Printer Friendly

Evaluacion serologica de herpesvirus bovino 1 y 5 en sistemas de cria en la Altillanura Colombiana.

Serological evaluation of bovine herpesvirus 1 and 5 in cattle-breeding systems on Colombia's high plains

INTRODUCCION

Las enfermedades virales constituyen una seria amenaza para los sistemas ganaderos en Colombia. Dentro de estas enfermedades, las producidas por los virus herpes tienen una importancia singular, ya que desde tiempo atras se han referenciado los efectos patogenos del herpesvirus bovino tipo 1 (BoHV1) y sus consecuencias traducidas como perdidas economicas (1). Ademas, recientemente se reconoce la presencia del herpesvirus bovino-5 (BoHV5) y sus consecuencias, particularmente en Suramerica (1).

Estos dos herpesvirus pertenecen a la familia Herpesviridae, subfamilia Alfaherpesvirinae, genero Varicelovirus (2,3). El genoma de BoHV es un DNA de doble cadena con un tamano de 135kpb para BoHV-1 y 138kpb para BoHV-5 (4). Los dos herpesvirus estan relacionados antigenica y geneticamente; presentando una identidad en su secuencia de nucleotidos del 85% y en la secuencia de aminoacidos de 82% (5). Esta diferencia puede explicar el hecho de que los dos virus difieran en su capacidad neuroinvasiva y en su neurovirulencia (6). El BoHV-1 se ha asociado con la presentacion de cuadros clinicos diversos como infecciones respiratorias (Rinotraqueitis infecciosa Bovina -IBR) (7), genitales (vulvovaginitis pustular infecciosa-balanopostitis pustular infecciosa (VPI/BPI)) (7), abortos (8) y ocasionalmente encefalitis (9). En Colombia, la forma genital (VPI/BPI) fue reportada por primera vez en ganado proveniente de los Llanos Orientales y con el tiempo, se ha constituido en una de las enfermedades que ocasiona mayores problemas, alcanzando una alta prevalencia en las principales regiones ganaderas del pais (10). Debido a su impacto economico, las formas IBR y VPI/ BPI han sido objeto de diversos estudios seroepidemiologicos, que han estado acompanados de aislamientos de cepas de campo en diversas regiones del pais: la Sabana de Bogota (11), Llanos Orientales y Cordoba (12) y Antioquia (10), junto con la caracterizacion molecular de uno de estos aislamientos.

Por su parte, el BoHV-5 es un patogeno cuyas manifestaciones clinicas se asocian con la meningoencefalitis, la cual induce elevada tasa de mortalidad (13), llegandose a considerar como una de las principales causas de encefalitis letal (14). Debido a las similitudes geneticas, antigenicas, y en la estructura entre los dos virus, el BoHV5 fue considerado inicialmente como una variante neuropatogena del BoHV-1, denominada BoHV-1 subtipo 1.3 (9). Sin embargo, estudios posteriores basados en analisis moleculares del genoma, en pruebas de neutralizacion cruzada y reactividad a anticuerpos monoclonales, demostraron que eran dos virus diferentes (15). Por lo anterior, el Comite Internacional de Taxonomia Viral en 1992 lo clasifico como un nuevo virus, al que se denomino BoHV-5 (16).

El BoHV5 fue aislado por primera vez en Australia en 1962, a partir de un brote de encefalitis que causo una alta mortalidad en terneros (17). Posteriormente, fue reportado en Europa (18) y Estados Unidos (19). En Suramerica se ha reportado en Argentina (20,21), Brasil (3,22) y Uruguay (23). En Colombia, no hay reportes de enfermedad ocasionada por BoHV-5, sin embargo, los primeros indicios de su presencia se mostraron en estudios sobre muestras de tejidos provenientes de animales muertos con sintomatologia nerviosa, donde el virus se detecto a traves de tecnicas moleculares como la PCR (24). En estudios posteriores de Pedraza F et al (25), se determino una sero-prevalencia de 58.4% a este virus en hatos de los llanos orientales. Sin embargo, Colombia carece de estudios que se aproximen a la situacion clinica y epidemiologica, la cual requiere ser abordada ya que la presencia de este virus esta demostrada en otros paises del continente desde hace mas de una decada. Por otro lado, se debe resaltar que la determinacion de la prevalencia de BoHV5 es laboriosa por metodos serologicos debido a que los dos herpesvirus son antigenicamente muy similares, y los dos virus comparten epitopes comunes, por lo tanto se ha observado reactividad cruzada tanto en hatos vacunados como infectados con BoHV-1, lo que lleva a pensar en un nivel de proteccion humoral cruzada. Por lo anterior, se ha establecido que en regiones donde se lleva a cabo la vacunacion rutinaria contra BoHV-1 el riesgo de presentacion de BoHV-5 es menor.

