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Veterinary Parasitology 148 (2007) 83–92

Quantification of vertical and horizontal transmission of

Neospora caninum infection in Dutch dairy herds

Chris J.M. Bartels a,*, Irene Huinink a, Marten L. Beiboer b,Gerdien van Schaik a, Willem Wouda a, Thomas Dijkstra a,

Arjan Stegeman c

a Animal Health Service Ltd., P.O. Box 9, 7400 AA, Deventer, The Netherlandsb Veterinary Software Design, Koekoeksbloem 1, 98801 LW Zuidhorn, The Netherlands

c Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University,

Yalelaan 7, 3584 CL Utrecht, The Netherlands

Received 29 January 2007; received in revised form 20 May 2007; accepted 4 June 2007

Abstract

Ninety-six of 108 randomly selected Dutch dairy herds had one or more cows with a positive serostatus for N. caninum. In these

96 herds, we have quantified the probabilities of vertical transmission (VT) and horizontal transmission (HT) of N. caninum

infection by combining serostatus and pedigree data in 4091 dam-daughter pairs. The probability of animals infected by vertical

transmission during pregnancy (Prob(VT)) was calculated as the proportion of seropositive daughters among daughters of

seropositive dams. The probability of animals infected by horizontal transmission (Prob(HT)) was the proportion of seropositive

daughters among daughters of seronegative dams. These probabilities were calculated after the frequencies of observed dam-

daughter combinations were corrected for (1) imperfect test-characteristics, (2) underestimation of horizontal transmission in

situations that seronegative dams were horizontally infected after the birth of their daughters and (3) overestimation of vertical

transmission in situations that seronegative daughters born from seropositive dams were horizontally infected. The incidence rate

for horizontal transmission was calculated based on Prob(HT) and the average age of the animals in these herds.

Based on the analysis of dam-daughter serology, Prob(VT) was 61.8% (95% CI: 57.5–66.0%) and Prob(HT) was 3.3% (95% CI:

2.7–3.9%). After adjusting the observed frequencies for imperfect test-characteristics, underestimation of horizontal transmission

and overestimation of vertical transmission, Prob(VT) decreased to 44.9% (95% CI: 40.0–49.9%) while Prob(HT) increased to

4.5% (95% CI: 3.9–5.2%). Prob(HT) corresponded with an incidence rate for horizontal transmission of 1.4 (95% CI: 1.2–1.7)

infections per 100 cow-years at risk.

When stratifying herds for the presence of farm dogs, Prob(HT) was higher (5.5% (95% CI: 4.6–6.4%)) in herds with farm dogs

than in herds without farm dogs (2.3% (95% CI: 1.5–3.4%)). When stratifying for within-herd seroprevalence, Prob(HT) was higher

(10.3% (95% CI: 8.6–12.2%)) in herds with high (�10%) within-herd seroprevalence compared with herds with low (<10%)

within-herd seroprevalence (2.0% (95% CI: 1.5–2.6%)). Although there was this relation between Prob(HT) and within-herd

seroprevalence (crude ORPREV = 5.7 (95% CI: 4.0–7.9)), in herds without farm dogs, this relationship was no longer statistical

significant (ORPREVjDOG- = 1.9 (95% CI: 0.7–5.5)). It indicated that the association between seroprevalence and Prob(HT)

depended largely on the presence of farm dogs.

In addition, when looking for the presence of specific age-groups with significantly higher seroprevalence compared with the rest

of the herd, there were 7 herds in which two or more horizontally-infected animals were present in specific age-groups. This was an

indication of a recent point-source exposure to N. caninum.

* Corresponding author. Tel.: +31 570660380.

E-mail address: c.bartels@gddeventer.com (C.J.M. Bartels).

0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetpar.2007.06.004

C.J.M. Bartels et al. / Veterinary Parasitology 148 (2007) 83–9284

These results reiterate the current control strategies to apply strict dog-management measures as well as to minimize within-herd

seroprevalence by monitoring serostatus of animals.

