Praca oryginalna Original paper
The equine herpesvirus 1 (EHV-1) is the most important pathogen of all the equine herpesviruses discovered so far, which, unlike other types of viruses, may cause clinically diverse medical conditions, such as mild infections of the upper respiratory tract and sometimes lungs, abortions, foal deaths in the neonatal period, myeloencephalopathy and chorioretinopathy (10, 23, 24, 33-35, 37, 53, 58). The clinical diversity of EHV-1 infections stems from the ability of this microbe to induce cell-associated viraemia, thanks to which the virus may spread in various organs and systems (9, 35, 42, 45, 51, 53).
EHV-1 infections are common in equine popula-tions all over the world. The inducement of aborpopula-tions in pregnant mares and neurological disorders are the
most important health and economic consequences (14, 15, 35, 56). Infected mares may abort as early as in the 5th month of pregnancy, but most frequently abortion takes place between the 8th month and the estimated date of foaling (1). An increased susceptibility to infec-tions in mares and aborinfec-tions during the final period of gestation has not yet been convincingly accounted for (12). One possible explanation for this phenomenon assumes the existence of unidentified host factors that activate the latent virus located in the leucocytes (52, 54). More often than not, the abortions are abrupt and not preceded by any symptoms whatsoever, whereas their course is rather quick (15, 53-56). After abortion, there are usually no complications with the mares’ reproductive system (19, 20, 53).
Impact of natural EHV-1 infection and
non-specific immunostimulation of mares
on the reproductive effect
KATARZYNA HUMELT, ZBIGNIEW GRĄDZKI, ŁUKASZ JAROSZ Department of Epizootiology and Clinic of Infectious Diseases, Faculty of Veterinary Medicine,
University of Life Sciences in Lublin, Głęboka 30, 20-612 Lublin, Poland
Received 25.11.2014 Accepted 15.01.2015
Humelt K., Grądzki Z., Jarosz Ł.
Impact of natural EHV-1 infection and non-specific immunostimulation of mares on the reproductive effect
Summary
EHV-1 infections are common in equine populations all over the world. The inducement of abortions in pregnant mares and neurological disorders are the most important health and economic consequences. Currently used prophylactic schemes with inactivated vaccines have contributed to a reduction in mass abortions. Most horses, however, despite having been vaccinated, are prone to infections. A method alternative to specific prevention programmes is the use of immunostimulatory preparations stimulating the effector functions of the immune system, which leads to an anti-infective response. This is particularly important in case of an emergency abortion on breeding stud farms on which a specific EHV-1 infection prophylaxis has not been applied. The purpose of the study was to evaluate the impact of a natural EHV-1 infection and non-specific stimulation of the immune system in pregnant mares with the use of isoprinosine on the reproductive effect. The study was conducted in a group of 28 pregnant mares between 5 and 16 years of age, in a comparable gestation period. The control group consisted of 13 mares, which were not subjected to veterinary procedures. In the experimental group, comprising 15 mares, Isoprivet (Vet-Agro, Lublin) was administered at a dose of 10 mg/kg of body mass twice at a 4-day interval. Studies showed that isoprinosine significantly reduced the abortion rate, as well as the percentage of mares returning to oestrus or showing symptoms of genital tract infections. The improvement in reproductive rates testifies to an increase in the activity of Th cells and the monocyte-macrophage system, which prevent the development of the disease and stop the virus from entering the cells of the reproductive system. The results indicate the effectiveness of nonspecific immunostimulation in reducing losses associated with EHV-1 infection in pregnant mares.
The EHV-1 virus has a powerful effect on the host’s immune system, which results in the development of immunity to the infection (35). In recent years, some of the mechanisms of humoral and cell-mediated immunity engaged in fighting off EHV-1 infections have been discovered (2, 51). The most important role is played by the T cells which block virus replica-tion, among which cells with the CD8+ phenotype are responsible for cytotoxic activity (CTL) restricted to class I MHC (29, 32, 36).
Currently used prophylactic schemes with inac-tivated vaccines have contributed to a reduction in mass abortions. Most horses, however, despite having been vaccinated, are prone to infections. The complex EHV-1 development cycle and the use of specific mechanisms that enable the virus to survive in infected cells, such as the latency inducement ability, are a major obstacle in developing efficacious vaccines that could provide protection against infections and reduce the intensity of clinical symptoms (55). This fact, together with the lack of the possibility of etiotropic treatment, fosters the search for alternative methods of affecting the immune system. One of such methods is the use of immunostimulatory preparations stimulating the effector functions of the immune system, which leads to an anti-infective response (17, 40).
