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Evidence of post-natal transmission of Neospora caninum in Dutch dairy herds
International Journal for Parasitology 31 (2001) 209±215
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Evidence of post-natal transmission of Neospora caninum in Dutch dairy herds Th. Dijkstra a,*, H.W. Barkema a, M. Eysker b, W. Wouda a a
b
Animal Health Service, P.O. Box 361, 9200 AJ Drachten, The Netherlands Division of Parasitology and Tropical Veterinary Medicine, Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.165, 3508 TD Utrecht, The Netherlands Received 25 September 2000; received in revised form 28 November 2000; accepted 28 November 2000
Abstract Eighteen dairy herds with neosporosis-associated abortions were analysed for antibodies against Neospora caninum. Blood samples of all cows, heifers and calves were collected on the same day for each farm. A total of 2430 heads of cattle were examined. For each herd, the seropositive and seronegative animals were plotted against month of birth. Analysis of seroprevalence in relation to age showed an equal distribution of seropositives in all age-groups in 10 herds. In contrast, in eight herds an age-group could be identi®ed which had a signi®cantly higher seroprevalence than the other animals in the herd. Most seropositive animals in the high seroprevalence age-groups had either seronegative dams or seronegative offspring, whereas there was a strong relationship between the serostatus of dams and offspring in the other animals in the herd. Aborting animals were mainly part of the high seroprevalence age-group. These ®ndings strongly indicate a post-natal infection of the animals in the high seroprevalence age-groups, probably due to a point source exposure to N. caninum. q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Abortion; Cattle; Epidemiology; Neospora caninum transmission
1. Introduction Neospora caninum is an apicomplexan protozoa, which causes neuromuscular disease in dogs and abortion in cattle. In many countries N. caninum is the most frequently diagnosed cause of bovine abortion (Dubey and Lindsay, 1996). In The Netherlands N. caninum is diagnosed in 17% of the aborted bovine foetuses (Wouda et al., 1997). Like all closely related protozoa (phylum Apixomplexa; family Sarcocystidae) N. caninum has a two-host life cycle (McAllister, 1999). Only recently it has been demonstrated that the dog can act as a de®nitive host, in which a sexual development may occur, which leads to faecal shedding of oocysts (McAllister et al., 1998). All other hosts, including cattle, are regarded as intermediate hosts, which only harbour asexual stages of the parasite (tachyzoites and encysted bradyzoites). In cattle, vertical or congenital transmission of N. caninum tachyzoites is generally considered to be the most
* Corresponding author. Tel.: 131-512-570700; fax: 131-512-520013. E-mail address: [email protected] (T. Dijkstra).
important mode of transmission (Anderson et al., 1997; BjoÈrkman et al., 1996; Davison et al., 1999b; Pare et al., 1996; Schares et al., 1998; Wouda et al., 1998b). Studies using pre-colostral blood samples showed that 81±95% of N. caninum-infected cows transmit the infection to their offspring (Davison et al., 1999b; Pare et al., 1996; Pare et al., 1997; Wouda et al., 1998b). However, as congenital transmission occurs in less than 100% of the cases, N. caninum infection should eventually disappear from cattle herds without some form of post-natal infection (French et al., 1999), unless seropositive animals are positively selected for breeding (BjoÈrkman et al., 1996). Few studies have provided suggestive evidence of post-natal transmission of N. caninum, mainly based on the lack of association between the serological status of dams and daughters (Dyer et al., 2000; Patitucci et al., 1999; Thurmond et al., 1997; Waldner et al., 1999), particularly in relation to abortion outbreaks (Patitucci et al., 1999; Thurmond et al., 1997; Waldner et al., 1999). A low grade of post-natal infection, less than 8.5%, has been reported in longitudinal studies (Davison et al., 1999b; Hietala and Thurmond, 1999; Pare et al., 1997). In a study involving 50 dairy herds with epidemic neosporosis, Wouda et al. (1999a) found equal
0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(00)00160-0
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seroprevalences across all age-groups in most herds. This suggested that the infection had been perpetuated by congenital transmission before the onset of abortion storms. In six of the 50 herds a higher seroprevalence was found in the dairy cows compared with the young stock. This was thought to be attributable to superimposed post-natal infection in these herds (Wouda et al., 1999a). However, in that study seroprevalence was based on a 20% herd sample, and dam-daughter pairs could not be compared. The objective of the present study was to obtain more detailed information on the extent of congenital and postnatal transmission of N. caninum infection in dairy herds by analysing whole herd serology and all dam-daughter relations. 2. Materials and methods 2.1. Herds and animals Eighteen dairy herds with a history of N. caninum associated abortions were selected from records of the Animal Health Service. A standard protocol was used for diagnostic evaluation of aborted foetuses, including bacterioscopic examination of abomasal content, culture from abomasum and lung on blood agar, and sampling from brain, heart and liver for histologic examination (Wouda et al., 1997). Diagnosis of N. caninum infection was based on histopathological examination of aborted foetuses (Wouda et al., 1997) or the presence of N. caninum antibodies in sera of aborting animals (Wouda et al., 1998a). These herds were subjected to a complete herd check for N. caninum antibodies between October 1998 and December 1999. A total of 2430 animals, including calves and heifers, were sampled. Mean number of animals per farm was 135, ranging from 36 to 289. All animals were of the Holstein Friesian breed. Purchase of animals, dam-daughter relations, and birth dates were available for all herds in the Dutch Identi®cation and Registration system (Nielen et al., 1996). On all 18 farms one or more dogs were present. On all farms each calf was fed only colostrum from its own dam. 2.2. De®nitions Abortion was de®ned as termination of pregnancy by an observed expulsion of the foetus between 100 and 260 days post-insemination. Animals at risk were de®ned as all heifers and cows between 100 and 260 days of pregnancy. If the annual abortion rate was less than 3% of the animals at risk the abortions were de®ned as sporadic. The term endemic abortions was used if the annual abortion rate was more than 3% of the animals at risk without an obvious peak of abortions (Davison et al., 1999a). The abortion pattern was de®ned as epidemic if at least 12.5% (one of eight) of the animals at risk aborted within a period of 2 months (Wouda et al., 1999a).
