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Neospora caninum: Deteccin de ADN en sangredurante la primera gestacin de vaquillas

infectadas naturalmente

Neospora caninum: DNA detection in blood during

rst gestation of naturally infected heifers

Recibido el 26 de junio de 2009 y aceptado el 12 de abril de 2010.

* Instituto Tecnolgico El Llano Aguascalientes, Km 18, carretera Aguascalientes-San Luis Potos, El Llano, Aguascalientes, Apartado Postal

74-2, Apartado Postal 2, C. P. 20041, Aguascalientes, Aguascalientes, Mxico, Tel. (449) 9161251, correo electrnico: cruva18@yahoo.com.

mx

** Agropecuaria Tepetatillo, S. A. de C.V., km 30, carretera Aguascalientes-San Luis Potos, 20330, Aguascalientes, Mxico.

Abstract

The aim o the study was to detect the DNA presence o N. caninum in naturally inected animals, at two moments o their frstgestation and at parturition, as well as to record the presentation o abortions. Twenty emales between 12 to 14 months o

age, seropositive to ELISA test, were selected rom a dairy arm with presence o this parasitosis. The emales were artifcially

inseminated and blood samples were taken in the frst and second third o gestation and during parturition; DNA was isolated

and it was analyzed by a single tube nested PCR with specifc primers. In the sampling corresponding to the frs t third o gestation,

7/20 positive cases were observed (35%), in the second 15/20 (75%) and during parturition 10/20 positive cases (50%) . From the

total o the animals included in this study, three stayed negative to the test in the three samplings (15%), our were always positive

(20%), eight were positive in the second sampling but negative in frst (40%) and fve were positive in the second and negative in

frst and the third sampling (25%). All animals remained seropositive during the study; our aborted in the last third o gestation.

All the live born calves were seropositive to N. caninum.

Key words: NEOSPORA CANINUM, DNA, BLOOD, PCR, DAIRY HERD, MEXICO.

Resumen

El objetivo del trabajo ue detectar la presencia de ADN de N. caninum en animales inectados naturalmente, en dos tiempos

de su primera gestacin y al parto, as como registrar la presentacin de abortos. Se seleccion, en un establo, con presencia

de parasitosis, un lote de 20 hembras de entre 12 y 14 meses de edad, seropositivas en ELISA, las hembras ueron inseminadas

artifcialmente y se tomaron muestras de sangre en el primero y segundo tercios de gestacin y al parto; se aisl ADN y se someti

a PCR anidado en un solo tubo con iniciadores especfcos. En el muestreo correspondiente al primer tercio de la gestacin,

se observaron 7/20 casos positivos (35%), en el segundo 15/20 (75%) y al parto 10/20 casos positivos (50%). De los animales

incluidos en el estudio, tres se mantuvieron negativos a la prueba en los tres muestreos (15%), cuatro ueron siempre positivos

(20%), ocho ueron positivos en el segundo muestreo pero negativos en el primero (40%) y cinco ueron positivos en el segundo

y negativos en el primero y tercero muestreos (25%). No se present seroconversin en ningn animal durante el estudio; cuatro

de ellos presentaron aborto en el ltimo tercio de gestacin. Todas las cras nacidas vivas resultaron seropositivas a N. caninum

Palabras clave: NEOSPORA CANINUM, ADN, SANGRE, PCR, GANADO LECHERO, MXICO.

Omar Ivn Santana* Carlos Cruz-Vzquez* Leticia Medina-Esparza*Miguel Ramos Parra* Ciro Castellanos Morales** Daniel Quezada Gallardo**

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Introduction

Bovine neosporosis is a disease caused by aparasite Apicomplexa, Neospora caninum,that can cause abortions between the thirdand ninth month o gestation, although this occursmore requently during the th and sixth month; itaects mainly dairy cattle and is considered to be an

important cause o abortions in Europe, South Arica,Japan, Australia, New Zealand and dierent countrieso America, including Mexico.1-5

Parasitosis transmission is carried out by two ways:vertical transmission (endogenous), rom an inectedmother to its etus; and horizontal transmission(exogenous), where the bovine must ingest eed or water contaminated with sporulated oocysts o theparasite, that are excreted by dogs the main endcarriers oN. caninum.