A synthetic immunostimulant frequently used in clinical practice is isoprinosine (methisporinol, inosine pranobex), which proves to have immunomodula-tory and antiviral effects (47). Isoprinosine works by increasing the proliferative activity of the T cells and macrophages, which results in an enhanced immune response (28). With regard to viral infections, it has been established that isoprinosine increases the concentration of specific antibodies and stimulates the mitogen-induced blastic transformation of lym-phocytes (28). The efficacy of isoprinosine has been repeatedly proven in clinical studies (25, 26, 48), but the mechanism of the antiviral activity of the prepara-tion in relaprepara-tion to herpesviruses is not fully known (18, 39). There are indications, however, that it stems from the modulation of the immune reactions of the body in response to contact with the germ, and not from a direct antiviral effect.
The purpose of the study was to evaluate the impact of a natural EHV-1 infection and a non-specific stimu-lation of the immune system of pregnant mares by isoprinosine on the reproductive effect.
Material and methods
Stud farm. The study was performed over a period of
one breeding season between January and April on a Thor-oughbred stud farm with a stock of 130 horses, including 60 breeding mares. In the study period, there were in total 50 pregnant mares in the breeding stable, aged from 5 to 16 years, at different stages of gestation. At the beginning of the breeding season, between December and March, 22 mares gave birth to healthy foals. The births that preceded
an abortion storm took place on the estimated dates of foal-ing, without complications. The newly born foals sucked colostrum and milk unaided, and their body mass growth was appropriate for their age bracket. From among the stock of 50 mares in the breeding stable, 48 were the property of the stud farm. At the beginning of March, the stock was supplemented with two pregnant mares from outside, which had no clinically discernible symptoms of the disease at the moment of being introduced into the stable. Mares before foaling, mothers with newly born foals and the newly intro-duced mares were kept in separate boxes on the premises of the common foaling stable and fed according to accepted standards. The analysis of the zoohygienic conditions in the stable, technical conditions of the premises and the working style of the staff ruled out any stress factors that might have possibly affected the condition of the animals or the potential of their immunity. In previous breeding seasons, regular herd healthcare programmes were run, which included prophylactic vaccinations against influenza, tetanus and virus-induced abortions. For reasons beyond the control of the farm stud owner, in the season when the study was conducted vaccinations against herpesvirus infections (EHV-1 and EHV-4) in pregnant mares were not performed.
The course of the abortion storm on the stud farm.
From the beginning of December until the outbreak of the disease on 12 March, 22 mares gave birth to healthy foals on the estimated date of foaling. At the moment of the out-break the other 28 mares were about to foal. The abortion storm began on 12 March, when two mares aborted during one night, one and two months before the estimated date of labour. Within the next 4 days, until 16 March, 4 further mares aborted, one week, one month (two mares) and one and a half months before the estimated date of foaling. In the meantime, on 15 March, a foal was born with discernible signs of acute asthenia, jaundice and respiratory disorder, and died within 24 hours, whereas on 16 March one of the mares gave birth to a stillborn foal. A flow chart showing the abortion storm in the given period is shown in Table 1. One of the two mares newly introduced into the stable was among the mares that also aborted. No symptoms preceded the abortion, and there were no complications in mares after the expulsion of the foetuses. Clinical observations of the mares in the post-abortive period indicated that most of them had oestrus symptoms, and there were new instances of mating. However, in some mares it was found that the return to oestrus was indicative of fertility disorders.
Groups of animals. The study was conducted in a group
of 28 pregnant mares between 5 and 16 years of age, in
Tab. 1. The course of the abortion storm on the stud farm
Time left to parturition Condition of the foetus/foal Number of mares 2 months dead 1 1.5 month dead 1 1 month dead 3
1 week dead 1
Due date alive* 1 Due date dead 1
Explanation: * – a foal with acute asthenia, dead within 24 hours of birth
a comparable gestation period with individual dif-ferences of up to 3 weeks. The mares were divided into 2 groups. The control group, group 1, comprised 13 mares with no veterinary procedures applied. In the study period, there were abortions and due date births of feeble or stillborn foals in this group. The study group, group 2, comprised 15 pregnant mares kept in boxes on the premises of a common breeding stable. Taking into account that no specific prophy-laxis of herpesvirus infection had been implemented on the stud farm, as well as the cases of abortion occurring in the breeding stables, the mares in that group were administered Isoprivet (Vet-Agro, Lublin), which is a 20% aqueous solu-tion of isoprinosine for injecsolu-tion. The immunostimulant was administered in intramuscular injections at a dose of 10 mg/ kg of body mass, twice at a 4-day interval.