El objetivo del presente estudio fue establecer la presencia de anticuerpos neutralizantes a BoHV-1 y BoHV-5, asi como tambien determinar el nivel de inmunidad cruzada contra los dos herpesvirus en fincas de la altillanura colombiana.

MATERIALES Y METODOS

Ubicacion geografica y muestreo. Este estudio se realizo en fincas ubicadas en los municipios de Puerto Lopez y Puerto Gaitan departamento del Meta, con una temperatura promedio de 26 y 29[grados]C respectivamente, y una altitud de 149 msnm. Como elemento discriminativo, ninguna de las fincas objeto de estudio reporta historia de vacunacion contra BoHV-1. Se realizo un muestreo por conveniencia, empleando 23 fincas, y realizando un muestreo al azar en cada finca para un total de 488 muestras, sin discriminacion de raza o sexo. Este muestreo se realizo por sangrado mediante puncion en la vena coccigea empleando el sistema de Vacutainer[R] (Becton Dickinson NJ, USA) y se realizo en animales mayores de un ano para evitar interferencia con anticuerpos maternales. Las muestras de sangre obtenidas fueron centrifugadas a 3.500 rpm durante 10 minutos y el suero se almaceno a -20[grados]C hasta su procesamiento.

Prueba de sero-neutralizacion viral.

Inicialmente, los sueros fueron inactivados a 56[grados]C durante 30 minutos; posteriormente, con el fin de determinar la mas alta dilucion de suero capaz de neutralizar los virus, se realizaron diluciones dobles seriadas de los sueros (1/2 hasta 1/256) con medio de cultivo MEM en placas de cultivo celular de 96 pozos. Cada una de las diluciones de los sueros se dejo en contacto con 50ul de una concentracion constante de virus de referencia [100TCID.sub.50] (50% de dosis infectiva en cultivo celular) de BoHV-1 (Cepa Colorado) y de BoHV-5 (Cepa EVI88/95), esta ultima fue donada por el Dr. Fabricio Campos de la Universidad Federal de Rio Grande de Sur (UFRGS), Brasil. Cada suero fue evaluado de manera independiente para cada uno de los virus. La sero neutralizacion se realizo por duplicado para cada uno de los sueros. La mezcla de sueros y virus se incubo a 37[grados]C al 5%C[O.sub.2] durante 24 horas y posteriormente se adiciono una suspension de celulas MDBK a un volumen de 50ul y una concentracion 2 x [10.sup.4] celulas/ pozo. Un periodo de incubacion mayor de 24 horas aumenta la probabilidad de detectar anticuerpos neutralizantes (Manual de la OIE sobre animales terrestres, 2012). Finalmente, las placas se incubaron a 37[grados]C al 5% C[O.sub.2] durante 72 horas, tiempo en el cual se establecio el titulo sero-neutralizante mediante la determinacion del efecto citopatico caracteristico del alfa herpesvirus. Los resultados fueron discriminados como: positivo cuando el titulo de anticuerpo neutralizante era [greater than or equal to] 2 y como negativo titulo de anticuerpo neutralizante < 2. Como controles se empleo un pozo independiente para cada uno de los sueros y se evaluo solo con las celulas con el fin de determinar si tenia algun toxico que pudiera alterar la viabilidad celular, igualmente se emplearon controles de celulas y controles de virus. Se realizo titulo parcial (256) con el fin de determinar titulos neutralizantes.

Analisis estadistico. Los datos fueron evaluados con estadistica descriptiva (media y desviacion estandar). Todos los datos fueron analizados con el software Statistix version 8.0 (Analytical Software, Tallahassee, FL).

RESULTADOS

Mediante la tecnica de sero-neutralizacion viral se evidencio la presencia de anticuerpos neutralizantes en el 87% (425/488) contra herpesvirus. De estos sueros positivos, el 84% (410/488) presentaron anticuerpos para BoHV-1 y el 60% (297/488) anticuerpos para BoHV-5 (Figura 1), (95% intervalo de confianza (IC)= 77-90 para BoHV-1 y (IC)=49-70 para BoHV5). De los 488 sueros evaluados solo 13% (63/488) fueron negativos para los dos herpesvirus. Se encontro reactividad para los dos virus en un mismo suero en el 57.7% (282/488). En cuanto a la evidencia de anticuerpos neutralizantes especificos para BoHV-1 se encontro 26% (127/488) y para BoHV-5 un 3% (15/488). La prueba de seroneutralizacion viral mostro una sensibilidad de 96.4% para BoHV-1 y del 88% para BoHV 5 con una especificidad de 83.5% y de 85% respectivamente. El coeficiente Q de Yule fue de 0.99 para BoHV-1 y de 0.95 para BoHV-5.