# 2007 Elsevier B.V. All rights reserved.

Keywords: Cattle; Epidemiology; Neospora caninum; Horizontal and vertical transmission of infection

1. Introduction

One of the leading causes of bovine abortion is the

protozoan parasite Neospora caninum. N. caninum has a

heteroxenous life cycle in which cattle are important

intermediate hosts and dogs and coyotes are the only

recognized definitive hosts hosts (Gondim et al., 2004;

McAllister et al., 1998). The parasite has a worldwide

prevalence and may cause abortion both after a primary

infection and as a result of recrudescence of a persistent

infection (Dubey et al., 2006). Apart from abortions,

economic losses due to infection with N. caninum are

primarily caused by premature culling (Bartels et al.,

2006b; Tiwari et al., 2005; Thurmond and Hietala,

1996) and possibly decreased milk production (Bartels

et al., 2006b; Romero et al., 2005; Hobson et al., 2002;

Hernandez et al., 2001; Thurmond and Hietala, 1997).

Current strategies to control neosporosis focus on a

reduction of the seroprevalence in cattle and on

separating dogs and dog-faeces from cattle to avoid

new infections in cattle (Dijkstra et al., 2005; Frossling

et al., 2005).

Both vertical and horizontal transmission routes

play a role in the infection of cattle. Vertical

transmission is responsible for the spread of infection

from a persistently-infected dam to her offspring

during pregnancy and contributes significantly to the

persistence of N. caninum infection in a herd by

propagating the infection to successive generations

(Bjorkman et al., 1996; Anderson et al., 1997; Schares

et al., 1998; Wouda et al., 1998). Reported vertical

transmission probabilities range from 41% (Pan et al.,

2004) to 95% (Davison et al., 1999b). Despite the

efficiency of vertical transmission, it is evident that

infection with N. caninum cannot be sustained in cattle

herds without horizontal transmission and this was

modelled by French et al. (1999). Horizontal transmis-

sion occurs when cattle ingest sporulated N. caninum

oocysts. There is convincing evidence that horizontal

transmission can be associated with N. caninum

abortion outbreaks, suggesting a point source exposure

(McAllister et al., 1996; Thurmond et al., 1997;

Mainar-Jaime et al., 1999; Waldner et al., 1999; Dyer

et al., 2000; Dijkstra et al., 2001). A few studies found

evidence for ongoing horizontal transmission of N.

caninum in cattle herds following a point-source

infection (Bjorkman et al., 2003; Dijkstra et al.,

2002b). In several other studies, there was a low

incidence of seroconversion in endemically-infected

herds suggesting a low level of horizontal transmission

(Davison et al., 1999b; Frossling et al., 2005; Hietala

and Thurmond, 1999; Schares et al., 1998; Wouda and

Brinkhof, 1998). These above-mentioned studies were

all based on herds with a history of clinical neosporosis.

To our knowledge, no studies have measured vertical

and horizontal transmission probabilities based on a

random sample of herds.

Within-herd seroprevalence and presence of farm

dogs are putative risk factors for N. caninum-associated

abortions (Bartels et al., 1999; Pare et al., 1998; Schares

et al., 2004; Wouda et al., 1999) and thus N. caninum

infection. As definitive hosts for N. caninum, dogs are

known to spread oocysts leading to horizontal

transmission in cattle. The relation between within-

herd seroprevalence and horizontal transmission is less

clear. Biologically, it can be hypothesized that increased

within-herd seroprevalence might lead to increased

horizontal transmission if cow to cow transmission

would be possible. For this reason, we were interested to

know if seroprevalence in itself is related to the

probability of horizontal transmission.

The objective of this study was to quantify the

probabilities of vertical and horizontal transmission of

N. caninum infection in Dutch dairy herds in general.

Additionally, we compared the probability of horizontal

transmission between herds with and without farm

dogs, and between herds with high versus low within-

herd seroprevalence. This was done by combining

serological data with pedigree data of seropositive

animals from an earlier seroprevalence study.

2. Materials and methods

2.1. Selection of herds

As part of a prevalence study (Bartels et al., 2006a),

108 dairy herds were randomly selected from the Dutch

dairy-herd population and blood was collected from all

female cattle above 3 months of age (11,672 animals).

In 96 herds (10,350 animals), 1 or more animals tested

C.J.M. Bartels et al. / Veterinary Parasitology 148 (2007) 83–92 85

Table 1

Contingency table used for calculating the probability of horizontal

and vertical transmission of N. caninum infection

Status of daughters Status of dam

Seropositive Seronegative

Seropositive a b

Seronegative c d

a + c b + d

seropositive and these herds were included in the

present study.

