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Arquivo Brasileiro de Medicina Veterinária e Zootecnia

Print version ISSN 0102-0935On-line version ISSN 1678-4162

Arq. Bras. Med. Vet. Zootec. vol.52 n.3 Belo Horizonte June 2000

https://doi.org/10.1590/S0102-09352000000300003 

Use of an ELISA system for detection of equine herpesvirus 1 (EHV-1) antibodies in non-symptomatic pregnant mares and neonatal foals

[Uso de um sistema de ELISA para detecção de anticorpos contra herpesvírus eqüino 1 (HVE-1) em éguas gestantes assintomáticas e potros recém-nascidos]

 

R. Carvalho1, L.M.F. Passos2*, A.M.G. Gouvea2, M. Resende3
A.S. Martins3, G.C. Franco4

1Ministério da Agricultura e do Abastecimento
2Escola de Veterinária da Universidade Federal de Minas Gerais
Caixa Postal 567
30123-970 – Belo Horizonte, MG
3Instituto de Ciências Biológicas da UFMG
4Instituto de Ciências Exatas da UFMG
Belo Horizonte, Minas Gerais, Brazil

 

Recebido para publicação, após modificações, em 2 de dezembro de 1999.
*Corresponding author
E-mail: lygia@dedalus.lcc.ufmg.br

 

 

ABSTRACT

An ELISA system was applied to study the prevalence of EHV-1 in vaccinated and non-vaccinated Brazilian horses. Serum samples were taken from non-symptomatic pregnant mares, before and after the parturition, and from their neonatal foals. The antigen was prepared from a purified virus fraction (HVE-1 AB1 strain). The ELISA absorbance of 0.3516 was determined as the threshold of negative values. This cut-off level was defined from a selected interval, which determined a sensibility of 100% and a specificity of 94.7%, from a serum collection obtained from pregnant mares and fetuses. In this study, a strong correlation was found between the ELISA and serum neutralization for specific EHV-1 antibody detection.

Keywords: Horse, herpesvirus, ELISA

 

RESUMO

Um sistema de ELISA foi utilizado para se estudar a prevalência do herpesvirus eqüino 1 (HVE-1) em plantéis de cavalos brasileiros vacinados e não vacinados. Amostras de soros foram coletadas de éguas gestantes assintomáticas, antes e após o parto e de seus respectivos potros recém-nascidos. O antígeno foi preparado de uma fração viral purificada (HVE-1 amostra Ab1). O ponto de corte (absorvância de 0,3516) foi determinado considerando o limite máximo para os valores negativos. Esse valor determinou sensibilidade de 100% e especificidade de 94,7%, utilizando um banco de soros de éguas gestantes e fetos. No estudo observou-se alta correlação entre o ELISA e a soro-neutralização para detecção de anticorpos específicos contra o EHV-1.

Palavras-chave: Eqüino, herpesvirus, ELISA

 

 

INTRODUCTION

Horses are frequently infected by equine herpesvirus 1 (EHV-1; equine abortion virus), which is considered a major equine pathogen and endemic amongst horse populations throughout the world. In the past, EHV-1 isolates were identified as two subtypes of a single virus (Burrows & Goodrigde, 1973) and, only after 1981, EHV-1 and EHV-4 were genetically differentiated as two types (Sabine et al., 1981; Studdert et al., 1981). EHV-1 is associated with respiratory and neurological symptoms, abortion and neonatal death, while EHV-4 (equine rhinopneumonitis) is mostly associated with respiratory disease (Studdert et al., 1984).

The epidemiology of EHV-1 is complicated, particularly because the latent virus may be present in animals that are serologically negative and also due to difficulties in distinguishing EHV-1 from EHV-4 by conventional serum diagnosis, as a result of their antigenic cross-reactivity (Allen & Bryans, 1986). Although the magnitude and duration of the immune response increase after successive natural or experimental infections, no association has been found between the level of virus-neutralizing antibodies and the protective mechanism involved in re-infection (Doll & Bryans, 1963; Mumford et al., 1987).

The most common diagnostic methods used to detect EHV-1 infection in equines have been traditionally performed by examination of tissues using immune fluorescence staining, virus isolation and serological testing (i.e., serum neutralization and ELISA) (Thomson et al., 1976; Dutta et al., 1983).

Abortion in the last term of pregnancy has been a common occurrence among Brazilian stud farms frequently reported by veterinarians. However, few epidemiological studies on abortion or respiratory equine herpesvirus diseases have been published in recent years (Vargas & Weiblen, 1991; Weiblen et al., 1994). In addition, the Brazilian information system on incidence of equine viral diseases has provided limited and non-accurate data, mainly due to the fact that specialized laboratory facilities are available to encourage equine owners and veterinarians to send aborted fetuses tissues, blood, nasal swabs or serum samples to local diagnostic laboratories. As reviewed by Powell (1996), another reason for the low level of regional information about the prevalence of EHV-1 abortion in many countries is that horse has not been considered as an economic "agricultural animal", and this has limited the alocation of funds to investigate important equine infectious diseases.