2.3. Blood sample collection All animals in a herd were bled on the same day. Blood samples were taken from the coccygeal or the jugular vein, using disposable needles and 8.5 ml SSTe Gel and Clot Activator Vacutainer w Plus serum-tubes (Becton±Dickinson Vacutainer Systems Europe). All samples were immediately transported to the laboratory of the Animal Health Service (Drachten, The Netherlands) and processed within 24 h. Serum was removed after centrifugation at 2000 £ g for 10 min. 2.4. Serology All blood samples were tested for antibodies to N. caninum using the ELISA of the Animal Health Service (Drachten, The Netherlands). This ELISA is based on a detergent lysate of whole sonicated tachyzoite antigens and detects all Ig classes. This test had a sensitivity of 98% (95% CI 93± 100%) using post-abortion sera and a speci®city of 92% (95% CI 85±98%) using non-suspect sera (Wouda et al., 1998a). 2.5. Analyses Analysis of the data was performed using Statistix for Windows 2.0. Statistical signi®cance was declared at P # 0:05. For each herd, all seropositive and all seronegative animals were plotted against the month of birth. High seroprevalence age-groups were detected by scrolling a 6month time window, starting at the month of birth of the oldest animal present, with a 1 month interval. If prevalence of N. caninum antibodies of the animals included in the 6 month interval was signi®cantly higher compared to the other animals of the herd, this interval was de®ned as being part of a high seroprevalence age-group. To correct for multiple testing the signi®cance value of 0.05 was divided by the number of tests per herd (Bland, 1995). The cut-off dates of a high seroprevalence age-group were determined by inclusion of the adjacent months that had a N. caninum seroprevalence higher than other months and exclusion of the months included in the 6 month time window that had a lower seroprevalence. A herd could have more than one high prevalence age-cluster. If in a herd a second age-group mainly consisted of daughters of the ®rst detected high seroprevalence age-group, the ®rst age-group was de®ned as the high seroprevalence agegroup. All dam-daughter relations were recorded. Differences in prevalence of N. caninum serostatus of dams and daughters were compared using x 2 analysis on contingency tables (Fleiss, 1981). Odds ratios were calculated from these contingency tables (Martin et al., 1987). In case of embryo transfer the recipients and not the donors were considered as dams, since transmission of N. caninum occurs after placentation (Thurmond and Hietala, 1995).
T. Dijkstra et al. / International Journal for Parasitology 31 (2001) 209±215
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Table 1 Seroprevalence of Neospora caninum antibodies in 18 dairy herds in relation to abortion history Herd a
Number of animals
Seroprevalence (%)
Number of abortions per year b
Abortion pattern
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Total
36 127 111 184 289 126 75 73 117 277 151 103 106 114 129 110 198 104 2430
13.9 50.4 14.4 66.8 21.1 56.3 78.7 27.4 35.0 13.0 34.4 36.9 27.4 47.4 58.9 52.7 47.0 58.7 39.4
2 6 6 8 5 11 15 12 7 15 13 (7) 8 (6) 5 (3) 11 (4) 6 (4) 9 (9) 25 (25) 11 (11) 174
Endemic Endemic Endemic Endemic Sporadic Endemic Epidemic Epidemic Endemic Endemic Endemic Endemic Endemic Epidemic Endemic Endemic Epidemic Epidemic
a b
Herds 1±10 had an equal age-distribution of seropositive animals. Herds 11±18 showed an age related clustering of seropositive animals. Abortions during 12 months prior to date of sampling, numbers in parentheses represent aborting animals in the high seroprevalence age-group.
3. Results 3.1. Seroprevalence Of the 2430 animals sampled, 39.4% were seropositive for N. caninum, ranging per herd from 13.0 to 78.7% (Table 1). In 10 herds (Herds 1±10) seropositive animals were equally distributed across all age-groups. In eight herds (Herds 11±18) a high seroprevalence age-group could be detected by the 6 months time window scrolling procedure (Table 2). In Herds 11±15 an age-group with a high seroprevalence was present amidst younger and older animals with a signi®cantly lower seroprevalence. Fig. 1 shows an example of such a herd (Herd 12). The extent of the high seroprevalence age-groups varied from 7 months (Herd 13) to 29 months (Herd 15). In Herds 16±18 a remarkable difference in seroprevalence could be distin-
guished between younger and older animals with varying cut-off dates per herd. Fig. 2 shows the age-distribution of seropositive and seronegative animals in one of these herds (Herd 17). 3.2. Dam-daughter serology In all herds there was a high degree (P , 0:0001) of association between N. caninum serological status of dams and daughters. In the herds with an equal age-distribution of seropositives, 207 (83.5%) of the 248 seronegative dams, had seronegative daughters, and 41 (16.5%) had seropositive daughters. Of the 248 seronegative daughters, 41 (16.5%) had seropositive dams, and 207 (83.5%) had seronegative dams (Table 3). In one herd (Herd 1) all ®ve seropositive animals belonged to one family. In the herds with a high seroprevalence age-group (Herds 11±18), more sero-
Table 2 Seroprevalence of Neospora caninum antibodies of the high seroprevalence age-group versus the other animals in the herd Herd
High seroprevalence age-group Age-group a(months)
11 12 13 14 15 16 17 18 Total
23±30 23±34 32±38 22±34 12±40 . 24 .9 . 27
a
Age at blood sampling.
No. of seropositives
Seroprevalence %
Other animals No. of seropositives
Seroprevalence %
22 19 13 15 56 51 84 43 303
69 91 87 88 93 63 59 80 71
30 19 16 39 20 7 9 18 158
27 23 18 40 29 25 16 36 27
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Fig. 1. Age-distribution of N. caninum seropositive and seronegative animals in herd 12 at the date of blood sampling (05-01-1999). There was a cluster of 19 seropositive animals born between March 1996 and February 1997 among generally seronegative animals born earlier or later. Six animals of this seropositive cohort aborted between April and December 1998 during their ®rst pregnancy. Five of the six seropositive animals born between August 1998 and January 1999 were daughters of this seropositive cohort.
negative dams had seropositive daughters (OR 2:2; P 0:0004), and more seronegative daughters had seropositive dams compared with Herds 1 to 10 (OR 3:1; P , 0:0001). Of the 195 seronegative dams in Herds 11± 18, 135 (69.2%) had seronegative daughters, and 60 (30.8%) had seropositive daughters. Of the 217 seronegative daughters, 82 (37.8%) had seropositive dams, and 135 (62.2%) had seronegative dams (Table 3).
Of the 58 seronegative dams with daughters in the high seroprevalence age-groups 48 (82.8%) had seropositive daughters, compared with 12 (8.8%) of the 137 seronegative dams with seropositive daughters outside the high seroprevalence age-group (Table 4) (OR 50:0; P , 0:0001). Of the 82 seropositive dams with seronegative daughters, 64 (78.0%) dams were part of the high seroprevalence agegroups (Table 4).
Fig. 2. Age-distribution of N. caninum seropositive and seronegative animals in herd 17 at the date of blood sampling (06-07-1999). There was a remarkable difference in seroprevalence between the animals born before October 1998 compared with the younger animals. A low seroprevalence group can be distinguished between October 1998 and May 1999. Twenty-four seronegative animals of the low seroprevalence group had a seropositive dam. In the high seroprevalence age-group also seronegative daughters with seropositive dams were present. Fifteen animals of the high seroprevalence group aborted in June 1999.
T. Dijkstra et al. / International Journal for Parasitology 31 (2001) 209±215 Table 3 Comparison of Neospora caninum serological status of dams and daughters in 10 dairy herds with an equal age-distribution of seropositive animals (Herds 1±10) and eight herds with a high seroprevalence age-group (Herds 11±18) Serostatus dam
Serostatus daughter
Herds 1±10
Herds 11±18
Positive Positive Negative Negative
Positive Negative Positive Negative
163 41 41 207
108 82 60 135
3.3. Abortion history All herds had a history of sporadic or endemic abortions for several years. However, for most herds (Herds 6-18) the farmers had noticed an increase of the number of abortions during the last 2 years. Five herds (Herds 7, 8, 14, 17 and 18) had experienced acute abortion epidemics (Table 1) involving 15.2±33.3% of the animals at risk. Within a period of 2 months eight, 11, 11, 15, and 10 animals aborted in Herds 7, 8, 14, 17, and 18, respectively. In Herd 8, ®ve of the 12 aborting animals had been introduced in the herd during the last year. No dam-daughter relations of the ®ve purchased aborting animals of this herd were available. In the eight herds with a high seroprevalence age-group, 78.4% (69 of the 88 animals) of the aborting animals were in the high seroprevalence age-groups (Table 1). In Herds 11±14, 20, 29, 20 and 25%, respectively of the animals in the high seroprevalence age-groups aborted during their ®rst pregnancy. In Herd 15, data on ®rst pregnancy abortions of the animals in the age-group 12 to 40 months were not available. In three herds (Herd 16, 17 and 18) with a remarkable difference in seroprevalence between younger and older animals, 11, 27 and 16% of the animals at risk aborted.