2.2. Serological testing of animals

Serum samples were tested using the Animal Health

Service (AHS) in-house serum ELISA (Wouda and

Brinkhof, 1998). An S/P-ratio of >0.5 was defined as

positive. The diagnostic sensitivity was 96.9% (95% CI:

94.5–99.4%) and the diagnostic specificity was 97.3%

(95% CI: 95.5–99.0%) (Von Blumroder et al., 2004).

2.3. Calculation of vertical and horizontal

transmission probabilities

Pedigree information of cattle was obtained from the

Dutch Identification and Registration (I&R, Royal

Dutch Dairy Syndicate, Arnhem, The Netherlands). A

software program Neospora# (Beiboer, Veterinary

Software design, Zuidhorn, The Netherlands, 2002)

was used to facilitate tracing dam-daughter relations

and linking serological-test results.

Data on dam-daughter relations were compiled in

2 � 2 tables (Table 1). The fraction of animals infected

by vertical transmission during pregnancy was calcu-

lated as the proportion of seropositive daughters among

the daughters of seropositive dams (a/a +c). The

Table 2

Algorithms used to convert observed frequencies of dam-daughter combinat

was done using the positive (PV+) and negative predictive (PV�) terms

Observed Corrected

Dam + daughter+ Dam + daugh

Dam + daughter+ (cell aa) a � PV+ � PV+ a � PV+ � (1

Dam + daughter� (cell b) b � PV+ � (1 � PV�) b � PV+ � P

Dam � daughter+ (cell c) c � (1 � PV�) � PV+ c � (1 � PV�(1 � PV+)

Dam � daughter� (cell d) d � (1 � PV�) �(1 � PV�)

d � (1 � PV+

a References to cell a–d relate to Table 1.

fraction of animals infected by horizontal transmission

was the proportion of seropositive daughters among the

daughters of seronegative dams (b/b + d).

As this was a cross-sectional study, the observed

frequencies of dam-daughters combinations were

adjusted for imperfect test characteristics, for possible

underestimation of horizontal transmission (i.e. some

positive-tested dams may have been infected after they

gave birth to a daughter) and overestimation of vertical

transmission (i.e. some positive-tested daughters from

positive dams may have been infected after being born

as negative daughter).

2.3.1. Adjustment for imperfect test characteristics

It is likely that due to imperfect test-characteristics a

proportion of animals were wrongly classified as

seropositive or seronegative, and as a consequence the

routes of transmission were assigned incorrectly. The

extent to which this occurred was calculated based on the

prevalence of infection in the study population and the

point-estimates of sensitivity and specificity of the AHS-

inhouse test. The predictivevalue for a positive test (PV+)

result was 78.4% (95% CI: 75.9–80.7) and for a negative

test result (PV�) 99.7% (95% CI: 99.5–99.8).

Assignment of vertical-infection status (Dam +

daughter+, cell a in Table 1) is the result of 4 different

probabilities. The fraction of animals correctly assigned a

vertical-transmission status was calculated as the

product of the probability of a truly-seropositive

daughter (PV+ = 0.784) times a truly-seropositive dam

(PV + = 0.784) which amounts to 0.615. The fraction of

animals incorrectly assigned to the combination

Dam + daughter + (1 � 0.615a) was divided over three

other possible dam-daughter combinations (0.169 for

Dam + daughter�, 0.169 for Dam � daughter+ and

0.047 for Dam � daughter�) according to the algorithms

given in Table 2.

ions into frequencies corrected for imperfect test characteristics. This

ter� Dam � daughter+ Dam � daughter�

� PV+) a � (1 � PV+) � PV+ a � (1 � PV+) �(1 � PV+)

V� b � (1 � PV�) �(1 � PV+)

b � (1 � PV�) � PV�

) � c � PV� � PV+ c � PV� � (1 � PV+)

) � PV� d � PV� � (1 � PV�) d � PV� � PV�

C.J.M. Bartels et al. / Veterinary Parasitology 148 (2007) 83–9286

Table 3

Conversion from observed to corrected frequencies of 4091 dam-daughter combinations. This was based on the algorithms in Table 2 and the point-

estimates for predictive values for positive (PV+ = 0.752) and negative (PV� = 0.997) test results

Observed Observed frequencies Corrected

Dam + daughter+ Dam + daughter� Dam � daughter+ Dam � daughter�

Dam + daughter+ (cell a) 325 200 55 55 15.2

Dam + daughter� (cell b) 201 0.5 157 0.1 43.3

Dam � daughter+ (cell c) 117 0.3 0.08 91.5 25.2

Dam � daughter� (cell d) 3448 0.03 10.3 10.3 3427

Corrected frequencies 201 223 157 3510

The corrected numbers of dam-daughter pairs were

summed (Table 3) and subsequently used for the

additional adjustments (Table 4, line b).