Over the last years, a variety of inactivated vaccines have been licensed in Brazil, in an attempt to prevent EHV-1 infections. Thus, vaccination programs, requiring repeated administrations in mares during pregnancy, are regularly adopted in susceptible Brazilian horse farms. However, no information is available regarding the levels of vaccine-induced immunity among Brazilian horses and the benefits of vaccination in controlling the disease.

To address these demands, this study had the objective to optimize an indirect ELISA and to evaluate its efficiency in detecting EHV-1 specific antibodies in sera, colostrum and milk from non-symptomatic mares and neonatal foals in vaccinated and non vaccinated horses in Brazil.

 

MATERIALS AND METHODS

In group 1, positive and negative sera were randomly collected from 36 pregnant mares (6-12 years old) and 38 fetuses at 6-10 months of gestation, respectively, at an abattoir. The ante-mortem and post-mortem examination of slaughtered animals did not evidentiate typical lesions of any disease. Autopsies of carcasses included a careful examination of internal viscera and head. These animals were from three different Brazilian States.

In group 2, serum samples were collected in a stud farm (farm 1) from non-symptomatic pregnant mares, regularly vaccinated against EHV-1. Blood was obtained from 18 mares on day 290 of pregnancy, from 27 on day 305, from 29 on day 320, and from 32 mares soon after the parturition. Sera were also obtained from 20 and 22 of their neonatal foals, respectively before 6 and 36 hours after birth. Additionally, colostrum and milk samples were collected from seven mares of this group, soon after parturition and 10 days later, respectively.

In group 3, six foals from the latter EHV-1 vaccinated study farm (farm 1) were sampled several times from birth up to 16 months of age.

Archived sera from both groups (1 and 2) were collected from January, 1995 to August, 1996 and stored at –20oC for approximately two years. The veterinary records did not show any clinical abnormality.

In group 4, sera were collected from 11 non-symptomatic dams, during the birth season, approximately one month before and after parturition, and from their foals before 12 hours after birth and at the end of the first month, in another study farm (farm 2). These samples were collected from November, 1997 to February, 1998. The animals were clinically healthy during the whole collection period and no case of abortion had been reported during the two previous breeding seasons. This horse population was not vaccinated against EHV-1 and EHV-4 infections.

The collection of specimens was carried out aseptically and all sera were diluted 1:2 in PBS containing antibiotics. The immunoglobulins present in the aqueous fraction of the colostrum or milk were obtained by coagulation at room temperature, followed by a brief centrifugation to clarify the aggregates and a simple immunoglobulin precipitation procedure, as described by McKinney & Parkinson (1987).

Serumneutralization test (SNT) was carried out using EHV-1 AB1 strain (kindly provided by Dr. R. A. Killington, Department of Microbiology, University of Leeds). Neutralization of EHV- Ab1 was determined by incubating the virus in the presence of antibodies and then adding RK13 cells at a concentration that gave a confluent monolayer after an overnight incubation at 37ºC, as previously described by Thomson et al. (1976). Each serum dilution, ranging from 1:2 to 1:32, was tested in triplicate.

Negative cells and positive horse serum for EHV-1 were used as controls. Complement was not added in the test, and the cytopathic effect (CPE) was evaluated during five days. Specific positive results were expressed as the reciprocate of the highest serum dilution ³ 1:4 showing no CPE in all wells. Negative and positive results below the serum dilution 1:4 were re-tested and the positives with titers below 4 were considered suspicious.

Purified virus was prepared following basically the protocol described by Meredith et al. (1989). The antigen for ELISA was obtained from infected culture supernatants prepared by inoculating EHV-1 AB1 strain into RK13 monolayers. The purified virus pellet was resuspended in sterilized ultra-pure Milli-Q water in approximately 0.1ml per culture bottle. Protein concentration of purified virus was determined according to Lowry et al. (1951). Control antigen was prepared following the same procedure, using uninfected RK13 cells. Checkerboard titration showed that the antigen concentration of 12.5 mg/ml was the lowest concentration which demonstrated a positive reaction throughout the serum dilutions. The optimum serum dilution was chosen based on the lowest dilution of the positive serum, i.e. the highest antibody concentration, tested with negative control antigen, which showed OD values less or equal those presented by negative serum tested with viral antigen at the same concentration.