4. Discussion Congenital transmission of N. caninum was found in all herds of the present study based on the presence of seropositive dam-daughter pairs and seropositive family lines in each of these herds. In one herd (Herd 1), congenital infection apparently was the only transmission route, as all seropositive cattle belonged to one family. In nine herds (Herds 2±10), congenital transmission was the predominant transmission route. This was based on an equal distribution of
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seropositive animals across the different age-groups and a strong correlation between serological status of dams and daughters. However, in these nine herds, mismatches in dam-daughter serostatus were also present, suggesting the occurrence of post-natal infection as well. A low rate of seroconversion suggesting post-natal infection has been observed by others in endemically infected herds (Davison et al., 1999b; Hietala and Thurmond, 1999; Pare et al., 1997), but further evidence is required to con®rm that these animals were truly infected post-natally (Davison et al., 1999b). Part of the mismatches in dam-daughter serostatus in our study may have been due to false positive or false negative test results of either dams or daughters. Because the reported sensitivity of the ELISA used is based on post-abortion sera, which usually have high levels of antibodies (Wouda et al., 1998a), it can be expected that in a cross-sectional study the sensitivity of the test will be lower. Therefore, part of the seronegative dams with seropositive daughters may have been false negatives. A proportion of 16.5% seronegative daughters with seropositive dams as we found in these herds, is not unexpected, because a congenital transmission rate as low as 81% has been found previously based on pre-colostral calf sera (Pare et al., 1996). In eight herds (Herds 11±18) of our study, post-natal infection with N. caninum appeared to have contributed signi®cantly to the prevalence of infected animals, as speci®c age-groups could be identi®ed, which had a signi®cantly higher seroprevalence compared with that of the other animals in the herd. In addition, the number of dam-daughter pairs with different serostatus was disproportionally high. Clustered seropositive animals in these eight herds were either seropositive daughters with seronegative dams (as exempli®ed in Fig. 1) or seropositive dams with seronegative offspring (as exempli®ed in Fig. 2). The ®rst situation indicates postnatal infection of the daughters, and the second situation indicates postnatal infection of the dams after the birth of the seronegative daughters. The age related clustering of post-natally infected animals strongly supports a point source exposure of these animals. Evidence of a point source exposure has been previously based on the epidemic curve of the abortions during N. caninum-associated abortion storms (McAllister et al., 1996; McAllister et al., 2000; Schares et al., 1997; Yaeger et al., 1994). This would imply that epidemic abortions were the effect of a post-natal infection, probably due
Table 4 Comparison of Neospora caninum serological status of dams and daughters in eight dairy herds with a high seroprevalence age-group Serostatus dam
Serostatus daughter
Daughters in high seroprevalence age-group
Daughters outside high seroprevalence age-group
Positive Positive Negative Negative
Positive Negative Positive Negative
43 15 48 10
65 67 12 125
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to oocyst exposure. This is supported by our results. In all eight herds with evidence of post-natal infection there had been an increase in abortions during the last 2 years and in three of these herds (Herds 14, 17 and 18) an abortion epidemic had occurred. In addition, the abortions in these herds mainly occurred in the animals of the high seroprevalence age-groups. However, epidemic abortions were also seen in two herds without clear evidence of post-natal infection (Herds 7 and 8). The abortion storms in these herds may have been due to a synchronised recrudescence of N. caninum infection following an event causing immune suppression in a number of animals, as has been speculated previously (Atkinson et al., 2000; Wouda et al., 1999a). The present study shows the power of a single serological screening for neosporosis of all animals (including calves and heifers) in the herd. An analysis of the agedistribution of the seropositives combined with a comparison of dam-daughter serostatus is a practical approach to study the mode of transmission of N. caninum in a herd. Cut-off dates of age-groups in this study were dependent on the seroprevalence data per herd. Usually age-groups are de®ned ®rst and then the seroprevalence is compared among the groups (Dyer et al., 2000; Schares et al., 1998). However, in this way age related differences in seroprevalence within herds may be underestimated and congenital infection rates, if based on seroprevalences per year group, may be overestimated. If herds are analysed in one dataset without considering the herd-speci®c age-distribution of seroprevalence (Davison et al., 1999a; Mainar-Jaime et al., 1999), probably age-clustering will be underestimated or even not be found. The results of this study indicate that it is important to include young animals in the analysis. Generally, about 12.5% of a herd consists of animals younger than 6 months and the dams of these calves are usually still present in the herd. If this young age-group is included, the number of dam-daughter pairs that can be evaluated increases considerably. Congenitally infected calves usually have high antibody levels (Anderson et al., 1997; Davison et al., 1999b) and few false negative test results. Positive serology of young animals can be used for the interpretation of the serologic status of their dams, which may have ¯uctuating antibody titers (Dannatt, 1997; Schares et al., 1999; Stenlund et al., 1999). Calves can be seropositive due to colostral N. caninum antibodies. In all the 18 herds of the present study the calves were fed only colostrum of their own dams so there were no false-positive results due to the feeding of pooled colostrum. The fact that we found strong evidence of post-natal infection in eight of 18 dairy herds, indicates that this mode of infection is far from rare in cattle in The Netherlands. However, most herds of this study were problem herds and therefore the results may not be representative for the all over importance of post-natal N. caninum infections in The Netherlands.