2.3.2. Adjustment for horizontal infection in dams

Horizontal transmission tended to be underestimated

when using cross-sectional data. This happened when a

seronegative dam had been infected after its daughter

was born. Romero and Frankena (2003) provided a

method to adjust for this. They assumed that the

probability of horizontal transmission in dams occurred

at the same probability as in daughters. Of seronegative

dams, the probability of daughters that was infected

horizontally was calculated as (b/b + d) (referring to

Table 1) and then the frequencies of a, c and (a + c)

(Table 1) were reduced according to this proportion

while the subtracted numbers were added to b, d and

(b + d), respectively (Table 4, line c).

Table 4

Frequencies of dam-daughter pairs and probabilities of vertical (Prob(VT) an

Dutch dairy herds with 1 or more seropositive animal to N. caninum. Startin

adjustments are illustrated: (1) adjusting for imperfect test characteristics (b)

(c) and (3) adjustment for postnatal infection in seronegative daughters from s

is given as the proportion of seropositive daughters among daughters from ser

as the proportion of seropositive daughters among daughters from seroneg

Status of

daughters

Sta

Se

a Observed dam-daughter combinations Seropositive 32

Seronegative 20

b Dam-daughter combinations adjusted for

imperfect test characteristics

Seropositive 20

Seronegative 22

c Dam-daughter combinations adjusted for

postnatal infection of dams after

birth of daughter

Seropositive 19

Seronegative 21

d Dam-daughter combinations adjusted for

postnatal infection of daughters born

from seropositive dams after birth

Seropositive 18

Seronegative 22

2.3.3. Adjustment for horizontal infection in

daughters from a seropositive dam

Overestimation of vertical transmission may have

occurred when a seronegative daughter born from a

seropositive dam had been infected horizontally.

Adjustment for this kind of overestimation was done

using the following algorithm:

a0 ¼ ðaþ cÞ ProbðVTÞ þ ðaþ cÞ

� ð1� ProbðVTÞÞ ProbðHTÞ (1)

where a0 is equal to a after the abovementioned adjust-

ment for horizontal infection in dams. It is the sum of the

number of vertically-infected daughters born from ser-

opositive dams ((a + c)Prob(VT)) plus the number of

seropositive dams with daughters (originally not infected

by vertical transmission) that were infected by horizontal

transmission ((a + c)(1 � Prob(VT)(Prob(HT)). Conse-

d horizontal (Prob(HT) transmission of 4091 dam-daughter pairs in 96

g with the observed dam-daughter frequencies (a), the effects of three

; (2) adjustment for postnatal infection in dams after birth of daughters

eropositive dams (d). The vertical transmission probability (Prob(VT))

opositive dams and the horizontal transmission probability (Prob(HT))

ative dams

tus of dams Prob(VT)%

(95% CI)

Prob(HT)%

(95% CI)ropositive Seronegative

5 117 61.8 (57.5–66.0) 3.3 (2.7–3.9)

1 3448

1 157 47.4 (42.7–52.4) 4.3 (3.6–5.0)

3 3511

2 166 47.4 (42.6–52.3) 4.5 (3.9–5.2)

3 3521

2 166 44.9 (40.0–49.9) 4.5 (3.9–5.2)

3 3521

C.J.M. Bartels et al. / Veterinary Parasitology 148 (2007) 83–92 87

quently, we assumed that after failure of vertical trans-

mission the probability of horizontal transmission in

daughters of seropositive dams was the same as that

probability in daughters of seronegative dams.

In this algorithm (a + c) and Prob(HT) are known

(see adjustment of postnatal infections in dams

after giving birth). Moreover, since Prob(VT) equals

a/(a + c), a can be solved by rewriting Eq. (1) as:

a ¼ a0 � ðaþ cÞ ProbðHTÞ1� ProbðHTÞ (2)

The result of this adjustment on the frequencies of

dam-daughter combinations is given in Table 4, line d.