Vertical transmission is recognized as the elementresponsible or inection perpetuation in the herd.4-7In chronically inected cows, transmission to the etus

during gestation happens as a consequence o latentinection exacerbation due to immunodepressiongenerated by gestation;8-10 in consequence parasitemiaallows the inecting parasite orms to invade theplacenta and other etal tissues. In these cases, thenewborn is clinically healthy although inected, andsometimes there are abortions.4,11-13

Bovine neosporosis diagnosis may be carried out incattle by dierent indirect serological techniques suchas enzyme linked Immunosorbent assay (ELISA) andindirect immunofuorescence, while in the abortedetuses direct detection methods are used, such ashistopathology, immunohistochemistry and recentlyPCR assay, using mainly brain, heart and liver thatare the most commonly aected organs;7,14 also, ithas been reported that by PCR it is possible to detectDNA rom the parasite in leucocytes, lymphocytes andblood, which directly demonstrates the presence o theparasite in live animals with natural or experimentalinections.15-18

The objective o this study was the detection oN.caninumDNA by single tube nested PCR in naturallyinected animals at two moments o their rst gestationand at parturition, as well as to record the presentationo abortions.

This study was carried out on a dairy arm located inAguascalientes, Mexico, in the northern central regiono Mexico; the arm maintains Holstein cattle lodged inopen pens and managed with reedom o movement,19eed consists o a ration that includes maize silage,alala, grain mix and mineral complements; thisarm had already been detected as seropositive withabortions associated with N. caninum.3,20

A lot was selected o 20 heiers, 12 to 14 months

Introduccin

La neosporosis bovina constituye una enermedadcausada por un parsito Apicomplexa, Neosporacaninum, que puede provocar abortos entre eltercero y noveno meses de gestacin, aunque es mscomn que suceda entre el quinto y sexto meses; aectaprincipalmente al ganado lechero y se considera

importante causa de aborto en Europa, Sudrica,Japn, Australia, Nueva Zelanda y dierentes pases deAmrica, incluyendo Mxico.1-5

La transmisin de la parasitosis se realiza mediantedos ormas: la transmisin vertical (endgena), de unamadre inectada a su eto, y la transmisin horizontal(exgena), en la cual el bovino debe ingerir alimentoo agua contaminados con ooquistes esporulados delparsito, que excreta el perro, principal portadordenitivo de N. caninum.

La transmisin vertical se reconoce como responsablede la perpetuacin de la ineccin en el hato.4-7 En vacas inectadas de orma crnica, la transmisin al

eto durante la gestacin sucede como consecuenciadel recrudecimiento de la ineccin latente, debidoa la inmunodepresin generada por la gestacin;8-10la parasitemia consecuente permite que las ormasinectantes del parsito invadan la placenta y dierentestejidos etales. En estos casos, generalmente la cra naceinectada pero clnicamente sana, aunque el abortotambin puede presentarse.4,11-13

El diagnstico de la neosporosis bovina se puederealizar en el ganado mediante dierentes tcnicasserolgicas indirectas, como el inmunoensayo enzimtico(ELISA) y la inmunofuorescencia indirecta, mientrasque en los etos abortados se usan mtodos de deteccindirectos, como la histopatologa, la inmunohistoqumica y recientemente las pruebas de PCR, utilizandoprincipalmente cerebro, corazn e hgado, que son losrganos comnmente ms aectados;7,14 asimismo, seha inormado que mediante PCR es posible detectar ADN del parsito en leucocitos, linocitos y sangre, locual demuestra la presencia del parsito de maneradirecta en animales vivos con inecciones naturales oexperimentales.15-18