Our results show that this type of study can help to de®ne the most probable period of a point source infection, that is the period during which the high seroprevalence age-group was housed or fed as a separate group. Such information is essential to get a better understanding of the epidemiology of neosporosis in cattle. The dog has been shown to be a risk factor for N. caninum-associated abortion storms (Bartels et al., 1999). A few dogs present at farms of the present study were incorporated in a previous seroepidemiological study, which indicated a relationship between N. caninum infections in dogs and cattle (Wouda et al., 1999b). Acknowledgements The authors thank Albert Winkel for help with data collection, and C.J.M. Bartels, J.W. Hesselink and Y.H. Schukken for valuable comments on the manuscript. This study was partially funded by the Dairy Commodity Board (Rijswijk, The Netherlands). References Anderson, M.L., Reynolds, J.P., Rowe, J.D., Sverlow, K.W., Packham, A.E., Barr, B.C., Conrad, P.A., 1997. Evidence of vertical transmission of Neospora sp. infection in dairy cattle. J. Am. Vet. Med. Assoc. 210, 1169±72. Atkinson, R.A., Cook, R.W., Reddacliff, L.A., Rothwell, J., Broady, K.W., Harper, P., Ellis, J.T., 2000. Seroprevalence of Neospora caninum infection following an abortion outbreak in a dairy cattle herd. Aust. Vet. J. 78, 262±6. Bartels, C.J.M., Wouda, W., Schukken, Y.H., 1999. Risk factors for Neospora caninum-associated abortion storms in dairy herds in The Netherlands (1995 to 1997). Theriogenology. 52, 247±57. BjoÈrkman, C., Johansson, O., Stenlund, S., Holmdahl, O.J.M., Uggla, A., 1996. Neospora species infection in a herd of dairy cattle. J. Am. Vet. Med. Assoc. 208, 1441±4. Bland, M., 1995. An Introduction to Medical Statistics, Oxford University Press Inc, New York. Dannatt, L., 1997. Neospora caninum antibody levels in an endemicallyinfected dairy herd. Cattle Pract. 5, 335±7. Davison, H.C., French, N.P., Trees, A.J., 1999a. Herd-speci®c and agespeci®c seroprevalence of Neospora caninum in 14 British dairy herds. Vet. Rec. 144, 547±50. Davison, H.C., Otter, A., Trees, A.J., 1999b. Estimation of vertical and horizontal transmission parameters of Neospora caninum infection in dairy cattle. Int. J. Parasitol. 29, 1683±9. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1±59. Dyer, R.M., Jenkins, M.C., Kwok, O.C., Douglas, L.W., Dubey, J.P., 2000. Serologic survey of Neospora caninum infection in a closed dairy cattle herd in Maryland: risk of serologic reactivity by production groups. Vet. Parasitol. 90, 171±81. Fleiss, J.L., 1981. Statistical Methods for Rates and Proportions, John Wiley & Sons, New York. French, N.P., Clancy, D., Davison, H.C., Trees, A.J., 1999. Mathematical models of Neospora caninum infection in dairy cattle: transmission and options for control. Int. J. Parasitol. 29, 1691±704. Hietala, S.K., Thurmond, M.C., 1999. Postnatal Neospora caninum transmission and transient serologic responses in two dairies. Int. J. Parasitol. 29, 1669±76. Mainar-Jaime, R.C., Thurmond, M.C., Berzal-Herranz, B., Hietala, S.K.,
T. Dijkstra et al. / International Journal for Parasitology 31 (2001) 209±215 1999. Seroprevalence of Neospora caninum and abortion in dairy cows in northern Spain. Vet. Rec. 145, 72±75. Martin, S.W., Meek, A.H., Willeberg, P., 1987. Epidemiologic Measures of Association. Veterinary Epidemiology Principles and Methods, Iowa State University Press, Iowa, pp. 128±34. McAllister, M.M., Huffman, E.M., Hietala, S.K., Conrad, P.A., Anderson, M.L., Salman, M.D., 1996. Evidence suggesting a point source exposure in an outbreak of bovine abortion due to neosporosis. J. Vet. Diagn. Invest. 8, 355±7. McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., McGuire, A.M., 1998. Dogs are de®nitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473±8. McAllister, M.M., 1999. Uncovering the biology and epidemiology of Neospora caninum. Parasitology Today. 15, 216±7. McAllister, M.M., BjoÈrkman, C., Anderson-Sprecher, R., Rogers, D.G., 2000. Evidence of point-source exposure to Neospora caninum and protective immunity in a herd of beef cows. J. Am. Vet. Med. Assoc. 208, 1441±4. Nielen, M., Jansen, F.C.M., van Wuijckhuise, L.A., Dijkhuizen, A.A., 1996. Dutch cattle identi®cation and registration (I&R) system: analysis of its use for controlling an outbreak of foot and mouth disease. Tijdschr. Diergeneeskd. 121, 576±81. PareÂ, J., Thurmond, M.C., Hietala, S.K., 1996. Congenital Neospora caninum infection in dairy cattle and associated calfhood mortality. Can. J. Vet. Res. 60, 133±9. PareÂ, J., Thurmond, M.C., Hietala, S.K., 1997. Neospora caninum antibodies in cows during pregnancy as a predictor of congenital infection and abortion. J. Parasitol. 83, 82±87. Patitucci, A.N., Charleston, W.A.G., Alley, M.R., O'Connor, R.J., Pomroy, W.E., 1999. Serological study of a dairy herd with a recent history of Neospora abortion. NZ. Vet. J. 47, 28±30. Schares, G., Peters, M., Wurm, R., Tackmann, K., Henning, K., Conraths, F.J., 1997. Neospora caninum causes abortions in cattle herds in North Rhine-Westphalia. Dtsch. TieraÈrztl. Wschr. 104, 208±12. Schares, G., Peters, M., Wurm, R., Barwald, A., Conraths, F.J., 1998. The ef®ciency of vertical transmission of Neospora caninum in dairy cattle analysed by serological techniques. Vet. Parasitol. 80, 87±98. Schares, G., Rauser, M., Peters, M., Zimmer, K., Peters, M., Wurm, R.,
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Dubey, J.P., de Graaf, D.C., Edelhofer, R., Mertens, C., Hess, G., Conraths, F.J., 1999. Serological differences in Neospora caninumassociated epidemic and endemic abortions. . J. Parasitol. 85, 688±94. Stenlund, S., Kindahl, H., Magnusson, U., Uggla, A., BjoÈrkman, C., 1999. Serum antibody pro®le and reproductive performance during two consecutive pregnancies of cows naturally infected with Neospora caninum. Vet. Parasitol. 85, 227±34. Thurmond, M., Hietala, S., 1995. Stragies to control Neospora infection in cattle. Bovine Pract. 29, 60±63. Thurmond, M., Hietala, S., Blanchard, P.C., 1997. Herd-based diagnosis of Neospora caninum-induced endemic and epidemic abortion in cows and evidence for congenital and postnatal transmission. J. Vet. Diagn. Invest. 9, 44±49. Waldner, C.L., Janzen, E.D., Henderson, J., Haines, D.M., 1999. Outbreak of abortion associated with Neospora caninum infection in a beef herd. J. Am. Vet. Med. Assoc. 215, 1485±90. Wouda, W., Moen, A.R., Visser, I.J.R., van Knapen, F., 1997. Bovine fetal neosporosis: a comparison of epizootic and sporadic abortion cases and different age-classes with regard to lesion severity and immunohistochemical identi®cation of organisms in brain heart and liver. J. Vet. Diagn. Invest. 9, 180±5. Wouda, W., Brinkhof, J., van Maanen, C., de Gee, A.L.W., Moen, A.R., 1998a. Serodiagnosis of neosporosis in individual cows and dairy herds, a comparison of three enzyme linked immunosorbent assays. Clin. Diagn. Lab. Immunol. 5, 711±6. Wouda, W., Moen, A.R., Schukken, Y.H., 1998b. Abortion risk in progeny of cows after a Neospora caninum epidemic. Theriogenology. 49, 1311±6. Wouda, W., Bartels, C.J.M., Moen, A.R., 1999a. Characteristics of Neospora caninum-associated abortion storms in dairy herds in The Netherlands (1995 to 1997). Theriogenology 52, 233±45. Wouda, W., Dijkstra, Th., Kramer, A.M.H., van Maanen, C., Brinkhof, J.M.A., 1999b. Seroepidemiological evidence for a relationship between Neospora caninum infections in dogs and cattle. Int. J. Parasitol. 29, 1677±82. Yaeger, M.J., Shawd-Wessels, S., Leslie-Steen, P., 1994. Neospora abortion in a midwestern dairy. J. Vet. Diagn. Invest. 6, 506±8.