2.4. Rate of horizontal infection

The incidence rate of horizontal infection (IR(HT))

was calculated based on the adjusted Prob(HT) and the

average age of animals. Based on the formula (Dohoo

et al., 2003):

ProbðHTÞ ¼ 1� exp�ðIRðHTÞTÞ (3)

where T is the average age of an animal, IR(HT) can be

calculated by converting algorithm (3) into:

IRðHTÞ ¼ �ln

�1� ProbðHTÞ

T

�(4)

assuming that the rate of infection is constant during an

animal’s lifetime.

2.5. Stratification

The study population was divided into herds with

(N = 66) and without (N = 30) farm dogs, and herds

with high prevalence (�10% within-herd prevalence,

N = 35) and with low prevalence (<10% within-herd

prevalence, N = 61). Crude odds ratios for farm-dog

presence (ORDOG) and seroprevalence (ORPREV) were

calculated to quantify the effect of these explanatory

variables. In addition, to assess the effect of farm-dog

presence on the relation between seroprevalence and

horizontal transmission, stratum-specific odds ratios

(ORPREVjDOG) and the Mantel-Haensel summary odds

ratio (ORMH_PREV) were calculated.

2.6. High seroprevalence age groups

For each herd, the number of horizontally-infected

animals was counted. Clusters of horizontally-infected

animals were determined according to the method of

Dijkstra et al. (2001). These researchers demonstrated

that the seroprevalence in specific age-groups was

significantly higher compared with the other animals in

the herd, indicating a point-source exposure to N.

caninum. They also demonstrated that such exposures

were found during a limited period of common housing

and feeding of animals (Dijkstra et al., 2002b). We

made use of this method by looking at a clustered

presence of seropositive animals within time periods of

�9 months. The time-window of �9-months was used

because (pregnant) young stock is traditionally grouped

together in specific age-groups in which the age

difference varies between 6 and 9 months.

2.7. Statistical analyses

Calculations for Prob(VT) and Prob(HT) were done

in Excel (Microsoft1 Excel, 2002). Ninety-five percent

confidence intervals for Prob(VT) and Prob(HT) were

calculated using the exact binomial distribution

(STATA/SE 8.2, 2004). Calculation of OR, stratified

OR and MH summary OR was done as described by

Dohoo et al. (2003) using Win Episcope 2.0 (Thrusfield

et al., 2001). We considered P � 0.05 to indicate

statistical significance.

3. Results

The average age of animals was 3.2 (S.D. 2.3) years.

Overall seroprevalence in the 96 seropositive study

herds was 11.3% (1173/10,350), and within-herd

seroprevalence ranged from 0.5 to 49.1%.

Of 4091 dam-daughter pairs, the serostatus of both

dam and daughter was known (Table 4) (29% of all

possible dam-daughters pairs in the sampled popula-

tion). Based on the unadjusted analysis of dam-daughter

serology, Prob(VT) was 61.8% (95% CI: 57.5–66.0%)

and Prob(HT) was 3.3% (95% CI: 2.7–3.9%). When

adjusting the observed frequencies for the predictive

values of positive and negative test results, under-

estimation of horizontal and overestimation of vertical

transmission, Prob(VT) decreased significantly to

44.9% (95% CI: 40.0–49.9%) while the Prob(HT)

increased to 4.5% (95% CI: 3.9–5.2%) (Table 4). Based

on this result and the average age of 3.2 years, the

incidence rate for horizontal transmission was calcu-

lated as 1.4 (95% CI: 1.2–1.7) infections per 100 cow-

years at risk.

In herds with farm dogs, Prob(HT) was higher (5.5%

(95% CI: 4.6–6.4%)) compared with herds without farm

dogs (2.3% (95% CI: 1.5–3.4%)) and the crude ORDOG

was 2.5 (95% CI: 1.7–3.9). Consequently, IR(HT) was

higher in herd with farm dogs (1.8 (95% CI: 1.5–2.2)

C.J.M. Bartels et al. / Veterinary Parasitology 148 (2007) 83–9288

Table 5

Frequencies of dam-daughter pairs, probabilities (Prob(HT) and incidence rates for horizontal transmission of 4091 dam-daughter pairs in 96 Dutch

dairy herds with 1 or more animals seropositive for N. caninum. Frequencies of dam-daughter pairs are given after adjustment for imperfect test

characteristics and horizontal infection of dams and daughters

Status of

daughters

Status of dams Prob(HT)%

(95% CI)