Los resultados obtenidos por la prueba de seroneutralizacion con los dos virus desafio BoHV-1 y BoHV-5, se muestran en la tabla 1. Al evaluar la reactividad serologica por fincas se encontro que todos los 23 predios (100%) presentaron sero-reactividad para BoHV-1 y 17 predios (73.9%) para BoHV-5. Cuando se evaluo la seroprevalencia en cada una de las fincas, se encontro que la reactividad para BoHV-1 fue mayor entre 47 al 100 %, mientras que para BoHV-5 fue entre 6 al 100% (Tabla 1).

La sero-prevalencia para BoHV-5 fue evidenciada en predios donde la reactividad para BoHV-1 fue alta (ej. Fincas 1, 4, 14, 16, 20); sin embargo, tambien se encontro reactividad para BoHV-5 en fincas donde la reactividad para el BoHV-1 era baja (ej. Finca 12, 15, 17, 19) (Figura 2). Estos resultados muestran que no existe un nivel alto de seroneutralizacion cruzada entre los dos herpesvirus.

El comportamiento de los titulos de anticuerpos evidencio que el 43.6% (213 sueros) presentaron anticuerpos neutralizantes mayores a la dilucion 1/64 para BoHV-1, de estos el 62.4% (133 sueros) presentaron titulos por encima a 256 (Titulo final 512). En cuanto a BoHV-5 se encontro que el 4.91% (21 sueros) presentaron titulos mayores de la dilucion 1/64, y de estos el 19% (4/21) presentaron titulos de anticuerpos mayores a 256 (Titulo final 1024).

DISCUSION

El objetivo de este estudio fue evaluar la presencia de BoHV-1 y BoHV-5 mediante serologia en granjas de la altillanura colombiana. Los datos confirman que BoHV-1 y BoHV-5 estan presentes en el pais como fue reportado previamente (25). Diferentes estudios en Colombia, han establecido una prevalecia para BoHV-1 que oscila entre 50 al 90%. A partir del presente estudio se confirma una alta sero-reactividad para este virus en hatos no vacunados, lo que demuestra la alta circulacion del virus en nuestra ganaderia. En cuanto al BoHV-5, Pedraza F et al (25) realizo un estudio en la misma region de pais (Puerto Gaitan -Meta) analizando 156 sueros provenientes de 2 granjas y determino reactividad serologica hacia los dos herpesvirus, con un porcentaje de sero-prevalencia para los dos virus: BoHV-1 (70.5%) y BoHV-5 (58.4%); ademas, de una reactividad cruzada del 56.4%. Estos resultados son similares con el presente estudio, en el cual se empleo un mayor numero de sueros (488) y un mayor numero de explotaciones (23). Asimismo, se encontro reactividad para los dos herpesvirus en un mismo suero en un porcentaje similar (57.7%). Sin embargo, el porcentaje de sero-positividad fue mas alto y el porcentaje de seronegativos a los dos herpesvirus fue mas bajo (disminuyo del 26 al 13%) comparado con el estudio de Pedraza et al, en 2011. Asi mismo, la especificidad para BoHV5 (anticuerpos exclusivos para BoHV5) aumento del 1 al 3%. Este estudio no solo valida la naturaleza endemica del BoHV1, sino que tambien resalta la circulacion de BoHV-5.

El empleo de un solo virus (BoHV-1 o BoHV-5) para realizar la prueba diagnostica de seroneutralizacion conduce a una baja sensibilidad, por lo tanto se recomienda realizar la prueba de forma independiente a los dos virus como lo mostro Varela A et al (3), en 2009. De esta manera se puede comenzar a discriminar los anticuerpos presentados para cada uno de los virus, determinar cual de ellos incide mas en la finca y si existe infeccion dual. Por otro lado, se debe senalar que la diferenciacion entre estos dos virus empleando metodos serologicos se dificulta, debido a que los dos herpesvirus son antigenicamente muy similares, comparten epitopes comunes, lo que puede mostrar reactividad cruzada en hatos vacunados o infectados con BoHV-1, generando falsos positivos. Sin embargo, este estudio mostro que no todas las fincas con anticuerpos altos para BoHV-1 presentaron altos titulos para BoHV-5 y viceversa, lo que lleva a pensar en que el nivel de proteccion humoral cruzada es limitado.

Por otro lado, la evaluacion de los anticuerpos neutralizantes podria permitir establecer si los animales han estado expuestos al virus, si estos son altos se puede inferir si los animales han estado expuestos recientemente o presentan una infeccion activa, y la reactividad a los dos virus evaluados de manera independiente nos puede indicar la presencia de los dos patogenos en hatos sin vacunacion. En este estudio se determino una circulacion activa de los dos virus por el elevado numero de seroreactores, al encontrarse animales con titulos mayores de 64 en un porcentaje de 43.6% para BoHV-1 y de 4.91% para BoHV-5. Asi mismo, se evidencio reactividad para los dos herpesvirus en un mismo suero, lo que puede deberse a que el ganado bovino este infectado con mas de un herpesvirus como lo menciono previamente Campos et al (1).

