ORIGINAL PAPER

Neospora caninum infection dynamics in dairy cattle

Marcelo Piagentini & Carla Fredrichsen Moya-Araujo &

Nereu Carlos Prestes & Izidoro Francisco Sartor

Received: 8 August 2011 /Accepted: 6 March 2012 /Published online: 13 May 2012# Springer-Verlag 2012

Abstract Neospora caninum is considered in manycountries as one of the key abortion agents in cattle. Thisstudy aims to investigate the parasite behavior in dairy cattlein the municipality of Avaré, SP, Brazil, where abortionsfrequently occur. An ELISAwas performed to compare twosamplings on a total of 615 animals; tests were performed inthe same herds with a gap of 3 years. An increase in thepercentage of reactive animals was observed, ranging from21.6% at the first sampling to 38.9% at the second sampling.Of the 176 animals tested at both samplings, 61.93%retained a non-reactive status, 15.9% retained a reactivestatus, 19.88% switched from non-reactive to reactive and2.27% switched from reactive to non-reactive. Of the 100animals with reproductive disturbances, 50% presented anti-Neospora antibodies, thereby indicating the presence of theprotozoa. When comparing cows and their respective femaleoffspring, a predominance of horizontal infection was ob-served. Moreover, considering the significant percentage ofanimals that switched from non-reactive to reactive and theabundant presence of dogs among the herds, the N. caninumtransmission may be attributed to presence of carnivores.

Introduction

Since Bjerkas et al. (1984) identified Neospora caninum indogs with neurological problems, it has been recognized as aprominent tissue cyst-forming parasite that induces congen-ital diseases and abortion, especially in cattle (Mugridge etal. 1999).

In vitro cultures and serological tests used to distinguishN. caninum from Toxoplasma gondii became available in1998 (Dubey et al. 1988a,b).

Although N. caninum and T. gondii are structurally sim-ilar, each exhibits distinct biological behaviors (Anderson etal. 1995, 2000). The infection caused by this Apicomplexais characterized by a chronic course, usually with an extend-ed persistence in the tissues of infected individuals(Björkman and Uglla 1999). N. caninum is one of the mostfrequently diagnosed causes of epidemic and endemic abor-tions in cattle (Schares et al. 1999) and utilizes canines asthe definitive hosts, which release the parasite oocysts intothe environment (McAllister et al. 1998).

Vertical transmission is the primary form of diseasetransfer, by which most congenital infections are estab-lished in apparently healthy calves (Björkman et al.1996; Paré et al. 1997; Davison et al. 1999; Hietalaand Thurmond 1999) as well as in herds with highseroprevalence (Hietala and Thurmond 1999; Dijkstraet al. 2001, 2002; Kashiwazaki et al. 2001; McAllisterand Latham 2002). Wouda et al. (1999) have reported astrong correlation between N. caninum seropositive dogsand the high prevalence of bovines with antibodiesagainst the parasite. Other possible modes of horizontaldisease transmission include parasite transfer from cowcolostrum and milk (Uglla 1998) as well as transmission fromthe placenta or the amniotic fluid of infected animals(McAllister et al. 1998).

M. Piagentini :N. C. Prestes : I. F. SartorSchool of Veterinary Medicine and Animal Science,Sao Paulo State University,Botucatu, SP, Brazil

C. F. Moya-AraujoSchool of Veterinary Medicine, FIO,Ourinhos, SP, Brazil

M. Piagentini (*)Department of Animal Reproduction, School of VeterinaryMedicine and Animal Science, Sao Paulo State University,Distrito de Rubião Jr., s/n,18618-000, Botucatu, SP, Brazile-mail: marcelovete@terra.com.br

Parasitol Res (2012) 111:717–721DOI 10.1007/s00436-012-2891-0

Infection by protozoa triggers an antibody response,which can be detected by various types of tests. The pres-ence of antibodies indicates that the animal was at somepoint contaminated. Diagnostic tests can be performed usingseveral methods, including histological techniques, immu-nohistochemistry and polymerase chain reaction (PCR)(Dubey and Lindsay 1996; Hemphill 1999).

Understanding the epidemiology and biology of this par-asite would not be possible without the application of sero-logical tests. Many tests can be performed to detect anti-N.caninum antibodies, such as ELISA, indirect immunofluo-rescence (RIFI) and direct agglutination testing (Björkmanand Uglla 1999), with the first two being the most common-ly used. RIFI is frequently used as reference test, althoughSchares et al. (1998) have observed more sensitivity andspecificity with the total crude antigen ELISA. Paré et al.(1995) have reported that an ELISA performed with tachy-zoite total lysate prepared from bovines presented 89%sensitivity and 97% specificity, while other laboratorieshave reported a sensitivity of 92–98% and a specificity of87–100% with a similar type of ELISA.

With this work, we aimed to describe the dynamics of N.caninum infection and its effect on reproduction disturban-ces in dairy cattle.