Incidence rate per 100

cow-years at risk (95% CI)Seropositive Seronegative

Herds with dog(s) present

(N = 2924 pairs in 66 herds)

Seropositive 160 141 5.7 (4.8–6.7) 1.8 (1.5–2.2)

Seronegative 185 2439

Herds with no dog(s) present

(N = 1167 pairs in 30 herds)

Seropositive 20 26 2.3 (1.5–3.4) 0.7 (0.5–1.1)

Seronegative 38 1083

High within-herd seroprevalence

(N = 1486 pairs in 35 herds)

Seropositive 136 124 10.3 (8.6–12.2) 3.4 (2.8–4.1)

Seronegative 146 1080

Low within-herd seroprevalence

(N = 2605 pairs in 61 herds)

Seropositive 32 49 2.0 (1.5–2.6) 0.6 (0.5–0.8)

Seronegative 78 2445

infections per 100 cow-years at risk) compared with

herds without farm dogs (0.7 (95% CI: 0.5–0.11)

infections per 100 cow-years at risk).

A similar effect was seen for seroprevalence. In herds

with high within-herd seroprevalence, Prob(HT) was

higher (10.3% (95% CI: 8.6–12.2%) compared with

herds with low seroprevalence (2.0% (95% CI: 1.5–

2.6%)). This resulted in a higher IR(HT) in high

prevalence herds (3.4 (95% CI: 2.8–4.1) infections per

100 cow-years at risk) compared with low prevalence

herds (0.6 (95% CI: 0.5–0.8) infections per 100 cow-

years at risk) (Table 5). The crude ORPREV was 5.7 (95%

CI: 4.0–7.9). However, the stratified ORPREVjDOG+ was

6.6 (95% CI: 4.2–9.8) while ORPREVjDOG- was 1.9 (95%

CI: 0.7–5.5). The Mantel-Haensel summary ORMH_PREV

Table 6

Descriptive information on seven herds with specific seropositive age-group

sample of 108 Dutch dairy herds (sampled in 2003)

Farm #Animals

sampled

#Seropos.

animals/#animals

excl. cluster

#Seropos.

animals/#animals

in cluster

1 89 3/83 2/6

2 67 22/60 7/7

3 55 19/46 8/9

4 170 10/126 3/10

4/12

5/14

3/8

5 146 20/130 14/16

6 127 21/107 10/20

7 128 27/110 16/18

was 5.6 (95% CI: 3.9–8.2) with a significant Chi-square

value for the Breslow-Day statistic (P-value = 0.03). This

indicated that the strength of the relation between

seroprevalence and horizontal transmission depends

largely on the presence of farm dogs(s).

Fifty-seven (59%) out of 96 seropositive herds had at

least 1 horizontally-infected animal and 27 herds had

�2 horizontally-infected animals. In seven herds,

specific age-groups with significantly higher seropre-

valence compared with the rest of the herd were present

(Table 6). In Fig. 1, the situation in herd 7 with 128

animals and 33.3% seroprevalence of N. caninum

infection is illustrated. A cluster of horizontally infected

animals was born between April and December 2000.

Sixteen out of the 18 animals born during this period

s indicative for point-source infection with N. caninum from a random

P-value #Horizontally

infected animals

in cluster

Period of birth-dates for cluster

0.03 2 November 2000

<0.01 2 November 1999–February 2000

<0.01 3 August 2000–January 2001

0.02 2 November–December 1999

<0.01 2 July 2000–January 2001

<0.01 2 July–November 2001

0.03 2 April–June 2002

<0.01 6 April 1998–January 1999

<0.01 7 May–December 2000

<0.01 7 April–December 2000

C.J.M. Bartels et al. / Veterinary Parasitology 148 (2007) 83–92 89

Fig. 1. Overview of serological testing for N. caninum in herd 7 (N = 128 animals) and assigned infection route of seropositive animals.

tested seropositive. Six of these had an unknown

infection route, three were assigned a vertical and seven

a horizontal infection route. Two dogs had been

purchased on this farm, the first at the end of 1995

and the second at the beginning of 2001.