Por otro lado, a pesar de la importancia que tienen las pruebas serologicas para determinar la presencia de anticuerpos frente a un patogeno determinado, es importante el empleo de pruebas moleculares como la PCR la cual permite la deteccion del genoma viral mediante la amplificacion de fragmentos especificos de cada uno de los virus para determinar con precision que virus esta actuando.

Se puede concluir que a partir del presente estudio se confirma la circulacion de BoHV-1 en las zonas evaluadas y en una mayor proporcion que el BoHV5. La presencia de titulos de anticuerpos para BoHV-5 en hatos donde los titulos para BoHV-1 son bajos es un indicativo que permite inferir sobre una posible circulacion de BoHV-5 en esta zona del pais. Las pruebas serologicas permiten establecer una aproximacion a la presencia de los dos virus en un hato. Se recomienda posteriormente determinar la presencia de los dos virus mediante tecnicas moleculares que permitan diferenciar especificamente entre los dos herpesvirus.

INTRODUCTION

Viral diseases represent a serious threat for livestock systems in Colombia, with those produced by herpesviruses having a singular importance. The pathogenic effects of bovine herpesvirus type 1 (BoHV-1) and its consequences lead directly to great economic losses and have been referred to for a long time (1). The presence of bovine herpesvirus-5 (BoHV-5) and its consequences has been more recently shown, particularly in South American countries (1).

Both these herpesviruses belong to the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus (2,3). BoHV has a double-stranded DNA genome, 135 Kbp long for BoHV-1 and 137.8 Kbp for BoHV-5 (4). Both herpesviruses are antigenically and genetically related, showing 85% nucleotide and 82% amino acid sequence identity (5). The sequence differences between the two, however, could explain why both viruses differ regarding their neuroinvasive ability and neurovirulence (6). BoHV-1 has been associated with various clinical syndromes, such as respiratory (infectious bovine rhinotracheitis (IBR)) (7) and/or genital infections (infectious pustular vulvovaginitis-infectious pustular balanopostitis (IPV/IPB) (7), abortions (8), and, occasionally, encephalitis (9). The genital form (IPV/IPB) was reported for the first time in Colombia in cattle from the Colombian eastern plains and has become one of the diseases causing the most problems, with a high prevalence reached in Colombia's main livestock regions (10). The IBR and IPV/IPB forms have been targeted by several sero-epidemiological studies due to their economic impact; this has been accompanied by isolates being taken from field strains from several areas of Colombia, such as the savannah around Bogota (11), the eastern plains, and the departments of Cordoba (12) and Antioquia (10), together with the molecular characterization of one these isolates.

BoHV-5 is a pathogen whose clinical manifestations are associated with meningoencephalitis, which induces high mortality rates (13) and is even considered as a major cause of lethal encephalitis in cattle (14). Due to genetic, antigenic, and structural similarities between both viruses, BoHV-5 was initially considered a neuropathogenic variant of BoHV-1, called BoHV 1, subtype 1.3 (4,9). However, later studies based on molecular analyses of the genome and reactivity to monoclonal antibodies tests showed that they are two different viruses (15). The International Committee on Taxonomy of Viruses (ICTV) thus classified it as a new virus in 1992, calling it BoHV-5 (16).

BoHV-5 was isolated for the first time in Australia in 1962 from an outbreak of encephalitis which caused a high rate of mortality in calves (17). It was then reported in Europe (18) and the USA (19). Regarding South America, it has been reported in Argentina (20,21), Brazil (3,22), and Uruguay (23). No reports of disease caused by BoHV-5 have been noted in Colombia; however, the first indications of its presence was shown in studies of tissue samples taken from dead animals having symptoms of nervous system disorders, from which the virus was detected by PCR (24). Later studies by Pedraza F et al (25), determined a sero-prevalence of 58.4% for this virus in herds from the eastern plains. Apart from the above reports, there is a lack of studies in Colombia dealing with the clinical and epidemiological situation, which needs to be analyzed, especially due to the presence of the virus in other countries of the region for more than a decade. It should be noted that determining BoHV-5 prevalence by serological methods is laborious, as both herpesviruses are antigenically very similar since they share common epitopes. Cross-reactivity has been observed for both viruses in vaccinated herds, as well as in cattle naturally infected with BoHV-1. The foregoing raises the question of whether there is a level of humoral cross-protection, which could lead to argumenting that there is a lower risk of BoHV-5 being present in regions where vaccination against BoHV-1 is routine.