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Evidence of post-natal transmission of Neospora caninum in Dutch dairy herds Th. Dijkstra a,*, H.W. Barkema a, M. Eysker b, W. Wouda a a
b
Animal Health Service, P.O. Box 361, 9200 AJ Drachten, The Netherlands Division of Parasitology and Tropical Veterinary Medicine, Institute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.165, 3508 TD Utrecht, The Netherlands Received 25 September 2000; received in revised form 28 November 2000; accepted 28 November 2000
Abstract Eighteen dairy herds with neosporosis-associated abortions were analysed for antibodies against Neospora caninum. Blood samples of all cows, heifers and calves were collected on the same day for each farm. A total of 2430 heads of cattle were examined. For each herd, the seropositive and seronegative animals were plotted against month of birth. Analysis of seroprevalence in relation to age showed an equal distribution of seropositives in all age-groups in 10 herds. In contrast, in eight herds an age-group could be identi®ed which had a signi®cantly higher seroprevalence than the other animals in the herd. Most seropositive animals in the high seroprevalence age-groups had either seronegative dams or seronegative offspring, whereas there was a strong relationship between the serostatus of dams and offspring in the other animals in the herd. Aborting animals were mainly part of the high seroprevalence age-group. These ®ndings strongly indicate a post-natal infection of the animals in the high seroprevalence age-groups, probably due to a point source exposure to N. caninum. q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Abortion; Cattle; Epidemiology; Neospora caninum transmission
1. Introduction Neospora caninum is an apicomplexan protozoa, which causes neuromuscular disease in dogs and abortion in cattle. In many countries N. caninum is the most frequently diagnosed cause of bovine abortion (Dubey and Lindsay, 1996). In The Netherlands N. caninum is diagnosed in 17% of the aborted bovine foetuses (Wouda et al., 1997). Like all closely related protozoa (phylum Apixomplexa; family Sarcocystidae) N. caninum has a two-host life cycle (McAllister, 1999). Only recently it has been demonstrated that the dog can act as a de®nitive host, in which a sexual development may occur, which leads to faecal shedding of oocysts (McAllister et al., 1998). All other hosts, including cattle, are regarded as intermediate hosts, which only harbour asexual stages of the parasite (tachyzoites and encysted bradyzoites). In cattle, vertical or congenital transmission of N. caninum tachyzoites is generally considered to be the most
* Corresponding author. Tel.: 131-512-570700; fax: 131-512-520013. E-mail address: [email protected] (T. Dijkstra).
important mode of transmission (Anderson et al., 1997; BjoÈrkman et al., 1996; Davison et al., 1999b; Pare et al., 1996; Schares et al., 1998; Wouda et al., 1998b). Studies using pre-colostral blood samples showed that 81±95% of N. caninum-infected cows transmit the infection to their offspring (Davison et al., 1999b; Pare et al., 1996; Pare et al., 1997; Wouda et al., 1998b). However, as congenital transmission occurs in less than 100% of the cases, N. caninum infection should eventually disappear from cattle herds without some form of post-natal infection (French et al., 1999), unless seropositive animals are positively selected for breeding (BjoÈrkman et al., 1996). Few studies have provided suggestive evidence of post-natal transmission of N. caninum, mainly based on the lack of association between the serological status of dams and daughters (Dyer et al., 2000; Patitucci et al., 1999; Thurmond et al., 1997; Waldner et al., 1999), particularly in relation to abortion outbreaks (Patitucci et al., 1999; Thurmond et al., 1997; Waldner et al., 1999). A low grade of post-natal infection, less than 8.5%, has been reported in longitudinal studies (Davison et al., 1999b; Hietala and Thurmond, 1999; Pare et al., 1997). In a study involving 50 dairy herds with epidemic neosporosis, Wouda et al. (1999a) found equal
0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(00)00160-0
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seroprevalences across all age-groups in most herds. This suggested that the infection had been perpetuated by congenital transmission before the onset of abortion storms. In six of the 50 herds a higher seroprevalence was found in the dairy cows compared with the young stock. This was thought to be attributable to superimposed post-natal infection in these herds (Wouda et al., 1999a). However, in that study seroprevalence was based on a 20% herd sample, and dam-daughter pairs could not be compared. The objective of the present study was to obtain more detailed information on the extent of congenital and postnatal transmission of N. caninum infection in dairy herds by analysing whole herd serology and all dam-daughter relations. 2. Materials and methods 2.1. Herds and animals Eighteen dairy herds with a history of N. caninum associated abortions were selected from records of the Animal Health Service. A standard protocol was used for diagnostic evaluation of aborted foetuses, including bacterioscopic examination of abomasal content, culture from abomasum and lung on blood agar, and sampling from brain, heart and liver for histologic examination (Wouda et al., 1997). Diagnosis of N. caninum infection was based on histopathological examination of aborted foetuses (Wouda et al., 1997) or the presence of N. caninum antibodies in sera of aborting animals (Wouda et al., 1998a). These herds were subjected to a complete herd check for N. caninum antibodies between October 1998 and December 1999. A total of 2430 animals, including calves and heifers, were sampled. Mean number of animals per farm was 135, ranging from 36 to 289. All animals were of the Holstein Friesian breed. Purchase of animals, dam-daughter relations, and birth dates were available for all herds in the Dutch Identi®cation and Registration system (Nielen et al., 1996). On all 18 farms one or more dogs were present. On all farms each calf was fed only colostrum from its own dam. 2.2. De®nitions Abortion was de®ned as termination of pregnancy by an observed expulsion of the foetus between 100 and 260 days post-insemination. Animals at risk were de®ned as all heifers and cows between 100 and 260 days of pregnancy. If the annual abortion rate was less than 3% of the animals at risk the abortions were de®ned as sporadic. The term endemic abortions was used if the annual abortion rate was more than 3% of the animals at risk without an obvious peak of abortions (Davison et al., 1999a). The abortion pattern was de®ned as epidemic if at least 12.5% (one of eight) of the animals at risk aborted within a period of 2 months (Wouda et al., 1999a).
2.3. Blood sample collection All animals in a herd were bled on the same day. Blood samples were taken from the coccygeal or the jugular vein, using disposable needles and 8.5 ml SSTe Gel and Clot Activator Vacutainer w Plus serum-tubes (Becton±Dickinson Vacutainer Systems Europe). All samples were immediately transported to the laboratory of the Animal Health Service (Drachten, The Netherlands) and processed within 24 h. Serum was removed after centrifugation at 2000 £ g for 10 min. 2.4. Serology All blood samples were tested for antibodies to N. caninum using the ELISA of the Animal Health Service (Drachten, The Netherlands). This ELISA is based on a detergent lysate of whole sonicated tachyzoite antigens and detects all Ig classes. This test had a sensitivity of 98% (95% CI 93± 100%) using post-abortion sera and a speci®city of 92% (95% CI 85±98%) using non-suspect sera (Wouda et al., 1998a). 2.5. Analyses Analysis of the data was performed using Statistix for Windows 2.0. Statistical signi®cance was declared at P # 0:05. For each herd, all seropositive and all seronegative animals were plotted against the month of birth. High seroprevalence age-groups were detected by scrolling a 6month time window, starting at the month of birth of the oldest animal present, with a 1 month interval. If prevalence of N. caninum antibodies of the animals included in the 6 month interval was signi®cantly higher compared to the other animals of the herd, this interval was de®ned as being part of a high seroprevalence age-group. To correct for multiple testing the signi®cance value of 0.05 was divided by the number of tests per herd (Bland, 1995). The cut-off dates of a high seroprevalence age-group were determined by inclusion of the adjacent months that had a N. caninum seroprevalence higher than other months and exclusion of the months included in the 6 month time window that had a lower seroprevalence. A herd could have more than one high prevalence age-cluster. If in a herd a second age-group mainly consisted of daughters of the ®rst detected high seroprevalence age-group, the ®rst age-group was de®ned as the high seroprevalence agegroup. All dam-daughter relations were recorded. Differences in prevalence of N. caninum serostatus of dams and daughters were compared using x 2 analysis on contingency tables (Fleiss, 1981). Odds ratios were calculated from these contingency tables (Martin et al., 1987). In case of embryo transfer the recipients and not the donors were considered as dams, since transmission of N. caninum occurs after placentation (Thurmond and Hietala, 1995).