4. Discussion

In the present study, we quantified the probability of

vertical and horizontal transmission of N. caninum

infection in Dutch dairy herds. We applied adjustments

to the observed frequencies of dam-daughter combina-

tions to account for imperfect test characteristics,

underestimation of horizontal and overestimation of

vertical transmission. Additionally, we defined a time at

risk, allowing for the conversion of probabilities into

rates. For vertical transmission the time at risk was one

pregnancy, while for horizontal transmission, we

converted Prob(HT) into IR(HT) by using the average

age of animals as the time at risk. In epidemiological

terms, the incidence rate is an important parameter and

it allows direct comparison between studies. For this

reason, it is preferably used in simulation models to

evaluate different disease control strategies.

The unadjusted probability of vertical transmission

(Prob(VT)) in the observed dam-daughter relations was

61.8%. This percentage decreased significantly to

44.9% when dam-daughter frequencies were corrected.

This drop in Prob(VT) was mainly caused by the effect

of test specificity in a population with moderate

seroprevalence, leading to relatively low predictive

value for positive test results. The adjustment related to

overestimation of vertical transmission had much less

effect on the final outcome of Prob(VT). When applying

adjustments on Prob(HT), this probability increased

from 3.3 to 4.5%. Again, the adjustment for imperfect

test characteristics had a greater effect on Prob(HT)

than adjusting for underestimation of horizontal

transmission. This probability corresponded with an

incidence rate for horizontal transmission of 1.4 per 100

cow-years at risk in a random group of seropositive

Dutch dairy herds.

Previously, a limited number of prospective studies

on dairies with N. caninum-associated abortion pro-

blems have been conducted, providing incidence rates

of horizontal transmission. These varied between less

than 1% per year (Hietala and Thurmond, 1999) to an

overall estimate of 1.9 horizontal infections per 100

heifer-years at risk (Davison et al., 1999b) and 8.5

horizontal infections per 100 cow-years at risk (Pare

et al., 1997). Comparison with our incidence rate needs

caution because these three studies were based on

herd(s) situations with N. caninum-associated abortion

problems.

There have also been studies looking into vertical

and horizontal transmission probabilities based on

cross-sectional data (Dijkstra et al., 2001; Pan et al.,

2004; Romero and Frankena, 2003). However, none or

limited adjustments such as described in our study were

carried out. Therefore, the presented probabilities for

vertical transmission will most likely be overestimated

and for horizontal transmission will be underestimated.

C.J.M. Bartels et al. / Veterinary Parasitology 148 (2007) 83–9290

Looking at the data of one of these studies based on

Dutch dairy herds with N. caninum-associated abortion

problems (Dijkstra et al., 2001), the presented Prob(VT)

and Prob(HT) were 68.8% and 22.8%, respectively.

When carrying out the adjustments described in our

study, Prob(VT) and Prob(HT) were 42.0% and 30.0%,

respectively. When assuming the same average age of

animals as in our study, the incidence rate for horizontal

transmission in these abortion-problem herds was 11.1

infections per 100 cow-years at risk, or eight times

higher as in the random herds studied in our study. Thus,

comparison between studies shows that differences in

vertical and horizontal transmission probabilities are

related to what is known about the herd situation. In

addition, it is plausible that vertical and horizontal

transmission probabilities vary between countries

because of variation in the presence of definitive hosts,

conditions for oocyst sporulation and virulence in

parasite strains.

Both farm-dog presence and high within-herd

seroprevalence had a strong statistical relation with

Prob(HT): the ORDOG for presence of farm dogs and

ORPREV for high within-herd seroprevalence were 2.5

and 5.7, respectively. A similar effect of the relation

between within-herd seroprevalence and horizontal

transmission was found by Romero and Frankena

(2003). This relationship was explained by an increased

exposure to environmental sources of infection,

including infected placentas, amniotic fluid or water

and food contaminated with N. caninum oocysts from

dogs. We were able to underscore the importance of

farm dogs on the occurrence of horizontal transmission

by stratification for farm-dog presence. The association

between within-herd seroprevalence and Prob(HT)

increased when farms had dogs while, on farms without

dogs, this relation became weaker (ORPREVjDOG = 1.9)

and was no longer statistically significant. This

illustrates that the effect of within-herd seroprevalence

on Prob(HT) depended for a large part on the presence

of farm dogs. In biological terms, it reinforced the

important role farm dogs play in spreading the

infection. In an environment of high within-herd

seroprevalence, dogs have a greater chance to become

infected and the probability of horizontal transmission

increases accordingly. When no farm dogs were present,

the probability of horizontal transmission tended to

increase with an increasing seroprevalence. This

indicated that cattle may acquire new infections by

sources other than farm dogs. In the Dutch situation,

where dogs are the only known definitive hosts, stray

dogs most likely act as additional sources of oocysts.