Anatomopathological examination. Nine aborted
foe-tuses were subjected to anatomopathological examination, along with two foals born on the estimated date of labour, one of which was a stillborn and the other a feeble foal, which died within 24 hours of birth. In the group of aborted foetuses, there was one 9-month abortion, four 10-month abortions and four 11-month abortions. The examination included the aborted foetus from the mare that had been newly introduced into the stable. All foetuses were well-developed in accordance with their age.
PCR examination for the presence of equine herpes-virus type 1 (EHV-1) genetic material. The material for
the isolation of nucleic acids was a homogenate of internal organs of the aborted foetus and fragments of the placenta. The standard extraction method included Proteinase K, phenol and chloroform. The isolated nucleic acid was stored frozen at a temperature of –20°C. Each time, parallel to the examination of samples, a DNA extraction control proce-dure was performed with the use of the reference strain suspension of the EHV1 Ab4 virus cultured in a continu-ous cell line, obtained from the collection of the National Veterinary Research Institute in Puławy. Primer sequences were selected based on the literature data (37) from two con-servative regions within the glycoprotein C (gC) gene and the 76 (g76) gene of the equine herpesviruses genome. The assumed reaction parameters made it possible to amplify the DNA simultaneously within both regions. As a result, reaction products of different size were obtained for each amplified region. This method made it possible to identify the EHV-1 DNA and to differentiate it from the closely related equine herpesvirus type 4 (EXV-4). The simultane-ous amplification of both regions also made it possible to confirm the reaction specificity. The amplification products were analysed electrophoretically in 1.5% agar gel (Sigma, USA) with the addition of the mass standard (100bp DNA, Fermentas, Lithuania). The result of the PCR reaction was regarded as positive in the presence of single DNA bands in the gel of a size defined by the location of the particular pairs of primers in the genome.
Results and discussion
The effect of the natural equine herpesvirus type 1 (EHV-1) infection and the non-specific stimulation of the immune system by isoprinosine on the reproductive
rate, measured as the number of births and abortions, as well as the number of stillborn or feeble foals, is summarised in Table 2.
According to the data in Table 2, in the group of mares that was not subjected to immunostimulation with the use of isoprinosine (gr. 1), 6 out of 13 mares aborted, one gave birth to a stillborn on the estimated date of labour, and one gave birth to a feeble foal, which died within 24 hours. All the aborted foetuses and the stillborn foal were subjected to clinical evaluation and anatomopathological examination, the results of which gave rise to the suspicion of equine herpesvirus type 1 infection. The anatomopathological examina-tion revealed that all foetuses had a strongly yellowed hypodermis and pericardial sac, large amounts of a yel-lowish exudate in the abdominal and thoracic cavities, ecchymosis of internal organs and fibrinous pleurisy (Fig. 3). On the liver surface, there were discoloured areas and fine, pinhead-sized grey-white subcapsular necrotic foci (Fig. 2). The results of the bacteriologi-cal and mycologibacteriologi-cal examination of internal organs were negative.
The suspicion of the disease was confirmed by the results of virological examination, as a PCR-based test revealed the presence of EHV-1 genetic material in sections from pathologically affected internal organs (Fig. 1).
Tab. 2. The impact of natural EHV-1 infection and isoprinosine on the reproductive effect
Group immunostimulationNon-specific Number of mares Number of births Number of abortions Number of foalssb./f. 1 – 13 5* 6 1 + 1
2 + 15 12* 3 0
Explanations: * – spontaneous birth on due date; sb. – stillborn; f – feeble
Fig. 1. Products of the PCR reaction obtained on the DNA matrix of wild-type EHV-1 strains with the use of primers complementary for the regions within the glycoprotein C (gC) gene and the 76 (g76) gene
Explanations: M – molecular mass marker (100 bp DNA ladder, MBI Fermentas, Lithuania); K– – negative control; K+ – positive control; no. 1, 2, 3, 4, 5, 6, 7 – DNA of EHV-1 strains from internal organs of the aborted foetuses and the placenta (no. 1, 4 – liver, no. 2, 5, 6 – lungs, no. 3, 7 – placenta)
In the group of 15 mares that had been subjected to immunostimulation (gr. 2), 12 mares gave birth to healthy foals on the estimated date of foaling (Tab. 2). The remaining 3 mares aborted within 3-4 days of receiving the first dose of the immunostimulant. The anatomopathological examination of the aborted foetuses and laboratory tests of the samples of their internal organs by the PCR method confirmed EHV-1 infection.