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Table 1 Seroprevalence of Neospora caninum antibodies in 18 dairy herds in relation to abortion history Herd a
Number of animals
Seroprevalence (%)
Number of abortions per year b
Abortion pattern
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Total
36 127 111 184 289 126 75 73 117 277 151 103 106 114 129 110 198 104 2430
13.9 50.4 14.4 66.8 21.1 56.3 78.7 27.4 35.0 13.0 34.4 36.9 27.4 47.4 58.9 52.7 47.0 58.7 39.4
2 6 6 8 5 11 15 12 7 15 13 (7) 8 (6) 5 (3) 11 (4) 6 (4) 9 (9) 25 (25) 11 (11) 174
Endemic Endemic Endemic Endemic Sporadic Endemic Epidemic Epidemic Endemic Endemic Endemic Endemic Endemic Epidemic Endemic Endemic Epidemic Epidemic
a b
Herds 1±10 had an equal age-distribution of seropositive animals. Herds 11±18 showed an age related clustering of seropositive animals. Abortions during 12 months prior to date of sampling, numbers in parentheses represent aborting animals in the high seroprevalence age-group.
3. Results 3.1. Seroprevalence Of the 2430 animals sampled, 39.4% were seropositive for N. caninum, ranging per herd from 13.0 to 78.7% (Table 1). In 10 herds (Herds 1±10) seropositive animals were equally distributed across all age-groups. In eight herds (Herds 11±18) a high seroprevalence age-group could be detected by the 6 months time window scrolling procedure (Table 2). In Herds 11±15 an age-group with a high seroprevalence was present amidst younger and older animals with a signi®cantly lower seroprevalence. Fig. 1 shows an example of such a herd (Herd 12). The extent of the high seroprevalence age-groups varied from 7 months (Herd 13) to 29 months (Herd 15). In Herds 16±18 a remarkable difference in seroprevalence could be distin-
guished between younger and older animals with varying cut-off dates per herd. Fig. 2 shows the age-distribution of seropositive and seronegative animals in one of these herds (Herd 17). 3.2. Dam-daughter serology In all herds there was a high degree (P , 0:0001) of association between N. caninum serological status of dams and daughters. In the herds with an equal age-distribution of seropositives, 207 (83.5%) of the 248 seronegative dams, had seronegative daughters, and 41 (16.5%) had seropositive daughters. Of the 248 seronegative daughters, 41 (16.5%) had seropositive dams, and 207 (83.5%) had seronegative dams (Table 3). In one herd (Herd 1) all ®ve seropositive animals belonged to one family. In the herds with a high seroprevalence age-group (Herds 11±18), more sero-
Table 2 Seroprevalence of Neospora caninum antibodies of the high seroprevalence age-group versus the other animals in the herd Herd
High seroprevalence age-group Age-group a(months)
11 12 13 14 15 16 17 18 Total
23±30 23±34 32±38 22±34 12±40 . 24 .9 . 27
a
Age at blood sampling.
No. of seropositives
Seroprevalence %
Other animals No. of seropositives
Seroprevalence %
22 19 13 15 56 51 84 43 303
69 91 87 88 93 63 59 80 71
30 19 16 39 20 7 9 18 158
27 23 18 40 29 25 16 36 27
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Fig. 1. Age-distribution of N. caninum seropositive and seronegative animals in herd 12 at the date of blood sampling (05-01-1999). There was a cluster of 19 seropositive animals born between March 1996 and February 1997 among generally seronegative animals born earlier or later. Six animals of this seropositive cohort aborted between April and December 1998 during their ®rst pregnancy. Five of the six seropositive animals born between August 1998 and January 1999 were daughters of this seropositive cohort.
negative dams had seropositive daughters (OR 2:2; P 0:0004), and more seronegative daughters had seropositive dams compared with Herds 1 to 10 (OR 3:1; P , 0:0001). Of the 195 seronegative dams in Herds 11± 18, 135 (69.2%) had seronegative daughters, and 60 (30.8%) had seropositive daughters. Of the 217 seronegative daughters, 82 (37.8%) had seropositive dams, and 135 (62.2%) had seronegative dams (Table 3).
Of the 58 seronegative dams with daughters in the high seroprevalence age-groups 48 (82.8%) had seropositive daughters, compared with 12 (8.8%) of the 137 seronegative dams with seropositive daughters outside the high seroprevalence age-group (Table 4) (OR 50:0; P , 0:0001). Of the 82 seropositive dams with seronegative daughters, 64 (78.0%) dams were part of the high seroprevalence agegroups (Table 4).
Fig. 2. Age-distribution of N. caninum seropositive and seronegative animals in herd 17 at the date of blood sampling (06-07-1999). There was a remarkable difference in seroprevalence between the animals born before October 1998 compared with the younger animals. A low seroprevalence group can be distinguished between October 1998 and May 1999. Twenty-four seronegative animals of the low seroprevalence group had a seropositive dam. In the high seroprevalence age-group also seronegative daughters with seropositive dams were present. Fifteen animals of the high seroprevalence group aborted in June 1999.
T. Dijkstra et al. / International Journal for Parasitology 31 (2001) 209±215 Table 3 Comparison of Neospora caninum serological status of dams and daughters in 10 dairy herds with an equal age-distribution of seropositive animals (Herds 1±10) and eight herds with a high seroprevalence age-group (Herds 11±18) Serostatus dam
Serostatus daughter
Herds 1±10
Herds 11±18
Positive Positive Negative Negative
Positive Negative Positive Negative
163 41 41 207
108 82 60 135
3.3. Abortion history All herds had a history of sporadic or endemic abortions for several years. However, for most herds (Herds 6-18) the farmers had noticed an increase of the number of abortions during the last 2 years. Five herds (Herds 7, 8, 14, 17 and 18) had experienced acute abortion epidemics (Table 1) involving 15.2±33.3% of the animals at risk. Within a period of 2 months eight, 11, 11, 15, and 10 animals aborted in Herds 7, 8, 14, 17, and 18, respectively. In Herd 8, ®ve of the 12 aborting animals had been introduced in the herd during the last year. No dam-daughter relations of the ®ve purchased aborting animals of this herd were available. In the eight herds with a high seroprevalence age-group, 78.4% (69 of the 88 animals) of the aborting animals were in the high seroprevalence age-groups (Table 1). In Herds 11±14, 20, 29, 20 and 25%, respectively of the animals in the high seroprevalence age-groups aborted during their ®rst pregnancy. In Herd 15, data on ®rst pregnancy abortions of the animals in the age-group 12 to 40 months were not available. In three herds (Herd 16, 17 and 18) with a remarkable difference in seroprevalence between younger and older animals, 11, 27 and 16% of the animals at risk aborted.