This was earlier hypothesized by Schares et al. (2004) in

a study comparable to the Dutch situation. Another

more speculative hypothesis is the uptake of infectious

stages other than sporulated oocysts such as tachyzoites.

In a study by Uggla et al. (1998), neonatal calves were

infected orally by colostrum spiked with Nc-SweB1

tachyzoites. In an experiment in which heifers were

challenged orally with tachyzoites, one out of eight

animals seroconverted (Weston et al., 2005). Possible

horizontal transmission in cattle with oral lesions was

suggested when these cattle ingest food or water

contaminated with tachyzoites such as from placenta or

vaginal discharge from cattle calving or aborting due to

N. caninum.

In 7 out of 96 herds evidence was found for a point-

source infection based on the existence of a specific age-

group of animals in which the seroprevalence for N.

caninum was higher compared with the rest of the herd.

Simultaneously, it indicated that point-source infections

did not necessarily lead to increased clinical neosporo-

sis but could occur without abortion problems.

Previously Dijkstra et al. (2002a) have described a

similar finding in one herd in which more than half of

the animals seroconverted without any signs of

abortion.

Whatever the unknown mechanisms of horizontal

transmission, our findings emphasize the appropriate-

ness of current control strategies to reduce seropreva-

lence by testing animals and subsequently deciding to

cull seropositive animals and/or their offspring. In

addition, strict dog-management measures are neces-

sary. It is particularly important to prevent dogs (both

dogs living on the premises and stray dogs) from being

present at calving and to prevent contamination of feed

and drinking water with dog faeces (Dijkstra et al.,

2005).

When using unadjusted cross-sectional data, prob-

abilities of vertical transmission tend to be over-

estimated and probabilities of horizontal transmission

tend to be underestimated. A cohort study in which both

dam and daughter were bled at regular intervals would

be a preferred study design. In such a study design, more

accurate probabilities for vertical and horizontal

transmission could be calculated because it would

allow for correction biases such as variation in antibody

titers by age and throughout pregnancy (Davison et al.,

1999a; Maley et al., 2001; Stenlund et al., 1999). In

addition, a cohort study would allow for accurate

calculation of ‘animal time at risk’ as denominator for

the horizontal-transmission rate. However, as cohort

studies require long study periods and subsequently

more financial means, these are applied scarcely and

often involving a limited number of herds. By adjusting

C.J.M. Bartels et al. / Veterinary Parasitology 148 (2007) 83–92 91

cross-sectional data according the methods described

here, we believe that cross-sectional studies can provide

reliable estimates for vertical and horizontal transmis-

sion probabilities.

In conclusion, the adjusted probabilities of vertical

and horizontal transmission in Dutch cattle were 44.9%

and 4.5%, respectively. The incidence rate for

horizontal transmission was 1.4 infections per 100

cow-years at risk. The adjustment for imperfect test

characteristics had a major impact on the estimated

probabilities. The observed relation between within-

herd seroprevalence and Prob(HT) was largely depen-

dent on the presence of farm dogs. Additionally, in 7%

of dairy herds we found a specific age-group with high

seroprevalence indicating a point-source infection.

Such point-source infections apparently may occur

without obvious clinical signs. This means that the

within-herd seroprevalence can increase unnoticed to

levels that pose a risk for an abortion epidemic.

Therefore, current control strategies based on contain-

ing seroprevalence and managing dogs on farm remain

important.

Acknowledgements

Serological data of the participating dairy herds were

obtained from previous studies, which were funded by

the Dairy Commodity Board (Rijswijk, The Nether-

lands) and the European Union (Research Project

QLK2-CT2001-0150 ‘‘Diagnosis and epidemiology of

Neospora caninum associated bovine abortions’’). The

authors thank Dr. Maarten Eysker and Ir. Wim Swart for

their valuable contribution to this manuscript and

participating farmers for allowing the use of herd data,

the NRS (the Dutch Cattle Improvement Organisation,

Arnhem, The Netherlands) for data supply.

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