El objetivo del presente trabajo ue detectar lapresencia de ADN de N. caninum mediante PCRanidado en un solo tubo, en animales naturalmente

inectados en dos momentos de su primera gestacin yal parto, as como registrar la presentacin de abortos.El estudio se realiz en un establo lechero ubicado

en Aguascalientes, Mxico, que se localiza en laregin centro-norte de Mxico; el establo mantienea los animales alojados en corrales abiertos bajo elsistema de estabulacin libre con ganado Holstein,19 y la alimentacin consiste en una racin integralque incluye silo de maz, alala, mezcla de granos y

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o age, seropositive to the ELISA test that identiesspecic N. caninumIgG; detected by commercial HerdCheck anti-N. caninum* with 100% sensitivity and98.9% specicity, used according to the procedurerecommended by the manuacturer. The test wascarried out with only one serum dilution (1:100),in order to detect positive and negative animals; thesera were run in pairs and the cuto point was 0.50,

considering as positive those with mean readings o >0.50. These animals were articially inseminated aterthey were selected or this study.

Two blood samples were taken rom the caudal vein with vacuum container tubes,** one withoutanticoagulant another with EDTA, on three occasions:during the rst third o gestation (when it wasconrmed by rectal palpation and ultrasound), onthe 55th day; on the second third o gestation on day160th and the last one when either calving or abortionoccurred. Calves born rom these animals were alsoincluded in the study by taking blood samples, withoutanticoagulant rom the jugular vein beore they took

colostrums.Blood samples collected without anticoagulant

were processed to obtain serum by centriugationat 1 000 gor 15 min, once obtained it was stored at20oC until their use. These samples were processedby ELISA, as described above, in order to record opticdensity values (OD), rom each o the three samples oevery animal included in the study.

Blood samples, with anticoagulant, were processedto extract DNA using the commercial packageUltraclean DNA BloodSpin,*** ollowing instructionso the manuacturer. DNA samples were subjectedto one tube nested PCR, similar to that described byEllis et al.,21 using previously established positive andnegative controls.3 DNA concentration o each samplewas veried by UV spectrophotometer and PCR assayswere applied on 5 L o the sample with 2 g DNA.Amplication products were analyzed in 2.5% agarosegels, running in each gel a molecular weight marker,to estimate the weight o the amplied product; thegels were stained with ethidium bromide and viewedwith an UV light lamp. Positive results were those thatshowed a product o 146 base pairs.

A chi2 test was applied (P < 0.05) to compareproportions o positive and negative results to the

nested PCR assay in each sampling.The 20 study animals included in the studymaintained their seropositive status during the ollow-up period, although they had OD value fuctuationsin the dierent samplings. Nevertheless, the highestOD values in most o the animals corresponded to thesecond third o gestation (Table 1).

DNA detection in blood was intermittent betweenanimals and between samplings (Table 2); thereore, it

complementos minerales; este establo ya haba sidoregistrado como seropositivo y con abortos asociadosa N. caninum.3,20

Se seleccion un lote de 20 vaquillas de entre 12 y14 meses de edad, seropositivas en la prueba de ELISAque detecta IgG especcas a N. caninum; aqullase mediante el uso del paquete comercial HerdCheck anti-N. caninum* con sensibilidad de 100% y

especicidad de 98.9%, de acuerdo con el abricante,siguiendo el procedimiento recomendado por esteltimo. La prueba se trabaj con una sola dilucinde suero (1:100), para detectar positivos y negativos;los sueros se corrieron pareados y el punto de corteue de 0.50, considerndose como positivos los quetuvieran lecturas medias > 0.50. Estos animales ueroninseminados articialmente en echas posteriores a suseleccin para el presente estudio.

Se tomaron dos muestras de sangre de la vena caudalcon tubos de ensaye al vaco,** una sin anticoagulantey otra con EDTA, en tres ocasiones: en el primer terciode gestacin (al conrmarse mediante palpacin rectal

y ultrasonido), al da 55 de sta; en el segundo terciode la gestacin, el da 160, y por ltimo al suceder elparto o la presentacin del aborto. Las cras nacidasde estos animales tambin se incluyeron en el estudio,tomando una muestra de sangre, sin anticoagulante,de la vena yugular antes de que tomaran calostro.