4. Discussion Congenital transmission of N. caninum was found in all herds of the present study based on the presence of seropositive dam-daughter pairs and seropositive family lines in each of these herds. In one herd (Herd 1), congenital infection apparently was the only transmission route, as all seropositive cattle belonged to one family. In nine herds (Herds 2±10), congenital transmission was the predominant transmission route. This was based on an equal distribution of
213
seropositive animals across the different age-groups and a strong correlation between serological status of dams and daughters. However, in these nine herds, mismatches in dam-daughter serostatus were also present, suggesting the occurrence of post-natal infection as well. A low rate of seroconversion suggesting post-natal infection has been observed by others in endemically infected herds (Davison et al., 1999b; Hietala and Thurmond, 1999; Pare et al., 1997), but further evidence is required to con®rm that these animals were truly infected post-natally (Davison et al., 1999b). Part of the mismatches in dam-daughter serostatus in our study may have been due to false positive or false negative test results of either dams or daughters. Because the reported sensitivity of the ELISA used is based on post-abortion sera, which usually have high levels of antibodies (Wouda et al., 1998a), it can be expected that in a cross-sectional study the sensitivity of the test will be lower. Therefore, part of the seronegative dams with seropositive daughters may have been false negatives. A proportion of 16.5% seronegative daughters with seropositive dams as we found in these herds, is not unexpected, because a congenital transmission rate as low as 81% has been found previously based on pre-colostral calf sera (Pare et al., 1996). In eight herds (Herds 11±18) of our study, post-natal infection with N. caninum appeared to have contributed signi®cantly to the prevalence of infected animals, as speci®c age-groups could be identi®ed, which had a signi®cantly higher seroprevalence compared with that of the other animals in the herd. In addition, the number of dam-daughter pairs with different serostatus was disproportionally high. Clustered seropositive animals in these eight herds were either seropositive daughters with seronegative dams (as exempli®ed in Fig. 1) or seropositive dams with seronegative offspring (as exempli®ed in Fig. 2). The ®rst situation indicates postnatal infection of the daughters, and the second situation indicates postnatal infection of the dams after the birth of the seronegative daughters. The age related clustering of post-natally infected animals strongly supports a point source exposure of these animals. Evidence of a point source exposure has been previously based on the epidemic curve of the abortions during N. caninum-associated abortion storms (McAllister et al., 1996; McAllister et al., 2000; Schares et al., 1997; Yaeger et al., 1994). This would imply that epidemic abortions were the effect of a post-natal infection, probably due
Table 4 Comparison of Neospora caninum serological status of dams and daughters in eight dairy herds with a high seroprevalence age-group Serostatus dam
Serostatus daughter
Daughters in high seroprevalence age-group
Daughters outside high seroprevalence age-group
Positive Positive Negative Negative
Positive Negative Positive Negative
43 15 48 10
65 67 12 125
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to oocyst exposure. This is supported by our results. In all eight herds with evidence of post-natal infection there had been an increase in abortions during the last 2 years and in three of these herds (Herds 14, 17 and 18) an abortion epidemic had occurred. In addition, the abortions in these herds mainly occurred in the animals of the high seroprevalence age-groups. However, epidemic abortions were also seen in two herds without clear evidence of post-natal infection (Herds 7 and 8). The abortion storms in these herds may have been due to a synchronised recrudescence of N. caninum infection following an event causing immune suppression in a number of animals, as has been speculated previously (Atkinson et al., 2000; Wouda et al., 1999a). The present study shows the power of a single serological screening for neosporosis of all animals (including calves and heifers) in the herd. An analysis of the agedistribution of the seropositives combined with a comparison of dam-daughter serostatus is a practical approach to study the mode of transmission of N. caninum in a herd. Cut-off dates of age-groups in this study were dependent on the seroprevalence data per herd. Usually age-groups are de®ned ®rst and then the seroprevalence is compared among the groups (Dyer et al., 2000; Schares et al., 1998). However, in this way age related differences in seroprevalence within herds may be underestimated and congenital infection rates, if based on seroprevalences per year group, may be overestimated. If herds are analysed in one dataset without considering the herd-speci®c age-distribution of seroprevalence (Davison et al., 1999a; Mainar-Jaime et al., 1999), probably age-clustering will be underestimated or even not be found. The results of this study indicate that it is important to include young animals in the analysis. Generally, about 12.5% of a herd consists of animals younger than 6 months and the dams of these calves are usually still present in the herd. If this young age-group is included, the number of dam-daughter pairs that can be evaluated increases considerably. Congenitally infected calves usually have high antibody levels (Anderson et al., 1997; Davison et al., 1999b) and few false negative test results. Positive serology of young animals can be used for the interpretation of the serologic status of their dams, which may have ¯uctuating antibody titers (Dannatt, 1997; Schares et al., 1999; Stenlund et al., 1999). Calves can be seropositive due to colostral N. caninum antibodies. In all the 18 herds of the present study the calves were fed only colostrum of their own dams so there were no false-positive results due to the feeding of pooled colostrum. The fact that we found strong evidence of post-natal infection in eight of 18 dairy herds, indicates that this mode of infection is far from rare in cattle in The Netherlands. However, most herds of this study were problem herds and therefore the results may not be representative for the all over importance of post-natal N. caninum infections in The Netherlands.