The economic significance of EHV1 infections stems from the possibility of inducing infrequent or massive abortions in pregnant mares (15, 20, 35, 48). An EHV-1-induced abortion is usually a single occur-rence, with very few instances of mares aborting in their subsequent pregnancies (53). This pattern seems to be corroborated by the analysis of the medical his-tory and documentation of the stud farm, which proved that none of the mares under study had an EHV-1-induced abortion in previous breeding seasons.
A productive EHV1 infection on stud farms usu-ally develops very quickly, so an immediate reaction to minimize economic losses is crucial here. In the period before abortion, the mares showed no symp-toms, which impeded the anticipation of the threat and delayed therapeutic management (53). Intervention measures in abortion threat emergencies should be aimed at reducing or inhibiting leucocyte-associated viraemia, which can be facilitated by an efficient immune system, especially cell-mediated cytotoxic mechanisms (2, 12, 36, 41).
The body defence system against an EHV-1 infec-tion is complex and consists of a multi-component response (2, 7, 8, 36). The response comprises local immunity mechanisms of the mucous membrane as well as systemic immunity with neutralising antibod-ies and cytotoxic T cells (CTL) (41, 57). The stimula-tion of all the above-menstimula-tioned lines of defence by the available vaccines has proved impossible (35, 51). A limited efficacy of EHV-1 vaccines has been partly demonstrated by the results of the authors’ own
research, which showed that, despite regular vacci-nations against herpesvirus infections performed in previous breeding seasons on the stud farm, in the first non-vaccination season an abortion storm caused foal losses in 22% of pregnant mares. It should be noted that a complete eradication of EHV-1 infections on stud farms is impossible, because of the sustainability of the virus in the organism, in a form beyond the immune control of the host (4-6, 13, 16, 21, 50, 51, 59). It is for this reason that greater attention is given to the possibilities of stimulating non-specific anti-infective immunity, which may provide an effective antiviral barrier in case of the introduction of the germ into the herd or the reactivation of latent infection. The reduction or inhibition of the cell-associated viremia should, in theory, constitute a crucial element of the preventive strategy against abortions and, presumably, also neurological disorders (22, 52, 53, 55).
The available literature fails to provide reports on the effects of isoprinosine on EHV-1 infections in horses. This medicine is commonly used to alleviate clinical symptoms and to fight off infections caused by
Herpes simplex in people. The efficacy of isoprinosine
in counteracting herpesviruses was proved, among others, by Gordon et al. (27). In the assessment of their research results, these authors are inclined to accept the commonly promoted thesis that the anti-herpesvirus properties of this medicine are related more to the modification of the function of the host’s ribosomes, which results in preventing or delaying the translation of viral mRNA, than to a direct disturbance of the translation process.
Isoprinosine administered to mares in the authors’ own research significantly reduced the abortion rate, as well as the percentage of mares returning to oestrus or showing symptoms of genital tract infections. The results obtained indicate that the non-specific immuno-stimulation of pregnant mares is effective in reducing losses associated with EHV-1 infections. In view of the available literature and the lack of similar
clini-Fig. 2. Small gray-white subcapsular necrotic foci in the liver Fig. 3. Yellowing pulmonary pleura and large amounts of a yellowish exudate accumulated in the thoracic cavity
cal observations and studies, it is to be assumed that the improvement in reproductive rates testifies to an increase in the activity of Th cells and the monocyte-macrophage system in the bodies of infected mares, which prevent the development of the disease and stop the virus from entering the cells of the reproductive system (30, 31, 38, 46, 49). Isoprinosine presumably had an effect on the stability of the response of the infected body by curtailing the inflammatory response thanks to the contribution of regulatory Treg cells and CD25+FoxP3+ lymphocytes, or the increase in broad-spectrum protective immune response which is not accompanied by the damaging of tissues (44). Isoprinosine, used in the study, probably stimulated the host’s natural antiviral mechanisms by inducing the release of cytokines, such as IFN-α, TNF-α and IL12, and by activating NK cells and T cells with the Th1 profile (3, 11, 12, 43). The validation of these hypotheses requires further research, as it cannot be ruled out that the observed positive effect of isoprino-sine may have been due to other factors on the part of the infected organism.
The results obtained indicate that isoprinosine has a favourable influence on the reproductive effect in mares with a natural EHV-1 infection. The reduction in mass abortions after isoprinosine administration in this study demonstrates that isoprinosine may supplement programmes of specific prophylaxis against EHV-1 infections on stud farms in surveillance zones.
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Corresponding author: Prof. dr hab. Zbigniew Grądzki, Głęboka 30, 20-612 Lublin, Poland; e-mail: zbigniew.gradzki@up.lublin.pl