Las muestras de sangre recolectadas sin anticoa-gulante, se procesaron para obtener el suero mediantecentriugacin a 1 000 g durante 15 min, el cual serecuper y almacen a 20C hasta su uso. Estasmuestras ueron procesadas mediante ELISA, como sedescribi anteriormente, con la nalidad de registrarel valor de las densidades pticas (DO), presentes enlos tres muestreos para cada animal incluido en elestudio.

Las muestras de sangre, con anticoagulante, seprocesaron para extraer ADN utilizando el paquetecomercial Ultraclean DNA BloodSpin,*** siguiendolas instrucciones del abricante. Las muestras de ADN se sometieron a una PCR anidada en un solotubo, similar a la descrita por Ellis et al.,21 utilizandotestigos positivos y negativos previamente denidos.3La concentracin de ADN en cada muestra uevericada por espectrmetro UV y en los ensayos dePCR se usaron 5 l de la muestra con 2 g de ADN.

Los productos de la amplicacin se analizaron engeles de agarosa al 2.5%, corriendo en cada gel unmarcador de peso molecular, para estimar el peso

*IDDEX Laboratories, Inc., Westbrook, Maine, EstadosUnidos de Amrica.**Venoject, Terumo Europe N.V, Blgica.*** MO BIO Laboratories, Inc., Carlsbad, Caliornia, EstadosUnidos de AmricaPhix 174 DNA Promega Co., Madison, Wisconsin, EstadosUnidos de Amrica.

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in the body o the animals, which indicates that

reactivation o inection during gestation o animals with chronic inection has a dynamic presentationbeginning rom the rst third o gestation although notin a continuous manner. Other authors 15,16 observeda similar situation in two year old animals. Previously,this phenomenon was only evident in live animals bythe elevation o antibody titers which was detectedmainly during the second third o gestation,4,5 that inturn is related to an increase in parasite multiplication,as indicated by the vertical transmission as well as theprobability o abortion.22

Several studies have reported that abortions byN.caninumare more requent between the th and sixth

months o gestation, that is to say, during the secondthird o gestation when cell mediated immunity islowered, and apparently inection reactivation isavored.4,8-10 In this study, during sampling made inthis period, parasite DNA was detected in 75% o theanimals; likewise, in this period the highest OD valueso the study were detected in most o the animals. Thereare literature reports that indicate more requency oabortions during the second third o gestation than in

incluidos en el estudio, tres se mantuvieron negativos a

la prueba de PCR anidado en los tres muestreos (15%),cuatro ueron siempre positivos (20%), ocho ueronpositivos en el segundo muestreo pero negativos en elprimero (40%) y cinco ueron positivos en el segundoy negativos en el primero y tercero muestreos (25%).

El aborto se present nicamente en cuatro animales,en todos los casos sucedi en el ltimo tercio de lagestacin; en dos de estas vacas se detect ADN en lostres muestreos y en una ms en el segundo tercio dela gestacin y al momento del parto (Cuadro 2); noue posible contar con muestras de suero sanguneode los etos abortados para determinar la presencia deanticuerpos anti-N. caninum. Todas las cras nacidas

vivas ueron seropositivas.Los resultados observados en el lote bajo estudiopermiten inerir presencia de ormas inectantesde N. caninum circulando por el cuerpo de losanimales, lo cual indica que el enmeno dereactivacin de la ineccin durante la gestacin enanimales crnicamente inectados se presenta deorma dinmica desde el primer tercio de gestacin,aunque no de orma continua; otros autores15,16