Our results show that this type of study can help to de®ne the most probable period of a point source infection, that is the period during which the high seroprevalence age-group was housed or fed as a separate group. Such information is essential to get a better understanding of the epidemiology of neosporosis in cattle. The dog has been shown to be a risk factor for N. caninum-associated abortion storms (Bartels et al., 1999). A few dogs present at farms of the present study were incorporated in a previous seroepidemiological study, which indicated a relationship between N. caninum infections in dogs and cattle (Wouda et al., 1999b). Acknowledgements The authors thank Albert Winkel for help with data collection, and C.J.M. Bartels, J.W. Hesselink and Y.H. Schukken for valuable comments on the manuscript. This study was partially funded by the Dairy Commodity Board (Rijswijk, The Netherlands). References Anderson, M.L., Reynolds, J.P., Rowe, J.D., Sverlow, K.W., Packham, A.E., Barr, B.C., Conrad, P.A., 1997. Evidence of vertical transmission of Neospora sp. infection in dairy cattle. J. Am. Vet. Med. Assoc. 210, 1169±72. Atkinson, R.A., Cook, R.W., Reddacliff, L.A., Rothwell, J., Broady, K.W., Harper, P., Ellis, J.T., 2000. Seroprevalence of Neospora caninum infection following an abortion outbreak in a dairy cattle herd. Aust. Vet. J. 78, 262±6. Bartels, C.J.M., Wouda, W., Schukken, Y.H., 1999. Risk factors for Neospora caninum-associated abortion storms in dairy herds in The Netherlands (1995 to 1997). Theriogenology. 52, 247±57. BjoÈrkman, C., Johansson, O., Stenlund, S., Holmdahl, O.J.M., Uggla, A., 1996. Neospora species infection in a herd of dairy cattle. J. Am. Vet. Med. Assoc. 208, 1441±4. Bland, M., 1995. An Introduction to Medical Statistics, Oxford University Press Inc, New York. Dannatt, L., 1997. Neospora caninum antibody levels in an endemicallyinfected dairy herd. Cattle Pract. 5, 335±7. Davison, H.C., French, N.P., Trees, A.J., 1999a. Herd-speci®c and agespeci®c seroprevalence of Neospora caninum in 14 British dairy herds. Vet. Rec. 144, 547±50. Davison, H.C., Otter, A., Trees, A.J., 1999b. Estimation of vertical and horizontal transmission parameters of Neospora caninum infection in dairy cattle. Int. J. Parasitol. 29, 1683±9. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1±59. Dyer, R.M., Jenkins, M.C., Kwok, O.C., Douglas, L.W., Dubey, J.P., 2000. Serologic survey of Neospora caninum infection in a closed dairy cattle herd in Maryland: risk of serologic reactivity by production groups. Vet. Parasitol. 90, 171±81. Fleiss, J.L., 1981. Statistical Methods for Rates and Proportions, John Wiley & Sons, New York. French, N.P., Clancy, D., Davison, H.C., Trees, A.J., 1999. Mathematical models of Neospora caninum infection in dairy cattle: transmission and options for control. Int. J. Parasitol. 29, 1691±704. Hietala, S.K., Thurmond, M.C., 1999. Postnatal Neospora caninum transmission and transient serologic responses in two dairies. Int. J. Parasitol. 29, 1669±76. Mainar-Jaime, R.C., Thurmond, M.C., Berzal-Herranz, B., Hietala, S.K.,
T. Dijkstra et al. / International Journal for Parasitology 31 (2001) 209±215 1999. Seroprevalence of Neospora caninum and abortion in dairy cows in northern Spain. Vet. Rec. 145, 72±75. Martin, S.W., Meek, A.H., Willeberg, P., 1987. Epidemiologic Measures of Association. Veterinary Epidemiology Principles and Methods, Iowa State University Press, Iowa, pp. 128±34. McAllister, M.M., Huffman, E.M., Hietala, S.K., Conrad, P.A., Anderson, M.L., Salman, M.D., 1996. Evidence suggesting a point source exposure in an outbreak of bovine abortion due to neosporosis. J. Vet. Diagn. Invest. 8, 355±7. McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., McGuire, A.M., 1998. Dogs are de®nitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473±8. McAllister, M.M., 1999. Uncovering the biology and epidemiology of Neospora caninum. Parasitology Today. 15, 216±7. McAllister, M.M., BjoÈrkman, C., Anderson-Sprecher, R., Rogers, D.G., 2000. Evidence of point-source exposure to Neospora caninum and protective immunity in a herd of beef cows. J. Am. Vet. Med. Assoc. 208, 1441±4. Nielen, M., Jansen, F.C.M., van Wuijckhuise, L.A., Dijkhuizen, A.A., 1996. Dutch cattle identi®cation and registration (I&R) system: analysis of its use for controlling an outbreak of foot and mouth disease. Tijdschr. Diergeneeskd. 121, 576±81. PareÂ, J., Thurmond, M.C., Hietala, S.K., 1996. Congenital Neospora caninum infection in dairy cattle and associated calfhood mortality. Can. J. Vet. Res. 60, 133±9. PareÂ, J., Thurmond, M.C., Hietala, S.K., 1997. Neospora caninum antibodies in cows during pregnancy as a predictor of congenital infection and abortion. J. Parasitol. 83, 82±87. Patitucci, A.N., Charleston, W.A.G., Alley, M.R., O'Connor, R.J., Pomroy, W.E., 1999. Serological study of a dairy herd with a recent history of Neospora abortion. NZ. Vet. J. 47, 28±30. Schares, G., Peters, M., Wurm, R., Tackmann, K., Henning, K., Conraths, F.J., 1997. Neospora caninum causes abortions in cattle herds in North Rhine-Westphalia. Dtsch. TieraÈrztl. Wschr. 104, 208±12. Schares, G., Peters, M., Wurm, R., Barwald, A., Conraths, F.J., 1998. The ef®ciency of vertical transmission of Neospora caninum in dairy cattle analysed by serological techniques. Vet. Parasitol. 80, 87±98. Schares, G., Rauser, M., Peters, M., Zimmer, K., Peters, M., Wurm, R.,
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Dubey, J.P., de Graaf, D.C., Edelhofer, R., Mertens, C., Hess, G., Conraths, F.J., 1999. Serological differences in Neospora caninumassociated epidemic and endemic abortions. . J. Parasitol. 85, 688±94. Stenlund, S., Kindahl, H., Magnusson, U., Uggla, A., BjoÈrkman, C., 1999. Serum antibody pro®le and reproductive performance during two consecutive pregnancies of cows naturally infected with Neospora caninum. Vet. Parasitol. 85, 227±34. Thurmond, M., Hietala, S., 1995. Stragies to control Neospora infection in cattle. Bovine Pract. 29, 60±63. Thurmond, M., Hietala, S., Blanchard, P.C., 1997. Herd-based diagnosis of Neospora caninum-induced endemic and epidemic abortion in cows and evidence for congenital and postnatal transmission. J. Vet. Diagn. Invest. 9, 44±49. Waldner, C.L., Janzen, E.D., Henderson, J., Haines, D.M., 1999. Outbreak of abortion associated with Neospora caninum infection in a beef herd. J. Am. Vet. Med. Assoc. 215, 1485±90. Wouda, W., Moen, A.R., Visser, I.J.R., van Knapen, F., 1997. Bovine fetal neosporosis: a comparison of epizootic and sporadic abortion cases and different age-classes with regard to lesion severity and immunohistochemical identi®cation of organisms in brain heart and liver. J. Vet. Diagn. Invest. 9, 180±5. Wouda, W., Brinkhof, J., van Maanen, C., de Gee, A.L.W., Moen, A.R., 1998a. Serodiagnosis of neosporosis in individual cows and dairy herds, a comparison of three enzyme linked immunosorbent assays. Clin. Diagn. Lab. Immunol. 5, 711±6. Wouda, W., Moen, A.R., Schukken, Y.H., 1998b. Abortion risk in progeny of cows after a Neospora caninum epidemic. Theriogenology. 49, 1311±6. Wouda, W., Bartels, C.J.M., Moen, A.R., 1999a. Characteristics of Neospora caninum-associated abortion storms in dairy herds in The Netherlands (1995 to 1997). Theriogenology 52, 233±45. Wouda, W., Dijkstra, Th., Kramer, A.M.H., van Maanen, C., Brinkhof, J.M.A., 1999b. Seroepidemiological evidence for a relationship between Neospora caninum infections in dogs and cattle. Int. J. Parasitol. 29, 1677±82. Yaeger, M.J., Shawd-Wessels, S., Leslie-Steen, P., 1994. Neospora abortion in a midwestern dairy. J. Vet. Diagn. Invest. 6, 506±8.