The present study was thus aimed at determining the presence of neutralizing antibodies against BoHV-1 and BoHV-5, as well as determining the level of cross-immunity against both herpesviruses in farms on Colombia's High Plains.

MATERIALS AND METHODS

Geographic location and sampling. This study was carried out on cattle farms located near the towns of Puerto Lopez and Puerto Gaitan in the department of Meta in Colombia. The area is located at 149 meters above sea level and has an average temperature between 26[grados]C and 29[grados]C. As a discriminative element, it should be stated that none of the farms in the study reported a history of vaccination against BoHV-1. Sampling was made by convenience. Twenty-three farms were involved in the study; 488 sera samples were taken by random sampling, without discriminating race or gender. Sampling involved bleeding by puncturing the coccygeal vein using the Vacutainer system (Becton Dickinson, NJ, USA) and was carried out on animals older than one year old to avoid interference with maternal antibodies. The blood samples were centrifuged at 3.500 rpm for 10 minutes and the sera were stored at -20[grados]C until being processed.

Viral seroneutralization test. Sera were initially inactivated at 56[grados]C for 30 minutes; then diluted in two-fold dilutions (1/2 to 1/256) in MEM culture medium on 96-well cell culture plates for determining the highest sera dilution able to neutralize the virus. Each sera dilution was left in contact with 50pl of a constant concentration ([100TCID.sub.50]) (i.e. 50% infective dose in cell culture) of reference BoHV-1 (Colorado strain) and BoHV-5 (EVI88/95 strain); the latter was kindly donated by Dr. Fabricio Campos from the Universidade Federal do Rio Grande do Sul (UFRGS) in Brazil. Each serum was evaluated independently for each virus. The virus neutralizations were done in duplicate. The sera-virus mixture was incubated at 37[grados]C for 24 hours in 5% C[O.sub.2] and then a suspension of 50ul of MDBK cells at a concentration of 2 x [10.sup.4] cells/well was added. It has been reported that a 24 hour incubation period may score up to 16 fold higher antibodies titers. The plates were then incubated at 37[degrees]C in 5%[ .sub.2] for 72 hours; this time was necessary to establish the sero-neutralizing titer by determining the characteristic alpha herpesvirus cytopathic effect. The results were considered positive when the neutralizing antibody titer was [greater than or equal to] 2, whereas values <2 gave a negative neutralizing antibody titer. A separate well containing a mixture of sera and cells was used as control for each sample in order to eliminate any toxic effect by the sera; in addition, cell and virus controls were also used. A partial titer of 256 was done in order to determine neutralizing antibodies.

Statistical analysis. Descriptive statistics (average and standard deviation) were used for evaluating the data. Statistix software, version 8.0 (Analytical Software, Tallahassee, FL) was used for analyzing the data.

RESULTS

The viral sero-neutralization test revealed an 87% (425/488) presence of neutralizing antibodies against herpesvirus; 84% (410/488) of these positive sera had antibodies against BoHV-1 and 60% (297/488) showed antibodies against BoHV-5 (Figure 1) (95% confidence interval, (CI) = 77-90 to BoHV-1 and (CI)=49-70 to BoHV5). Only 13% (63/488) of the 488 sera evaluated were negative for both herpesviruses. Reactivity for both herpesviruses in the same sera was found in 57.7% (282/488) of the samples. Regarding evidence for specific neutralizing antibodies, 26% (127/488) was found for BoHV-1 and 3% for BoHV-5 (15/488). The viral neutralization test showed a sensitivity of 96.4% for BoHV-1 and 88% for BoHV-5, with a specificity of 83.5% and 85%, respectively. The Q Yule coefficient was 0.99 for BoHV-1 and 0.95 for BoHV-5.

Table 1 gives the sero-neutralization test results for each farm, for BoHV-1 and BoHV-5, and for both simultaneously. It was found that all 23 farms (100%) had sero-reactivity for BoHV-1 and 17 farms (73.9%) for BoHV-5. Regarding serological reactivity per farm, this was greater for BoHV-1 (47-100%), while BoHV-5 showed a reactivity of 6-100%.

Reactivity for BoHV-5 was found on farms where reactivity for BoHV-1 was high (farms 1, 4, 14, 16, and 20); however, there was also reactivity for BoHV-5 in farms where reactivity for BoHV-1 was low [(farms 12, 15, 17, and 19) (Figure 2)]. Thus, these results do not show a correlation between cross-protection against both herpesviruses.

It was found that 43.6% (213 sera samples) of the farms had BoHV-1 neutralizing antibodies when antibody activity was evaluated at dilutions greater than 1/64; moreover, 62.4% (133 sera samples) of these had titers higher than 1/256 dilution. Regarding BoHV-5, it was found that 4.91% (21) had titers higher than 1/64 dilution and 19% (4/21) of these had antibody titers higher than 1/256 (Final titer 1024).