Materials and methods

This study was performed at the municipality of Avaré, SP,Brazil, a 1219.6-km2 area with an altitude of 766 m locatedat 23 05′55″ S and 48 55′33″ W. The region has a warmclimate with temperatures ranging from 12 to 33.9°C, withan average temperature of 22.95°C.

Serum samples taken from 615 Bos taurus cows of threedifferent breeds collected in two samplings (n10245; n20310) with a 3-year gap were serologically analyzed. Of thetotal animals analyzed in the first sampling, 176 remained intheir respective herd, thereby allowing for a comparativeanalysis of the immunological status between the two testperiods. Each animal became pregnant at least one timeduring the study.

An anti-Neospora serological status survey was per-formed on 100 animals, and the infection-associated repro-duction disturbances were assessed.

One of the herds tested was comprised of 60 cows andtheir respective daughters. This herd was used to comparethe serological status between cows and female offspring.

All herds were comprised exclusively of Holstein cows.Reposition was made with heifers of their own squad, withno new animals acquired during the study period. An inten-sive management system was employed, and the animalsreceived hay, silage and concentrate and remained in thepaddock at all times. A vaccination system was rigorously

developed, according to literature recommendations, withspecial attention to abortive diseases such as IBR/BVD,brucellosis and leptospirosis.

In addition to the dogs living on the farms, other strayand neighbor dogs also lived amongst the herds, as theproperties are located close to the urban area limits.

Anti-Neospora antibody detection was performed using atachyzoites total lysate ELISA test originally described byParé et al. (1995).

The laboratory procedures were performed according tothe manufacturer’s instructions, and the results wereexpressed as the ratio S/P (< 0.50 negative, > 0.50 positive).

The normal curve for the two proportions test (Hoel1981) was applied, considering the large sample sizes(n10245 and n20370), according to the method:

Z0 ¼ bρ1 � bρ2ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

bp:bq 1n1þ 1

n2

� �

r

Z0 critical value to be compared to tables (± 1.96)P estimated proportion of the casesq complement p to 1.00n sample size

Results

The results demonstrate that the seroprevalence ofN. caninumin properties at the municipality of Avaré was 21.6% at thefirst sampling and 38.9% at the second sampling (Table 1).

A significant difference between the two samples wasdetected (P00.22 and P00.39), indicating that the propor-tion of reactive animals observed in the second samplingwas significantly higher than that found in the firstsampling.

Of the 176 animals compared in both samplings viaELISA test, 35 non-reactive animals (19.88%) began topresent antibodies at the second test, four switched fromreactive to non-reactive (2.27%) status, 109 retained non-reactive status (61.93%) and 28 were reactive in both tests(15.90%), as shown in Table 2.

Table 1 Number and relative percentage of examined Bos taurusbreeds from the municipality of Avaré, SP and of Neospora caninumreactivity by ELISA at first and second samplings

Animals First Second Total

Number of examined animals 245 370 615

Number (percentage)of reactive animals

53 (21.6) 144 (38.9) 197 (32)

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In the population analyzed, 100 animals experiencedreproduction-related pathological events, from which 14%that were seronegative at the first sampling tested positive atthe second test, 26% displayed no anti-Neospora antibodiesin both tests and 9% were seroreactive in both tests. Of theanimals tested exclusively in the second sampling, 24%were not seroreactive and 27% were seroreactive (Table 3).

Applying the binomial distribution test for 50% of thereactive animals with reproductive disturbances and 50% ofthe non-reactive animals with reproduction disturbances, theH0 hypothesis (Hoel 1981) can be rejected, thereby suggest-ing that the two occurrences differ significantly.

Sixty animals, including cows and their respective femaleoffspring, were examined in both samples, of which nineseropositive female offspring were born of seropositivecows, nine seronegative female offspring were born of se-ropositive cows, 15 seropositive female offspring were bornof seronegative cows and 27 seronegative female offspringwere born of seronegative cows (Table 4).

Discussion

To monitor the behavior of N. caninum in cattle herds at themunicipality of Avaré, SP, a survey of seroprevalence of theprotozoa was performed to analyze the following: the im-munological status of animals at two different time points,the correlation between the serological status of animals andreproduction disturbances, and the relationship between the

serological status of cows and their respective femaleoffspring.

Serological tests were utilized due to their ease of usewith an epidemiological survey, and the ELISA test wasselected for the analysis due to its high sensitivity andspecificity. This type of test is constituted by total lysate oftachyzoites.

A significant increase in the percentage of infected ani-mals was observed in all of the farms studied, ranging from21.6% at the first sampling to 38.9% at the second sampling.The latter percentage was considered as a high seropreva-lence level, which was higher than the results observed byCox et al. (1998), Andreotti et al. (1999), Gondim et al.(1999) and Hasegawa et al. (1999) in beef cattle; it was alsosignificantly higher than the results described by Pituco etal. (1998), Ogawa et al. (1999), Rezende et al. (1999), Sartoret al. (1999), Stobbe and Côrtes (1999) and Melo et al.(2001) in dairy cattle. It is important to note that Pituco etal. (1998) and Cox et al. (1998) have worked in farms with adeclared abortion history. However, this observation waslower than the results observed by Belo et al. (1999),Stobbe and Côrtes (1999) and Corbellini et al. (2001), eachof which described an abortion history of the analyzedherds.