Falcon 96-well polyvinyl plates were passively adsorbed with 50ml of purified virus antigen per well diluted in carbonate buffer, pH 9.6. Plates were incubated overnight at 4oC. To remove unbound antigen, the plates were washed five times with PBS-Tween (0.05%, v/v). The blocking step was made by coating each well with 200ml of 2% (w/v) dried skimmed milk for 30 min, at 37ºC. This was followed by (i) 50ml of equine test serum diluted 1:20 in ELISA diluting buffer [0.05% Tween, 0.2% (w/v) dried skimmed milk in PBS] per well and incubation for 1 hour at 37oC; (ii) addition of 50ml of horseradish peroxidase rabbit anti-horse IgG (Sigma) diluted at 1:10,000 in ELISA buffer and incubation for 1 hour at 37ºC. The plates were washed five times after each incubation step. Then, 50ml of substrate solution [0.5mg/ml orthophenyldiamine (OPD) dissolved in 0.1M citric acid - 0.2M Na2HPO4 buffer, pH 5.0, plus 0.2 ml/ml of 30% H2O2] were added to appropriate wells and the plate was incubated in dark at 37ºC during 15 min. The reaction was interrupted by adding 25ml of 4M NH2SO4 per well. Negative controls included the use of PBS instead of primary antibody and wells without antigen. The positive control was a horse serum with a neutralizing titer of 32. Positive and negative controls were included in all reaction sets for detecting any variation in the ELISA test conditions. On each plate, one column was left blank. The absorbance values were read in an ELISA reader at wavelength 492nm (Sigma Diagnostic).

To minimize day-to-day variation, all serum samples were tested in duplicate on two different days and the results were expressed as the average of the four optical density (OD) values.

The ELISA cut-off value was determined from the OD values of 23 mares and 38 equine fetus sera collected in an abattoir, respectively diagnosed as positive (infected) and negative (uninfected) by SNT. A normality test showed that the distribution of the uninfected group could not be considered as Gaussian. Thus the percentile method was used to calculate the lowest limit for a positive result, in a way to obtain the highest value for both sensitivity and specificity. Therefore, it was selected an interval from 0.3516 to 0.6189, which gave a sensitivity of 100% and a specificity of 94.7%. Comparisons between the two independent groups were performed using either the t-test or the non-parametric Mann-Whitney test, the latter when the Gaussian distribution was not acceptable. Comparisons between mares and their foals were performed using the paired t-test or the non-parametric signal test. Finally, to detect changes in the OD average during consecutive periods of time, the F-test for dependent observations was employed. All tests were considered for statistical significance at the level of 5%.

 

RESULTS

A comparison between the OD means from both mares and fetuses groups, respectively positive and negative controls, used to determine the ELISA cut-off shows that they were significantly different (P=0.00). Only two negative sera intermixed with the positive; all negative sera presented OD means below 0.35 and the majority of the positive presented OD values above 0.66 (Fig. 1). Considering the interval between 0.3516 and 0.6189 (100% sensitivity and 94.74% specificity), all sera with an OD above 0.3516 were defined as positive for EHV-1, since the objective was to have a more sensitive test.

 

 

The indirect ELISA was optimized and compared with SNT as a method of monitoring the presence of specific antibodies for EHV-1 in pregnant mares and foals. Among mares of group 2, the agreement between ELISA and SNT was 84%, 93% and 96% for sera collected respectively on days 290, 305, 320 of pregnancy and 97% for those collected on the day of the parturition. The ELISA showed a significant quantitative decrease of specific antibodies for EHV-1 among these mares between the pregnancy period from day 290 to 305 (P=0.017), day 305 to 320 (P=0.00) and day 320 to parturition (P=0.00) (Fig. 2). The OD average presented by foal sera collected until six hours after birth was lower than that of their dams soon after the parturition (P=0.007). On the other hand, sera collected 36 hours after birth from these foals showed an OD average higher than their dams at parturition (P=0.001) and also than themselves at six hours after birth (P=0.014).

 

 

Table 1 shows that the agreement between the detection of EHV-1 antibodies by ELISA and SNT was very high for mare sera and it was also significant for dam colostrum and foal sera taken soon after birth. However, low agreement was observed, between the two tests, when antibody levels in milk samples were compared. The ELISA showed to be more sensitive than the SNT in detecting specific EHV-1 antibodies in milk.

 

 

The OD means observed for colostrum on the day of parturition were higher than their foal sera taken 24 hours after birth (p=0.002), but no significant differences were seen when they were compared with foal sera taken 48 hours after birth (P=0.21).