DISCUSSION

The aim of this research was to evaluate the occurrence of BoHV-1 and BoHV-5 in Colombia's High Plains using serology. Our data confirmed that BoHV-1 and BoHV-5 are present in the country as previously reported (25). All studies conducted to date agree that in Colombia the prevalence of BoHV-5 is lower than BoHV-1. Prior studies carried out in Colombia have established 50 to 90% prevalence for BoHV-1. The present study has corroborated this situation in unvaccinated herds, thereby, demonstrating that the virus is in widespread circulation in Colombian cattle. Regarding BoHV-5, Pedraza et al. (25) carried out a study in the same region of Colombia (Puerto Gaitan, Meta) as that used in this study; they analyzed 156 sera samples taken from two cattle farms and found serological reactivity against both herpesviruses, with a sero-prevalence percentage of 70.5% for BoHV-1 and 58.4% for BoHV-5, as well as 56.4% cross-reactivity. Their results were similar to those found in the present study, which involved a greater amount of sera samples (488) and farms (23). Reactivity for both herpesviruses in the same sera was found at a similar percentage (57.7%). However, the seropositive percentage found here was higher, while the seronegativity percentage for both herpesviruses was lower (13%) than that reported by Pedraza et al, 2011 (26%). BoHV-5 specificity (antibodies exclusively against BoHV-5) reported in their study was 1% compared to 3% in our study. The current study not only validated the endemic nature of BoHV-1, but also highlighted the circulation of BoHV-5.

An important aspect to be considered is that a diagnosis for BoHV by sero-neutralization using just one type of virus (BoHV-1 or BoHV5) leads to poor sensitivity; it is thus currently recommended to test for each type of virus independently, as shown by Varela et al (3) in 2009. The antibodies presented for each virus can thus be discriminated, determining which virus most affects a particular farm and thus establishing whether there is dual infection. Yet, using serological methods for differentiating between these two viruses is difficult, as they are very similar antigenically and share common epitopes; for this reason, we used a reference strain of BoHV-5 for reducing the possibility of non-specificity. However, the current study showed that not all farms with high antibody titers of BoHV-1 showed high titers for BoHV-5 and vice versa, suggesting that the level of humoral cross protection is limited.

It is clear from the foregoing that evaluating BoHV neutralizing antibodies led to establishing whether the animals had been exposed to the virus; such that if the neutralizing titers were high, it could have been inferred that the animals had been recently exposed or that they had an active infection. In addition, reactivity to both viruses, evaluated independently for each virus, indicated the presence of the two pathogens in herds without vaccination. The present study led to determining an active circulation of both viruses in the study area, since animals were found having titers higher than 1/64, with a seropositive percentage of 43.6% to BoHV-1 and 4.91% to BoHV-5. The study also showed reactivity for both herpesviruses in the same sera, therefore, cattle can be infected with more than one herpesvirus, as previously reported by Campos et al (1).

Although serological tests are important in determining the presence of antibodies against a given pathogen, the importance of using molecular tests such as PCR should be noted, as they can lead to the precise determination of viral types by detecting the viral genome through amplification of specific fragments of each virus. In conclusion, the present study confirms BoHV-1 and BoHV-5 circulation in the areas evaluated, the latter virus was found in a greater proportion than in previous reports. The presence of antibodies for BoHV-5 in herds where titers for BoHV-1 are low suggest BoHV-5 circulation in this region of the country. Serological tests allow to establish an approach to determining the presence of the two viruses in a herd, however, the use of molecular techniques is recommended to differentiate specifically between the two herpesviruses.

Agradecimientos

Este trabajo fue financiado por el departamento general de investigaciones de la Universidad de los Llanos (DGI), codigo del proyecto: CAMVZ-11/2010. Los autores agradecen al Dr. Fabricio Campos de la Universidad Federal de Rio Grande de Sur, Brasil por toda su colaboracion.

Acknowledgements

This work was financed by the General Research Department (DGI) of the Universidad de los Llanos, project code CAMVZ-11/2010. We would like to thank Dr. Fabricio Campos from the Universidade Federal do Rio Grande do Sul (UFRGS), Brazil, for his collaboration.

REFERENCES

(1.) Campos FS, Franco A, Hubner S, Oliveira MT, Silva AD, Esteves PA, et al. High prevalence of co-infections with bovine herpesvirus 1 and 5 found in cattle in southern Brazil. Vet Microbiol 2009; 139(1-2):67-73.

(2.) Davison A, Eberle R, Ehlers B, Hayward G, McGeoch D, Minson A, et al. The order Herpesvirales. Arch Virol 2009; 154:171-177.

