgG antibodies in serum by postinoculation day (PID) 7. Maximal IgG antibody activity occurred at PID 35 in nonpregnant heifers and at PID 14 in pregnant heifers. Thereafter, IgG antibody activity declined slowly in both groups of heifers. Maximal IgM antibody activity occurred at PID 14 in both groups of heifers and declined rapidly thereafter. The IgG antibody activity during primary immune responses was restricted to the IgG1 subclass. Secondary responses were characterized by anamnestic IgG antibody responses. Antibody activity was present within the IgG1 and IgG2 subclasses during secondary immune responses, but the increase in antibody activity during this period was primarily in the IgG2 subclass. Secondary IgM antibody formation was elicited by abortion induced by the intra-amniotic inoculation of BHV-1, but not by reexposure by the intranasal route. Abortion occurred in 1 heifer 28 days after intranasal BHV-1 inoculation. Abortion in this heifer was not associated with a secondary antibody response. The nature of BHV-1 antigenic exposure in the bovine determined the relative distribution of anti-BHV-1 antibody activity in serum IgM, IgG1, and IgG2. The formation of IgM antibody, with the exception of secondary intranasal exposure, indicated recent BHV-1 antigenic exposure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival of equine herpesvirus-4, feline herpesvirus-1, and feline calicivirus in multidose ophthalmic solutions

Objective To determine survival over time of infectious equine herpesvirus‐4, feline herpesvirus‐1, and feline calicivirus in three commercially available and commonly used ophthalmic solutions (eyewash, fluorescein, and proparacaine HCl). Sample population Viruses used in this study were originally isolated from eyes of animals referred to the University of Illinois. Equine herpesvirus‐4 was propagated in MDBK cells and feline herpesvirus‐1 and feline calicivirus in CRFK cells. Procedure After separately inoculating a designated solution with a specific titer of an individual virus, solutions were incubated per manufacturer's recommendations, either at 4 °C or 25 °C. Virus titers within solutions were subsequently measured at 1, 8, and 24 h and 3, 5 and 7 days post inoculation using either plaque or TCID₅₀ assays. Results Equine herpesvirus‐4, feline herpesvirus‐1, and feline calicivirus were present in eyewash for 7 days, 5 days, and 7 days, respectively. Eyewash did not decrease survival time of any virus when compared to controls. Equine herpesvirus‐4 and feline herpesvirus‐1, both enveloped viruses, were not recovered at any time ≥ 1 h post inoculation in fluorescein. Feline calicivirus, a nonenveloped virus, was present in fluorescein for 7 days. Equine herpesvirus‐4 and feline herpesvirus‐1 did not remain infectious in proparacaine at any time ≥ 1 h post inoculation, but feline calicivirus was recovered at up to 24 h post inoculation. Conclusions Equine herpesvirus‐4, feline herpesvirus‐1, and feline calicivirus may be readily transmissible via the eyewash solution used in this study. Risk of iatrogenic transmission of the three viruses used in this study was significantly reduced in both fluorescein and proparacaine solutions. Feline calicivirus, the only nonenveloped virus evaluated, remained viable longer in both fluorescein and proparacaine solutions.     

Serological responses of specific pathogen-free foals to equine herpesvirus-1: primary and secondary infection, and reactivation.

Serum antibody (virus neutralisation, complement fixation, IgM and IgG) responses to equine herpesvirus-1 (EHV-1) infection were measured in six foals which were initially free from EHV-1 and EHV-4 infection and maternally-derived antibodies. Following primary infection, high titres of virus neutralisation and complement fixation antibodies were detectable against EHV-1, however, corresponding antibody levels against EHV-4 were low or inapparent, although the two viruses share a number of cross-reactive epitopes. In addition, following the primary infection with EHV-1, IgM levels increased before those of IgG, virus neutralisation and complement fixation antibodies, peaked sooner and thereafter declined. Stimulation of IgM levels was observed on secondary infection with EHV-1 given 61 days later. In contrast, IgG, virus neutralisation and complement fixation antibodies following primary infection were more sustained and no increase in their levels was observed on secondary infection. No consistent changes in IgM or IgG levels were seen after administration of dexamethasone to reactivate latent virus.     

Safety and efficacy of a thymidine kinase negative equine herpesvirus-1 vaccine in young horses.

A drug induced equine herpesvirus-1 (EHV-1) mutant lacking thymidine kinase inducing activity was developed and evaluated as a vaccine. The safety and effectiveness of the vaccine to protect against experimentally induced EHV-1 respiratory disease were evaluated in weanling horses free of EHV-1 neutralizing antibody. The vaccine was safe when administered either intramuscularly or intravenously, and EHV-1 was not shed intranasally during the 12 days following administration. Intranasal challenge with virulent EHV-1 was used to evaluate vaccine efficacy. Following challenge, there was a significantly (p less than 0.05) greater increase in peak body temperatures and duration of nasal virus shedding in the nonvaccinates, and a significant (p less than 0.05) increase in serum neutralizing antibody titers in the vaccinates.     

Review of two viral agents of economic importance to the equine industry (equine herpesvirus-1, and equine arteritis virus)

Equine herpesvirus type-1 (EHV-1) and equine arteritis virus (EAV) are infectious agents that cause serious health risks to horse populations and are disbursed worldwide, which can lead to significant financial losses. In addition to being responsible for abortion and neonatal death, these viruses are associated with respiratory illness. Although previous research and reviews have been written on these viruses, both viruses still affect horse populations around the world and the vaccines currently available are not completely protective, especially against EHV-1 and equine herpes myeloencephalopathy (EHM). Moreover, EAV is considered a threat to the $102 billion equine industry in the United States. As a result, these viruses represent a huge threat to the horse industry and efforts geared towards preventing the outbreak of the disease are strongly encouraged. For this reason, updates about these viruses are necessary and require more and more discussion on the nature and characteristics of these viruses to know how to overcome them. Prevention and control of abortion and neonatal foal death caused by each of the two viruses depend on appropriate management strategies coupled with prophylactic vaccination. This review presents the latest detailed information on EHV-1 and EAV from several aspects such as transmission, clinical signs, pathogenesis, latest developments on the treatment of the diseases, vaccination, and finally challenges and future perspectives. The information presented herein will be useful in understanding EHV-1 and EAV and formulating policies that can help to limit the spread of these viruses within horse populations.