Among the foals in group 3, a significant increase in OD level (P=0.00) was observed 24 to 72 hours after birth. The differences were not significant (P=0.10) between the groups from 72 hours until 45 days after birth. A significant decrease (P=0.008) in OD level was observed from day 45 to 60 and remained in decline (P=0.003) from day 60 to 120. No changes were observed (P=0.143) from day 120 to 150. Due to management conditions, some foals were not sampled, making impossible the comparisons among data from day 180 to 480. However, the OD mean of four foals showed a decline from day 150 to 180, respectively from 0.4911 to 0.3571 and an increase from day 210 to 480, respectively from 0.4043 to 0.872 (Fig. 3).

 

 

Fig. 4 shows the comparative level of antibodies as detected by the SNT and the ELISA in foal sera collected over a period of 16 months after birth in the farm using vaccination (farm 1). The number of animals with neutralizing antibodies showed a swift decline after day 45 until these became undetectable from day 90 to 210. At day 480, all four sampled foals were positive by SNT, showing 100% of agreement with the ELISA results. The ELISA showed to be more sensitive than SNT in detecting EHV-1 antibodies in neonatal sera, as well as in the plasma portion of milk collected from vaccinated mares.

 

 

Regarding farm 2 (Table 2), there was a perfect agreement between both the ELISA and the SNT for positive serum samples collected from mares before and after parturition. Most of these sera had serum neutralization titers £ 1:8. The agreement between both tests was low for sera taken from foals from the day of parturition up to two months after birth in the non-vaccinated horse population. No agreement was observed for foals two months after birth.

 

 

DISCUSSION

In Brazil, few studies have estimated the prevalence and incidence of the EHV-1 and these are limited to the Southern areas (Vargas & Weiblen, 1991; Weiblen et al., 1994). In this study, the indirect ELISA test showed a high agreement with STN results regarding to vaccinated and non-vaccinated mares, as well as a significant agreement for neonatal foals born from vaccinated mare. The ELISA showed to be more sensitive than SNT in detecting EHV-1 antibodies in sera taken soon after parturition in foals born from either vaccinated or non-vaccinated mares and also in detecting antibodies in SNT negative milk specimens.

EHV-1 infection has been shown in the literature to induce a short-lived immunity (Doll, 1961). The literature shows that the protective immunity conferred by maternal antibodies disappears within approximately 180 days (Kendrick & Stevenson, 1979), period in which colostral antibodies are decreasing, marking the change from passive to active immunity in foals (Studdert, 1974). Our results from foals in farm 1 showed a decrease in seroprevalence of EHV-1 neutralizing antibodies from day 45 until its complete disappearance (day 90 to 210). The ELISA results demonstrated also a drop, showing a decrease of OD mean values from day 150 to 180. The last period was coincident with the period before and immediately after the foals had been weaned. ELISA detected a little increase of EHV-1 antibody levels for these foals in farm 1 on day 210. However, their OD mean was doubled on day 480 and coincident with seroconvertion by SNT in this period, which was approximately nine to ten months after weaning. The results observed in both tests agree with Studdert (1974) and also with Doll & Bryans (1962, 1963), who reported that 85% of foals in Kentucky seroconverted against equine rhinopneumonitis six to eight months after weaning. More recently, Gilkerson et al. (1998), using a specific ELISA to investigate the seroprevalence of EHV-1 in Australian Thoroughbred weaning foals, showed an increase of specific EHV-1 antibodies after weaning and the evidence of EHV-1 infection both before and after weaning. Foals obtain the passive immunity from the colostrum and the ingestion of colostrum from immune dams is correlated to protective levels of EHV-1 antibodies (Doll, 1961). In this study, the results showed that while the neutralizing antibody level was detected soon after birth up to day 60 in sera from foals born from vaccinated mares, it was undetectable in sera from foals born from non-vaccinated mares. Only the ELISA was able to detect EHV-1 antibodies in foals in the non-vaccinated farm.

The development of an indirect single dilution ELISA may provide a rapid and less costly new tool for the investigation of EHV-1 abortion outbreaks and serodiagnosis among the Brazilian equine population. Additionally, the development of an ELISA for EHV-1 detection based on purified virus should provide a standard immunodiagnostic test to implement new ELISA technologies for detecting both antigen and antibody and also for distinguishing between EHV-1 and EHV-4 (Yeargen et al., 1985; Crabb & Studdert, 1993).

 

ACKNOWLEDGMENTS

This work was supported by research grants from Ministério da Agricultura e do Abastecimento da República Federativa do Brasil, Núcleo de Genética Eqüídea da Universidade Federal de Minas Gerais (UFMG) and Pró-Reitoria de Pesquisa (PRPq-UFMG). We are grateful to Dr. R.C. Leite from Escola de Veterinária (UFMG) for providing the laboratory facilities, C.A.C. Vicente and D.F. Reis for excellent technical assistance and A.F. Silva Neto for providing the serum samples of vaccinated herd.

 

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