(3.) Varela A, Holz C, Cibulski S, Texeira T, Antunes D, Franco A, et al. 2009. Neutralizing antibodies to bovine herpesvirus types 1 (BohV1) and 5 (BoHV5) and its subtypes. Vet Microbiol 2009; 142(3-4):254-260.

(4.) Thiry J, Keuser V, Muylkens, Meurens F, Gogev S, Vanderplasschen A, et al. Ruminant alphaherpesvirus related to bovine herpesvirus 1. Vet Res 2006; 37: 169-190.

(5.) Traesel C, Silva M, Rosado F, Furtado E. Nucleotide sequencing and phylogenetic analysis of the 30 region of glycoprotein C gene of South American bovine herpesviruses 1 and 5. Res Vet Science 2013; 94(1):178-185.

(6.) Del Medico M, Ladelfa M, Kotsias F, Muylkens B, Thiry J, Thiry E, et al. Biology of bovine herpesvirus 5. Vet J 2010; 184(2):138-142.

(7.) Muylkens B, Thiry J, Kirten P, Schynts F, Thiry E. Bovine herpesvirus 1 infection and infectious bovine rhinotracheitis. Vet Res 2007; 38: 181-209.

(8.) Ackermann M, Engels M. Pro and contra IBR-eradication. Vet Microbiol 2006; 113(34):293-302.

(9.) Silva M, Brum M, Loreto E, Weiblen R, Flores E. Molecular and antigenic characterization of Brazilian bovine herpesvirus type 1 isolates recovered from the brain of cattle with neurological disease. Virus Res 2007; 129(2):191-199.

(10.) Ruiz J, Jaime J, Vera V. Serological prevalence and isolation of Bovine herpesvirus-1 (BoHV1) in cattle herds from Antioquia and Valle del Cauca. Rev Colomb Cienc Pecu 2010; 23(3):299-307.

(11.) Gongora O, Villamil L, Vera V, Parra J, Ramirez G, Lopez G. Aislamiento de un herpes virus bovino tipo-1 (HVB-1) de secrecion nasal y esmegma prepucial en un toro reproductor. Rev. Med. Vet Zoot 1995; 63(1):43-47.

(12.) Vera V, Betancur C. Isolation of Bovine herpes virus type 1 from cattle in Cordoba--Colombia. Rev MVZ Cordoba 2008; 13(3):1495-1503.

(13.) Brower A, Homb K, Bochsler P, Porter R, Woods K, Ubl S, et al. Encephalitis in aborted bovine fetuses associated with bovine herpesvirus 1 infection. J Vet Diagn Invest 2008; 20: 297-303.

(14.) Ferrari H, Luvizotto M, Rahal P, Cardoso T. Detection of bovine herpesvirus type 5 in formalin-fixed paraffin-embedded bovine brain by PCR: a useful adjunct to conventional tissue-based diagnostic test of bovine encephalitis. J virol methods 2007; 146(1-2): 335-340.

(15.) Arce R, Almeida R, Silva T, Franco A, Spilki F, Roehe P, Arns C. Restriction endonuclease and monoclonal antibody analysis of Brazilian isolates of bovine herpesvirus types 1 and 5.Vet microbiol 2002; 88: 315-324.

(16.) Davidson A. Herpesvirus systematics. Vet microbiol 2010; 143(1):52-69.

(17.) Rissi D, Rech R, Flores E, Kommers G, Barros K. Meningoencephalitis by bovine herpesvirus-5. Pesq Vet Bras 2007; 27(7): 251-260.

(18.) Maidana S, Ladelfa M, Perez S, Lomonaco P, Del medico M, Odeon A, et al. Characterization of BoHV-5 fields strains circulation and report of transient specific subtype of bovine herpesvirus in Argentina. Vet Res 2011(7):1-8.

(19.) d'Offay J, Mock R, Fulton R. Isolation and characterization of encephalitic bovine herpesvirus type 1 isolates from cattle in North-America. Am J Vet Res 1993; 54 (4): 534-539.

(20.) Carrillo B, Ambrogi A, Schudel A, Vazquez M, Dahme E, Pospischil A. Meningoencephalitis caused by IBR virus in calves in Argentina. Zbl Vet Med B 1983; 30 (1-10): 327-332.

(21.) Perez S, Vagnozzi A, Sur J, Odriozola E, Campero C, Odeon A. Retrospective analysis of cases with diagnosis of cerebrocortical necrosis and its relation with type 5 bovine herpesvirus. Rev Arg Microbiol 2003; 35 (2), 69-73.

(22.) Freitas D, Ortega L, Fernandes R, Trindade G, Barbosa E. Characterization of field ovine herpesvirus samples using random amplified polymorphic DNa (RAPD). J virol methods 2007; 140: 200-205.

