seroprevalence of neospora caninum infection in dairy and beef cattle in spain

Seroprevalence of Neospora caninum infection in dairy andbeef cattle in Spain

A. Quintanilla-Gozalo a, J. Pereira-Buenoa, E. TabareÂ s b, E.A. Innes c,R. GonzaÂ lez-Paniello a, L.M. Ortega-Morab, *

aDepartamento de Sanidad Animal, Facultad de Veterinaria, Universidad de LeoÂn, 24007 LeoÂn, SpainbDepartamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain

cMoredun Research Institute, International Research Centre, Pentland Science Park, Bush Loan, Penicuik, Edinburgh EH26 OPZ,

UK

Received 12 March 1999; received in revised form 3 June 1999; accepted 3 June 1999

Abstract

In recent years, neosporosis has been identi®ed as a major cause of abortion in dairy and beef cattle. Althoughthe disease has been described worldwide, there is a lack of information concerning the prevalence of this infection

in di�erent cattle production systems. The aim of this study was to investigate the seroprevalence of Neosporacaninum infection in a representative area of beef and dairy cattle production in Spain. A cross-sectional study wasundertaken in which herds constituted the initial sampling unit and two strata (dairy and beef herds) were

considered. Using a 95% level of con®dence and setting 5% (beef) and 5.4% (dairy) error limits, 216 beef and 143dairy herds were randomly selected and sampled. Nine animals (>1 year old) were randomly sampled in each herdto detect the presence of the infection. A herd was considered infected when at least one animal was seropositive. In

total, serum samples from 1121 dairy and 1712 beef animals were collected and tested for speci®c anti-N. caninumIgG using an ELISA. Speci®c antibodies were detected in 55.1% (119/216) beef and 83.2% (119/143) dairy herds.Individual prevalences obtained were 17.9% (306/1712) for beef and 35.9% (402/1121) for dairy animals. Presence

of N. caninum infection was higher in dairy than in beef herds and the association between infection and the cattleproduction system (dairy or beef) was statistically signi®cant [w2Y=29.21, P<0.001, OR=4.04 (2.35±6.99)]. Herdsize of dairy cattle did not appear to be associated with N. caninum infection. On the contrary, infection wasassociated with herd size in beef cattle (w 2=12.79, P<0.01). Finally, no association was found between

replacement or pasture management and infection in beef herds. # 1999 Australian Society for Parasitology Inc.Published by Elsevier Science Ltd. All rights reserved.

Keywords: Neospora caninum; Seroprevalence; Cattle; Dairy; Beef; Herd management factors

1. Introduction

Neospora caninum is a cyst-forming coccidian

parasite that was ®rstly characterised and

described in 1988 in dogs in the USA [1, 2].

International Journal for Parasitology 29 (1999) 1201±1208

0020-7519/99/$20.00 # 1999 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

PII: S0020-7519(99 )00084-3

* Corresponding author: Tel. 34-91-3943713; fax: 34-91-

3943908; e-mail: [email protected]

Neospora caninum infection has since then beendiagnosed in a wide range of animal species,including domestic and wild ruminants (cattle,goat, sheep, camel, water bu�alo and deer) andseveral reports have con®rmed the worlwide dis-tribution of this parasite [3±8].

The life cycle of N. caninum has been recentlyelucidated. Infective oocysts are shed in thefaeces of dogs and when they are ingested by anappropriate intermediate host the parasite ®nallydevelops into tissue cysts [9]. However, horizon-tal transmission is apparently infrequent and thevast majority of bovine infections are acquiredcongenitally by transplacental infection [3]. It hasalso been recently suggested that oral infectionÐvia colostrumÐmight be another possible routeof vertical transmission in newborn calves [10].

Neosporosis is recognised as one of the mostcommon and important causes of sporadic andendemic bovine abortion in dairy and beef cattleworldwide [3, 11±13]. Although the disease hasbeen diagnosed in the main dairy and beef cattle-producing countries, there is little information onthe prevalence of this infection in the di�erentcattle production systems around the world.

The aims of the present study were to investi-gate the seroprevalence of N. caninum infectionÐusing a randomly selected sample of beef anddairy herdsÐin a representative area of beef anddairy cattle production in Spain and to clarifyseveral aspects of the epidemiology of bovineneosporosis. In order to investigate the relation-ship between infection, based on a positive sero-logic result, and herd production system (dairyor beef) and the association between infectionand other herd parameters such as size, replace-ment management and pasture management werestudied.

2. Material and methods

2.1. Target and studied area

Cattle herds located in a cattle raising regionin NW Spain were the target of this study. Thisarea was selected because it was considered to berepresentative of dairy and beef cattle production

in other parts of Spain. A cattle census providedby the Local Veterinary Service for the year 1996showed that this area had an average populationof 11 460 animals, aged more than 1 year old,distributed over 749 cattle herds. Of these ani-mals, 7836 were beef cattle (corresponding tonearly 0.5% of the Spanish beef cattle census)and were distributed over 498 herds (0.6%Spanish beef herd census) located principally in amountain area (1000 m above sea level), and3624 were dairy cattle (corresponding to nearly0.3% of the Spanish dairy cattle census) andwere distributed over 251 herds (0.2% Spanishdairy herd census) and located in the valley area(800 m above sea level). Beef cattle were of theBrown Swiss breed and its crosses with Asturianade los Valles and Limousine and were managedmainly as pasture-based herds. Dairy herds weremanaged under an intensive system and cattlewere mainly of the Friesian/Holstein breed.

2.2. Sample size determination

A cross-sectional study was undertaken inwhich herds constituted the initial sampling unitand two strataÐdairy and beef herdsÐwere con-sidered. Sample sizes required to estimate herdprevalence and detect infection were calculatedusing the Episcope Computer Program [14]. Asthere is a lack of information concerning preva-lence of N. caninum infection in the di�erentcattle producing systems, the worst caseÐ50%prevalenceÐwas considered. On the basis of theformula n= Z 2 pq/L 2, using a 95% level of con-®dence and setting error limits (L) of 5% forbeef and 5.4% for dairy herds, the desiredsample size (n) was 216 and 143 beef and dairyherds, respectively. This sample size was distribu-ted according to the proportional cattle herdnumber in the di�erent municipalities in thestudy area using random number tables. In arecent study, an intraherd neosporosis seropreva-lence in dairy cattle of 23.4% was found(Quintanilla-Gozalo A, Pereira-Bueno J, PeÂ rez-PeÂ rez V, Ortega-Mora LM. Neospora caninuminfection in dairy herds in NW Spain. Proc VIIthInt Coccidiosis Conf and EU COST 820Workshop, Oxford, UK, 1±5 September 1997).

A. Quintanilla-Gozalo et al. / International Journal for Parasitology 29 (1999) 1201±12081202

On the basis of the formula n=[1-(1-CL)1/D][N-(D-1)/2], where n is the required sample size, CLis the con®dence level (95%), D is the supposednumber of infected cattle in each herd (23.4%)and N is the ¯ock size (between 10 and 500 ani-mals), nine animals were randomly sampled ineach herd to detect the presence of theinfection [15]. A herd was considered infectedwhen at least one animal was seropositive.

2.3. Samples and data collection

Blood samples were collected from 1121 dairycattle belonging to 143 herds and 1712 beef cattlefrom 216 herds by the Local Veterinary Servicebetween April and August 1997. Data collectedat the time of sampling included herd locationand size, herd production (dairy or beef), predo-minant breed, replacement management and pas-ture management.

2.4. Parasite production and antigen preparation

Neospora caninum tachyzoites from the Nc-1isolate [2] were cultured in Vero cells inMinimum Essential Medium (MEM) (Sebak)supplemented with 100 UI�mlÿ1 penicillin,100 mg�mlÿ1 streptomycin, 2.2 g�lÿ1 sodium bi-carbonate and 2% (v/v) heat-inactivated equineserum (Sebak), at 378C in a 5% CO2 humidi®edincubator. Tachyzoites were harvested fromtissue culture and washed three times in sterile0.3 M PBS, pH 7.4. Tachyzoites were separatedfrom host cells by passing the mixture through a27-gauge needle, followed by passage through a5 mm polycarbonate ®lter. Tachyzoites solubleproteins to be used in the ELISA were preparedas follows. Approximately 2�109 tachyzoiteswere pelleted and resuspended in 4 ml of 10 mMTris hydrochloride containing 2 mM of theenzyme inhibitor phenylmethylsulfonyl ¯uoride.The tachyzoites were disrupted by two cycles offreezing and thawing followed by ®ve to sixcycles of ultrasound treatment in an ice-bath; thematerial was centrifuged at 10 000� g for 30 min

at 48C and the supernatant recovered, aliquotedand cryopreserved at ÿ808C. The protein contentwas determined using the bicinchoninic acidmethod [16].

2.5. Detection of anti-Neospora caninumantibodies

In this study, a slightly modi®ed version of theELISA described by Osawa et al. [17] was usedto detect the existence of speci®c anti-N. caninumIgG. A comparison between this ELISA methodand the IFAT was also carried out. As positivecontrol a pool of sera from N. caninum naturallyinfected cows from a herd where Neospora abor-tion had been diagnosedÐusing IFAT andimmunohistochemical analysisÐwere used. Thenegative control consisted of a pool of sera fromcows that had never aborted belonging to a herdwhere Neospora abortion had never been diag-nosed. Serum samples with O.D. equal to orhigher than 0.4 were considered to be Neosporaantibody-positives [17]. For the IFAT, tachy-zoites were obtained from cell cultures asdescribed above and subsequently coated onto10-spot IFAT glass slides (Biomerieux) and airdried. Basically, the procedure was carried out asdescribed by Trees et al. [18]. For comparison,two di�erent cuto� points (1/512 and 1/1024)were estimated.

2.6. Analysis of data

To validate the ELISA, sensitivity, speci®cityand level of agreement (kappa statistics) betweenthis ELISA and IFAT were calculated [19] on231 adult cattle serum samples from three di�er-ent infected herds. Chi-squared tests of indepen-dence were used to analyse associations betweeninfection by N. caninum and other factors studiedin the epidemiological study. The odds ratio werecalculated to determine the degree of the associ-ation. For statistical analyis the STATCALC of theEPI INFO V.6 program computer package pro-gram was used [20].

A. Quintanilla-Gozalo et al. / International Journal for Parasitology 29 (1999) 1201±1208 1203

3. Results

3.1. Comparison IFAT and ELISA

Sensitivity and speci®city of the ELISA (cuto�point 0.4 O.D.) is detailed in Table 1. A higherdegree of agreement between ELISA and IFATwas obtained when an IFAT titre of 1/512 wasused.

3.2. Seroprevalence of Neospora caninuminfection in cattle herds

Serological evidence indicates that N. caninuminfection is widely present in dairy and beefherds in the sampled area (Table 2). The infec-tion was more common in dairy compared withbeef herds and an important degree of associ-ation between infection and herd production sys-tem (dairy or beef) was observed.

3.3. Association infection-herd management factors

Farm size was classi®ed into three categoriesÐA, B and CÐon the basis of the number of ani-mals present. Seroprevalences were similar in thethree size categories of dairy herds studied, but a

signi®cant association between prevalence ratesand beef herd size was showed (Fig. 1).

Signi®cant di�erences were not observedamong dairy herds grouped according to munici-palities (ranging from 72.0 to 89.1%) (Table 3).

Herd seroprevalences according to geographicallocation of beef herds ranged from 20 to 75%. Inthis case, the study of the association betweeninfection and location of the herd was not carriedout due to the large number of categoriesincluded.

Herds were classi®ed in two categories accord-ing to their replacement policy: mainly donefrom animals born in the herd or mainly carriedout buying animals from other places. Almost allthe dairy herds included in this study belonged tothe ®rst category. Herd seroprevalence accordingto replacement policy in beef cattle was verysimilar for both categories and no signi®cantassociation was observed (Table 4).

According to the grazing management of theherd two categoriesÐcorresponding to intensivemanagement or use of nearby independent pas-tures exclusives for the herd and when communalpastures were mainly used, at least during thegrazing seasonÐwere contemplated. Pasturemanagement of dairy herds was very uniform

Table 2

Herd and individual seroprevalence of Neospora caninum infection in dairy and beef cattle

Herd seroprevalencea (%) (seropositives/examined) Individual seroprevalence (%) (seropositives/examined)

Dairy cattle 83.2 (119/143) 36.8 (412/1121)

Beef cattle 55.1 (119/216) 17.9 (306/1712)

a � 2Y =29.21; P<0.001; OR=4.04 (2.35±6.99).

Table 1

Comparison between IFAT and ELISA to detect speci®c anti-Neospora caninum antibodies in adult cattle serum samples

IFATa Degree of agreement (k)b

Positives Negatives

Sensitivity Speci®city

ELISA

Positives 54 (45) 11 (20) 0.88 (0.76) 100% (100%) 93.8% (89.2%)

Negatives 0 (0) 166 (166)

a IFAT titre 1/520 (titre 1/1024).b 0.80< k<1, almost perfect agreement; 0.60< k<0.80, substantial agreement.


and all were included in the ®rst group. In beefherds, no signi®cant association was observedbetween herd seroprevalence and grazingmanagement (Table 4).

4. Discussion

To the best of our knowledge, this is the ®rstcross-sectional study designed to investigate theseroprevalence of N. caninum infection in dairyand beef cattle herds in the same area. Becauseof the large number of samples processed in ourstudy we chose a whole-tachyzoite lysate-basedELISA which has a high sensitivity and goodspeci®city [17] and in our conditions, an almostperfect agreement with IFAT was obtained whena cuto� point of 1/512 was used [11]. Sensitivityand speci®city of this ELISA was similar to thosereported for another two widely used ELISAwhen compared with IFAT [21, 22]. However, ifthe IFAT results are considered as a veri®cationof true status of infection by N. caninum, someof the cows classi®ed as infected in this study

should be considered as false positives (93.8%speci®city). Among other causes, this may be dueto some cross-reactivity with Sarcocystis cruziwhen soluble N. caninum tachyzoites proteins areused as antigen [23] and the fact that S. cruziinfection is widespread in Spain [24], although, ina previous study using this technique, sera fromcattle infected with S. cruzi were negative in theN. caninum ELISA [17].

Seroprevalence rates of 83.2% dairy and55.1% beef herds are very high and one impli-cation of these results is that neosporosis shouldbe included in a di�erential diagnosis of causesof abortion in both dairy and beef herds inSpain. N. caninum infection in individual cattle inthis study was 36.8% in dairy and 17.9% in beefanimals. Considering that seropositive cows(aborting or non-aborting) have a two- to three-fold higher risk of abortion than seronegativecows [25, 26], reproductive problems in these ani-mals could have serious repercussions on thefarm economy. Selective culling of these animalsshould be implemented to decrease future risk ofabortion [26] and N. caninum-infected calves

Fig. 1. Herd seroprevalence of Neospora caninum infection in dairy and beef cattle in relation to herd size (A, fewer than 11

animals; B, between 11 and 49 animals; and C, more than 50 animals).


(infected mainly through transplacental trans-

mission) should not be used as replacementstock [27].

Results show that herd seroprevalence of N.caninum infection is higher in dairy than in beef

herds and an important degree of association wasobserved between infection and herd production

(dairy or beef). In the sampled area, beef cattlewere mainly of the Brown Swiss and its crosses

with local (Asturiana de los Valles) and French

beef breeds (Limousine and small numbers ofothers such as Charolais) and were kept at pas-

ture during the grazing season (April to October)and remained indoors during winter time. Dairy

herds were mainly of the Friesian/Holstein breed

and were managed as drylots or used nearbyindependent pastures. Infection by N. caninum

had been mainly diagnosed in dairy cattlealthough neosporosis abortion had been induced

in beef cows [28] and seroprevalence of the infec-

tion in beef cattle had been reported to be highand associated with a reduction in reproductive

performance [13]. A higher individual seropreva-lence has also been observed in dairy compared

with beef cattle cows in a study carried out in

France in cattle with abortion problems [29]. Atleast two studies have found no signi®cant associ-

ation between breed and abortion attributable toinfection with N. caninum [30, 31]. We hypoth-

esise that di�erences observed in the presentstudy between herd seroprevalence in dairy and

beef cattle could be mainly due to di�erent man-

agement practices between both types of cattleand not to di�erent breed susceptibilities.

Possibly di�erent factors such as historical repla-cement policies and origin and number of ances-

tors, one breed compared with multiple breed

constitution of the herds and drylot vs pasturemanagement facilitate vertical and horizontal

transmission in dairy herds to a greater extent

than in beef herds. Beef cattle in this part ofSpain are, in general, raised extensively, being

out on pastures most of the time, and are thusless likely to be exposed to infective oocysts poss-

ibly present in feedstu�s. The relative importanceof the source of infection for cattle of contami-

nated pasture compared with stored feed, wildcanids (foxes) compared with domestic dogs, and

di�erent classes of dogs (pet, farm or hounds) isstill unknown [32].

Most epidemiological studies of N. caninum

have examined risk factors for an individual cowrather than for a herd. In our study several fac-

tors (herd size, replacement policy and grazingmanagement) a�ecting herd management were

considered. A signi®cant association was foundbetween N. caninum infection and herd size in

beef cattle. This association was not found indairy cattle, although a higher rate of infection

was observed in medium-size herds than in othercategories. We could not ®nd an explanation for

this ®nding. We can only hypothesise that thisresult could be related to the maintenance of

management practices in dairy herds independentof the herd size and presence of a variety of man-

agement practices in beef herds dependent ofherd size. Moreover, a recent study examining in-

¯uence of herd size in dairy cattle did not ®ndany di�erences between case and control

herds [30].

The last two factors considered were related tovertical transmission (purchase policy) and hori-

zontal transmission (feeding practices) of N. cani-num infection in cattle herds. Although postnatal

transmission existsÐdogs are a de®nitivehost [9]Ðcongenital infection has been found in

78±88% of calves born to seropositive cows andaccount for most of the infection in the herds

Table 3

Seroprevalence of Neospora caninum infection in dairy and beef cattle in relation to location of the herd

Beef cattle Dairy cattle

Municipalities 1 2 3 4 5 6 7 8 9 10 1 2 3 4

Herd seroprevalence (%) 75.0 57.1 42.8 50.0 67.7 20.0 59.2 58.3 23.8 65.4 79.0 72.0 86.6 89.1


investigated [25]. No association was foundbetween these two factors and N. caninum infec-tion in beef herds. Similar results have beenfound in dairy cattle [30], in which the presenceand number of dogs appears to be the only herdfactor related with infection. Similarly, in a NewZealand study, no signi®cant association wasfound between the acquisition of heifers on afarm and abortions attributable to infection withN. caninum [31]. However, replacement policyprobably plays an important role due to the im-portance of vertical transmission. An importantconsideration is that infected cows, if retained inthe herd, may continue to produce infected calvesbecause of the possibility of persistent or inter-mittent parasitemia [33]. If they are clinicallynormal and retained in the herd as breedingstock, an increase in the rate of prevalence in theherd could result in an increase in the future riskof abortion or congenitally infected calves in theherd [27]. Neospora caninum can be transmittedfrom dam to o�spring for several generationsand this congenital transmission could explainthe maintenance of infection in a population ofcattle [34].

Acknowledgements

This work has been supported by a researchgrant from the Spanish Government (CICYT-AGF98-0804-C02), and in part by the Accionesintegradas programme from the SpanishGovernment and the British Council (HB97-167).The study was part of the EU research collabor-ation COST 820.

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seroprevalence of neospora caninum infection in dairy and beef cattle in spain

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