Considering the small percentage of animals (15.9%) thatremained seroreactive since the first test, it is possible tohypothesize that this was due to the fluctuation of antibod-ies, which can occur in infected animals (Dannatt 1998;Schares et al. 1999).

Considering the immunological status of N. caninum-infected animals with reproduction disturbances, repre-sented almost exclusively by abortions, it was found that50% of the disturbances could be associated with N.

Table 2 Neospora caninum serological status tested by ELISA in twosamplings, and the number and percentage of animals of Bos taurusbreeds from the municipality of Avaré, SP

First Second Number of animals Percentage (%)

NR R 35 19.88

R NR 4 2.27

NR NR 109 61.93

R R 28 15.90

Total 176 100.00

R reactive, NR non-reactive

Table 3 Relationship betweenthe number of Bos taurus breedswith reproduction disturbancesand their serological statusassayed by ELISA tests (firstand second samplings) forNeospora caninum

R reactive, NR non-reactive

Serological status Number of animals Percentage (%)

NR at the first sampling and R at the second sampling 14 14

NR at the first sampling and NR at the second sampling 26 26

R at the first sampling and R at the second sampling 9 9

NR at the second sampling 24 24

R at the second sampling 27 27

Total 100 100

Table 4 Cross tabulation of serological status for Neospora caninumfor cows and female Bos taurus offspring based on ELISA testsperformed at a farm at the municipality of Avaré, SP

Seropositive cow Seronegative cow

Seropositive female calf 9 15

Seronegative female calf 9 27

Total 18 42

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caninum infection, while no anti-N. caninum antibodieswere detected in the other 50 animals (50%). In this group,it is not likely that reproduction disturbances are associatedwith the protozoan because this agent displays lifetimepersistence (Hietala and Thurmond 1999), and seroconver-sion rates were low (Davison et al. 1999).

The concept of vertical transmission is less recognizedthan horizontal transmission. With vertical transmission,dogs, the definitive host, represent a high risk factor becausethey are infected by fetal fluids and the placenta of sickanimals (Dijkstra et al. 2002). Hietala and Thurmond (1999)have found that in many herds with high N. caninum prev-alence and dogs living among the herd, post-natal infectionrates were lower than 1% per year. Björkman et al. (1996),Paré et al. (1997) and Schares et al. (1998) have alsodescribed vertical transmission as the predominant modeof infection.

Tests have shown that 81–85% of cows infected by N.caninum can transmit this disease to their offspring (Paré etal. 1997; Davison et al. 1999), while Thumond et al. (1997)have observed that during an abortion epidemy, most cowsthat aborted were infected during the post-natal period.Dijkstra et al. (2001) have observed a high incidence ofinfection by horizontal transmission in herds with highseroprevalence; similar results have been described byKashiwazaki et al. (2001).

When comparing the serological status of cows and theirrespective female offspring, our findings strongly suggestthat post-natal infection was the predominate mode of trans-mission amongst the studied herds. It is not possible toensure that the nine seropositive female offspring born ofseropositive cows were infected by vertical transmission;because they were tested as adults, horizontal transmissionwas also possible.

As the analyzed properties are located in the urban pe-rimeter, stray dogs that circle throughout the property areconsidered the main source of infection because theyrelease oocysts into the environment. An epidemiologi-cal survey on serum from rural dogs of the same region,which was conducted at the same time as this study,found that 58.9% of the dogs from farms in the munic-ipality were infected by N. caninum (Hasegawa et al. 1999).Of the 63 reactive animals from the second sampling, at least35 animals (19.88%) may have been infected by horizontaltransmission.

N. caninum infection negatively affects reproduction(Yaeger et al. 1994; Lopes 1999; Andreotti 2001). Duringthe period of this study, some animals had up to fourabortions. Twelve out of the 14 animals that switched fromnon-reactive in the first sampling to reactive in the secondsampling displayed reproductive disturbances (embryonicdeath and recurrence of estrus) at some point between thesamplings.

Conclusion

We observed a significant increase in the percentage ofanimals infected by N. caninum in herds sampled at twodistinct time points.

By testing the same animal at two time points, it wasobserved that a significantly higher number of animalsswitched from non-reactive to reactive status when com-pared with those that switched from reactive to non-reactive status. Moreover, of the 176 animals, includingcows and heifers, 67 animals (38.05%) became infected atsome point between the samplings.

From a sample of 100 animals, 50% of the cows withreproductive disturbances had anti-Neospora antibodies.

This provides clear evidence that horizontal transmissionwas the predominant mode of infection in the studied herds.

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