Cuadro 2

DETECCIN DE ADN DEN caninum EN SANGRE, MEDIANTE PCR ANIDADA,EN 20 VAQUILLAS SEROPOSITIVAS INFECTADAS NATURALMENTE

DETECTION OFN caninum IN BLOOD BY NESTED PCR, IN 20NATURALLY INFECTED SEROPOSITIVE HEIFERS

CowPhysiological status

1stThird gestation 2ndThird gestation Calving / abortion

1 - - +

2 + + +*

3 - - -

4 - + -

5 - + +

6 + + +

7 - - -

8 - - +*

9 - + +

10 - + -

11 - - +

12 - + +13 + + +*

14 + + -

15 + + -*

16 + + -

17 + + +

18 - + -

19 - + -

20 - - -

* aborted

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observaron una situacin similar en animales de dosaos de edad. Anteriormente este enmeno sloera evidente en animales vivos por la elevacin enel ttulo de anticuerpos, situacin que se presentaparticularmente en el segundo tercio de la gestacin,4,5y que se relaciona con aumento en la multiplicacindel parsito, y como indicio de la transmisin vertical,as como de la probabilidad de aborto.22

Diversos estudios inorman que los abortos porN. caninum suceden con mayor recuencia entreel quinto y sexto meses de gestacin; es decir, en elsegundo tercio de sta, periodo durante el cual se haobservado detrimento de la inmunidad mediada porclulas, lo cual parece avorecer la reactivacin de laineccin.4,8-10 En el presente estudio, en el muestreocorrespondiente a ese periodo, se detect ADN delparsito en 75% de los animales; de igual orma, eneste periodo se detectaron los valores de DO msaltos del estudio en la mayora de los animales; en laliteratura se reconoce que la probabilidad de abortaren el segundo tercio de la gestacin es mayor que

en otras etapas,5,23 mientras que en otro estudio seinorma que en dicha etapa es posible encontrar 0.155ng de ADN del parsito en sangre por cada 1 ng degenoma bovino, ello representa dos veces ms que elencontrado en la etapa temprana de gestacin.16

Por otra parte, los valores de DO altos se asociancon mayor parasitemia, y son consecuencia de mayorproduccin de anticuerpos, los cuales pueden ayudara proteger del aborto,9,24 ya que animales sanos noproducen anticuerpos especcos.17 En el presenteestudio, los cuatro animales que presentaron abortolo hicieron en el ltimo tercio de la gestacin y slouno de ellos ue negativo a la presencia de ADN almomento de presentarse el aborto, aunque sus valoresde DO ueron elevados; el aborto por N. caninumpuedepresentarse a partir de los tres meses de gestacin,4,5 y se ha observado que alrededor del momento enque ocurre el aborto es posible detectar ADN ensangre.15,16 El estado endmico de la ineccin por N.caninumconlleva a la presentacin de baja proporcinde abortos, pero stos se pueden desencadenarpor actores asociados con la idiosincrasia de cadaanimal.4,5,24

En la literatura se inorma que en hatos conineccin crnica endgena, la respuesta inmune es

suciente para prevenir el aborto mas no la transmisinvertical,24 lo que coincide con los resultados del presenteestudio, ya que toda la progenie ue seropositiva; losanimales con menor edad transmiten ms cilmenteel parsito, por lo que la transmisin es prcticamente100% eectiva.6,16

En conclusin, el presente estudio permitidetectar la presencia de ADN de N. caninum encirculacin sangunea en dierentes momentos de la

the other stages,5,23 while there is a study that reportsthat during this stage it is possible to nd in blood0.155 ng o ADN per each 1 ng o bovine genome,this represents twice the amount ound during earlygestation stage.16

On the other hand, high OD values are associated with a higher parasitemia, and are the consequenceo higher antibody production, that can help protect

animals rom abortion,9,24 since healthy animals donot produce specic antibodies.17 In this study, theour animals that aborted did so during the last thirdo gestation and only one o them was negative tothe presence o DNA at the time o abortion, eventhough OD values were high; abortion byN. caninummay happen rom the third month o gestation on,4,5and observations have established that it is possible todetect the presence o DNA in blood at the time oabortion.15,16 Endemic status o inection byN. caninumderives in a low proportion o abortions, but these maybe caused by actors associated with the idiosyncrasy oeach animal.4,5,24

Literature reports that in herds with endogenouschronic inection, immune response is sucient toprevent abortion but not vertical transmission,24 thiscoincides with the results obtained during this study,since all progeny was seropositive; younger animalseasily transmit the parasite; thereore, transmission ispractically 100% eective.6,16

In conclusion, this study detected the presenceoN. caninumDNA in circulating blood, at dierentgestation stages o naturally and chronically heiers which demonstrated that the inection reactivationphenomenon may be present since the rst stage ogestation as a mechanism used by the parasite to causeetus inection and nally cause abortions.

Acknowledgments

Special thanks to the Direccion General de EducacionSuperior Tecnologica o the Secretaria de EducacionPublica or nancing granted to carry out this study.

Referencias

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site relationship in bovine neosporosis. Vet ImmunolImmunopathol 2005;108:29-36.PARE J, THURMOND MC, HIETALA SK. CongenitalNeospora caninuminection in dairy cattle and associatedcal hood mortality. Can J Vet Res 1996;60:133-139.DAVINSON HC, OTTER A, TREES AJ. Estimation overtical and horizontal transmission parameters oNeos- pora caninuminections in dairy cattle. Int J Parasitol1999;29:1683-1689.ROMERO JJ, PEREZ E, DOLZ G, FRANKENA K. Fac-tors associated with Neospora caninumserostatus in cattleo 20 specialized Costa Rican dairy herds. Prev Vet Med2002;53:263-272.DUBEY JP, SCHARES G. Diagnosis o bovine neosporo-

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Agradecimientos

Se agradece a la Direccin General de EducacinSuperior Tecnolgica, de la Secretara de EducacinPblica, por el nanciamiento otorgado para larealizacin de este trabajo.

Neospora caninum inoculation o heiers. Vet Parasitol2006;135:197-203.SERRANO-MARTNEZ E, FERRE I, MARTNEZ A,OSORO K, MATEOS-SANZ A, DEL POZO I et al. Experi-mental neosporosis in bulls: Parasite detection in semenand blood and specic antibody and intereron-gammaresponses. Theriogenology 2007;67:1175-1184.CRUZ-VAZQUEZ C, RAMOS PM, VITELA MI, GARCIA-

VAZQUEZ Z, QUINTERO-MARTlNEZ MT. Relation-ships between stable fy inestation with some physicalacility characteristics and sanitation practices in severaldairy arms in the state o Aguascalientes, Mexico. VetParasitol 2007;149:246-250.GARCIA-VAZQUEZ Z, CRUZ-VAZQUEZ C, MEDINAEL, GARCIA TD, CHAVARRIA MB. Serological surveyo Neospora caninum inection in dairy cattle herds inAguascalientes, Mexico. Vet Parasitol 2002;106:115-120.ELLIS JT, MCMILLAN D, RYCE C, PAYNE S, ATKIN-SON R, HARPER PAW. Development o a single tubenested polymerase chain reaction assay or the detectionoNeospora caninumDNA. Int J Parasitol 1999;29:1589-1596.

PEREIRA-BUENO J, QUINTANILLA-GOZALO A, SEI- JAS-CARBALLEDO A, COSTAS E, ORTEGA-MORALM. Observational studies in Neospora caninuminecteddairy cattle: pattern o transmission and age-relatedantibody fuctuations. Int J Parasitol 2000;30:906-909.LOPEZ-GATIUS F, PABON M, ALMERIA S. Neosporacaninum inection does not aect early pregnancy indairy cattle. Theriogenology 2004;62:606-613. WILLIAMS DJ, TREES AJ. Protecting babies: vaccinestrategies to prevent oetophaty in Neospora caninum-inected cattle. Parasite Immunol 2006;28:61-67.

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