(23.) Guarino H, Nunez A, Repiso M, Gil A, Dargatz D. Prevalence of serum antibodies to bovine herpesvirus-1 and bovine viral diarrhea virus in beef cattle in Uruguay. Preventive Veterinary Medicine 2008; 85 (1-2): 34-40.

(24.) Pedraza F, Alessi A, Barbosa E. Detection of bovine herpesvirus 5 (BoHV-5) in formalin-fixed, paraffin-embedded bovine brain by nested PCR in Colombian cattle. Rev Colomb Cienc Pecu 2010; 23 (3): 292-298.

(25.) Pedraza F, Lopez J, Avila J, Alessi A, Furtado E. Serological evidence of bovine herpesvirus 5 (BoHV5) in cattle at the Colombia's Eastern plains. Revista Cientifica FCV-LUZ 2011; 21: 16-21.

Diana Vargas B, [1] * M.Sc, Alejandro Bohorquez G, [1] MV, Jorge Parra A, [2] M.Sc, Jairo Jaime C, [1] Ph.D, Agustin Gongora O, [3] Ph.D.

[1] Universidad Nacional de Colombia, Facultad de Medicina Veterinaria y de Zootecnia, Grupo de Microbiologia y epidemiologia, Bogota, Colombia. [2] Corporacion Colombiana de Investigacion Agropecuaria (Corpoica), Centro de Investigaciones La Libertad, Villavicencio, Meta, Colombia. [3] Universidad de los llanos. Grupo de investigacion en Reproduccion y Genetica. Villavicencio, Meta, Colombia, Correspondence: dsvargasb@unal.edu.co

Received: June 2015; Accepted: December 2015.

Caption: Figure 1. Sero-reactivity to bovine herpesviruses on all farms evaluated.

Caption: Figure 2. Antibody distribution on each farm evaluated for BoHV-1 and BoHV-5.
Table 1. Bovine herpesvirus 1 and 5 prevalence and
average of titer antibodies on each farm evaluated.

Farm    n           BoHV-1                BoHV-5

               +    Average     %       +    Average
                     titer                    titer

1       30    26     238.9    86.67    16     19.8
2       19     9     11.3     47.37     9       2
3       18    18     129.1    100.00   11       2
4       9      7     130.6    77.78     5      7.5
5       13    12     49.6     92.31     9      3.2
6       23    17     102.9    73.91     7      1.7
7       17    16     154.8    94.12     2      0.7
8       28    28     171.5    100.00   21       3
9       29    14     19.1     48.28    13       2
10      30    24     44.4     80.00    16      5.6
11      30    22     20.7     73.33    22      3.2
12      13    12     36.9     92.31    11     14.7
13      17    11      8.4     64.71    10      5.2
14      23    22     147.3    95.65    16     10.5
15      32    24     66.1     75.00    16     26.7
16      28    25      150     89.29    21     29.8
17      9      7       8      77.78     4      6.6
18      16    15     84.7     93.75     1       1
19      34    33     39.1     97.06    21     22.2
20      30    30     106.7    100.00   30       8
21      16    15      9.4     93.75    15      3.7
22      7      6      102     85.71     5      5.1
23      17    17     109.7    100.00   16      8.3
Total   488   410    84.4     84.02    297     8.3

Farm    BoHV-5     BoHV-1 and
                     BoHV-5

          %       +      %

1        53.3    16     53.3
2        47.3     7     36.8
3       61.11    11    61.11
4       55.56     5    55.56
5       69.23     9    69.23
6       30.43     7    30.43
7       11.76     2    11.76
8       75.00    21    75.00
9       44.83    10    34.48
10      53.33    16    53.33
11      73.33    18    60.00
12      84.62    10    76.92
13      58.82    10    58.82
14      69.57    16    69.57
15      50.00    15    46.88
16      75.00    21    75.00
17      44.44     4    44.44
18       6.25     1     6.25
19      61.76    21    61.76
20      100.00   30    100.00
21      93.75    15    93.75
22      71.43     4    57.14
23      94.12    16    94.12
Total   60.86    282   58.40
COPYRIGHT 2016 Universidad de Cordoba
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2016 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:ORIGINAL
Author:Vargas B., Diana; Bohorquez G., Alejandro; Parra A., Jorge; Jaime C., Jairo; Gongora O., Agustin
Publication:Revista MVZ (Medicina Veterinaria y Zootecnia)
Date:May 1, 2016
Words:6017
Previous Article:Estudio clinico e histopatologico de la dermatitis fototoxica en terneros cebu en pastoreo de Brachiaria decumbens.
Next Article:Marcadores geneticos del pelaje del gato domestico Felis catus (Felidae) del suroccidente Colombiano.

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters