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Veterinary Parasitology 128 (2005) 195–200

IgG avidity pattern in cattle after ingestion of

Neospora caninum oocysts

C. Bjorkmana,*, L.F.P. Gondimb, K. Naslundc, A.J. Treesd, M.M. McAllisterb

aDepartment of Clinical Sciences, Swedish University of Agricultural Sciences, P.O. Box 7019, SE-75007 Uppsala, SwedenbDepartment of Veterinary Pathobiology, University of Illinois, 2001 South Lincoln Avenue, Urbana Illinois, IL 61802, USA

cDepartment of Parasitology (SWEPAR), National Veterinary Institute and Swedish University of Agricultural Sciences,

SE-75189 Uppsala, SwedendVeterinary Parasitology, Liverpool School of Tropical Medicine and Faculty of Veterinary Science, University of Liverpool,

Pembroke Place, Liverpool L3 5QA, UK

Received 22 June 2004; received in revised form 15 November 2004; accepted 26 November 2004

Abstract

The avidity (functional affinity) of specific antibodies are being used to estimate duration of bovine Neospora caninum

infection. Here, we report for the first time the avidity pattern in cattle orally inoculated with N. caninum oocysts. In all, 16

pregnant cows and 7 calves were administered N. caninum oocysts. In the cows, the avidity increased during the early course of

infection. In all but one, the avidity was �35 during the first 6 weeks after infection and no cow had an avidity value >50 until

week 9. The calves were sampled either week 6 (n = 3) or week 9 (n = 9) after infection, and by then had avidities between 2 and

17. The results are in agreement with results from previous investigations of naturally infected cattle, and calves that were

experimentally infected with tachyzoites. They further validate the ability of the N. caninum iscom avidity ELISA to accurately

assess the duration of infection.

# 2004 Elsevier B.V. All rights reserved.

Keywords: Avidity; Cattle; Neospora caninum; Oocysts; ELISA; Humoral immunity

1. Introduction

The protozoan parasite Neospora caninum is

increasingly recognised as an important pathogen

causing abortion and stillbirth in pregnant cattle

(Dubey, 1999). In many parts of the world, it is

* Corresponding author. Tel.: +46 1867 1778;

fax: +46 1867 3545.

E-mail address: camilla.bjorkman@kv.slu.se (C. Bjorkman).

0304-4017/$ – see front matter # 2004 Elsevier B.V. All rights reserved

doi:10.1016/j.vetpar.2004.11.030

considered one of the most common infectious causes

of bovine abortion. The most commonly recognised

route of infection in cattle is congenital transmission

of the parasite from an infected dam to her fetus (Pare

et al., 1996; Davison et al., 1999b). However, cattle

can also be infected postnatally by ingesting oocysts

shed by a dog or coyote, which are definitive hosts for

the parasite (McAllister et al., 1998; Gondim et al.,

2004d). Infection seems to be life-long, and infected

females can transmit the infection to their offspring

.

C. Bjorkman et al. / Veterinary Parasitology 128 (2005) 195–200196

during consecutive pregnancies (Bjorkman et al.,

1996). Congenital infection can result in abortion or

stillbirth although the majority of infected fetuses are

born clinically healthy but chronically infected.

Congenitally infected heifers can in their turn give

birth to infected calves and the infection can thus,

be kept in a cattle herd for many years without

involvement of a definitive host (Bjorkman et al.,

1996; Anderson et al., 1997). Abortion outbreaks in

cattle herds have been connected with recent infection

in the pregnant heifers and/or cows, whereas endemic

abortion patterns are thought to be consistent with

presence of chronically (persistently) infected dams

(Thurmond et al., 1997; Davison et al., 1999a;

McAllister et al., 2000; Dubey, 2003).

Demonstration of specific antibodies in serum from

an animal is indicative of infection (Bjorkman and

Uggla, 1999; Dubey, 2003). However, as the antibody

levels fluctuate during pregnancy in persistently

infected animals (Stenlund et al., 1999; Guy et al.,

2001), antibody levels or titers cannot be used to

discriminate between acutely and chronically infected

animals. For this purpose, an avidity ELISA was

developed, based on the fact that the first antibodies

synthesised after an antigenic challenge have lower

affinity for the antigen than those produced later on

(Bjorkman et al., 1999). This IgG avidity test utilised

membrane proteins incorporated into iscoms as antigen,

and was the first of its kind to be reported. Since then,

other avidity tests based on tachyzoite extracts (Maley

et al., 2001; Sager et al., 2003) and the N. caninum

membrane protein NcSRS2 (Schares et al., 2002) have

been described. The avidity of N. caninum specific IgG

antibodies increased with time in calves experimentally

infected by subcutaneous or intravenous injections of N.

caninum tachyzoites (Bjorkman et al., 1999). Even

though both the mode of transmission and the parasite

life stage used in these experiments differ from what is

seen under natural conditions, these results suggested

that low IgG avidity levels give an indication whether an

animal has been recently exposed to the infection

(Bjorkman et al., 1999). As far as is known, the only

natural route of postnatal infection in cattle is ingestion

of feed or water contaminated with oocysts shed by a

definitive host. Lactogenic infection with tachyzoites

has been shown experimentally in newborn calves

(Ugglaetal.,1998;Davisonetal.,2001)but isnotknown

to occur naturally.

This investigation was performed in order to further

elucidate the development of IgG avidity during N.

caninum infection by analysing samples from preg-

nant cows and young calves orally inoculated with N.

caninum oocysts. Samples were also analysed from

calves born to the inoculated cows.

2. Materials and methods

2.1. Animals and samples

Serum samples collected from pregnant cows and

from young calves were used in this study.

A) T

hirteen mixed breed beef cows that were derived

from a previous study that had—at that time point—

included 19 cows (Gondim et al., 2004b). The cows

were inoculated orally with 1500–115,000 sporu-

lated oocysts of different N. caninum isolates at

different time points during gestation. Blood

samples were collected by venous puncture before

and up to 24 weeks after inoculation. Not all cows

werekeptuntilcalving; therefore,only5calveswere

born to the 13cows. Included in thepresent studyare

the two calves shown to be seropositive before

ingestion of colostrum, one of which was stillborn

and blood sampled at necropsy. The identification

numbers of the cows and calves are the same as in

Gondim et al. (2004b).

B) T

hree 10–12-year-old Hereford cross Friesian

cows were at 70 days gestation infected orally with

600 sporulated oocysts of the Nc-Liv isolate and

blood samples were collected up to 30 weeks after

inoculation. This experiment has been described

in detail by Trees et al. (2002).

C) A

lso included were samples from seven 2–14-day-

old heifer and bull calves orally given 1400–

29,000 oocysts of Nc-Illinois (Gondim et al.,

2004a), Nc-beef (McAllister et al., 1998; McAll-

ister et al., 2000) or Nc-deer (Gondim et al.,

2004c). Blood samples were collected before and

6–9 weeks after inoculation.

2.2. Antibody analyses

N. caninum antibodies were detected by iscom

ELISA using tachyzoite antigens incorporated into

C. Bjorkman et al. / Veterinary Parasitology 128 (2005) 195–200 197

immunostimulating complexes (iscoms) as antigen

and a monoclonal anti-bovine IgG1 antibody as

conjugate (Bjorkman et al., 1997; Frossling et al.,

2003). All absorbance values were correlated to a

positive control serum with a mean absorbance value

of 1.0, and sera with corrected absorbance �0.20 were

considered positive.

Sera with absorbance values >0.35 in the iscom

ELISA were further tested for IgG avidity by the

method of Bjorkman et al. (1999). Briefly, the sera

were diluted in five-fold serial dilutions from 1:50 and

applied in duplicate microtitre wells. After incubation

with serum, 1 well for each serum was treated with

urea to release low avidity antibodies from the

antigen-antibody complex. Absorbance was measured

in treated and untreated wells after incubation with

conjugate and substrate. The IgG avidity was

calculated using the formula:

IgG avidity ¼ end point titer with urea

end point titer without urea

� �� 100

IgG avidity values could only be calculated who had

endpoint titers �1:50.

Fig. 1. Neospora caninum IgG avidity in pregnant cows following

oral inoculation with sporulated oocysts of different N. caninum

isolates at different time points during gestation. The cows were

administered (A) 1500–15,000 oocysts or (B) 41,000–115,00 oocysts.

Fig. 2. Neospora caninum IgG avidity in pregnant cows following

oral inoculation with 600 sporulated N. caninum oocysts of the Nc-

Liv isolate at 70 days gestation.

3. Results

A positive iscom ELISA result was seen in 10 cows

in group A, 3–4 weeks after inoculation and eight had

developed absorbance values higher than 0.35, which

made avidity measurement possible. In all 13 cows,

there was an increase in avidity during the early course

of infection (Fig. 1a and b). In one cow (no. 6), the

avidity was 37 by week 4 when an avidity value could

first be established, and it rose slowly to reach 53 at the

last sampling, 10 weeks after infection. In the

remaining cows, avidities �35 were detected during

the first 6 weeks after infection. None of the 13 cows

had an avidity value >50 until week 9. Three cows,

number 12, 13 and 14, were sampled until week 24

after inoculation. By then, cow 13 had such low

absorbance values that the avidity could not be

measured. By week 22, cow 12 had reached an avidity

value of 81, whereas the avidity did not exceed 45 in

cow 14.

The cows in group B showed the same initial

increase in avidity as seen in group A (Fig. 2).

Thereafter, cow 44 had a considerable fluctuation in

C. Bjorkman et al. / Veterinary Parasitology 128 (2005) 195–200198

avidity values between week 8 and 20. Although the

sampling continued until calving 30 weeks post

infection, the antibody levels decreased from week 16

and onwards and no avidity could be estimated later

than 20 weeks after infection in any cow.

Two calves born to group A cows were seropositive

at birth when tested by iscom ELISA with absorbance

values �0.97. One of them was stillborn and had an

avidity of 16. The dam had been inoculated

approximately day 130 in pregnancy. The other

seropositive calf was born to a cow inoculated when

164 days pregnant and had an IgG avidity of 32. Both

dams had avidities >50. The calves born to cows in

group B were all seronegative.

Four of the seven orally infected calves were blood

sampled 9 weeks after inoculation and their avidity

values were 8–17. The remaining three calves were

kept only until week six post inoculation and the

avidity values by then were 2, 3 and 16, respectively.

The iscom ELISA absorbances were 0.83–1.11.

4. Discussion

Here, we report for the first time the avidity pattern

in cattle orally inoculated with N. caninum oocysts. In

the pregnant cows there was an increase in IgG avidity

during the early course of infection similar to that

found in animals that had been injected with

tachyzoites (Bjorkman et al., 1999; Maley et al.,

2001; Schares et al., 2002). There was large individual

variation in avidity pattern, but all calves and most of

the cows had avidity levels <35 during the first 6–7

weeks post infection. The avidity levels in the calves

were considerably lower then the avidities previously

measured in calves infected with tachyzoites using the

same IgG avidity iscom ELISA (Bjorkman et al.,

1999). If this difference in avidity level was a result of

the infection mode, dose or any other circumstances is

not known. They were not, however, associated with

the N. caninum antibody levels. In the earlier study

(Bjorkman et al., 1999), the iscom ELISA absorbance

values of the tachyzoite inoculated calves at week 6

was 0.82–1.00 (not previously reported), and thus, of a

similar magnitude as those reported here.

In cow 44, a temporary drop in avidity, from 58 to

36, occurred between week 12 and 16, while the

ELISA absorbance values increased from 0.60 to 0.80.

A majority of persistently N. caninum infected cows

have consistently high avidity IgG antibodies (Guy

et al., 2001; Bjorkman et al., 2003), although in certain

individuals low avidity antibodies may persist for

many months. Drops in avidity were reported by Sager

et al. (2003) who applied a method based on a tachyzoite

antigen. However, a drop in avidity, as occurred in cow

44, has not previously been observed by the iscom

ELISA test. These sera were re-tested with similar

results. The avidity data from this cow are outliers

compared to other data in this and previous studies.

Indirect evidence that IgG avidity is low in recently

oocyst infected animals has been reported from

investigations performed in naturally infected cattle

herds after putative point source horizontal spread of

the parasite. When the avidity test was applied in a

long-term study in such a herd the mean avidity in the

cows was 30 during the outbreak and increased to 74

during the following 3 years (Bjorkman et al., 2003).

The mode of avidity in the herd during the same period

moved from 21–40 to 61–80. Similar results were

reported by Dijkstra et al. (2002) studying a dairy herd

in which almost 50% of the animals seroconverted

during a period of 6 months between two blood

samplings. However, individual naturally infected

cows can maintain their low avidity in spite of being

seropositive for many years (Bjorkman et al., 2003).

In accordance with previous findings, avidities >50

were only seen in animals that had been infected

longer than 8 weeks. Such overall high avidities have

been detected in herds in which congenital infection is

the principle route of parasite transmission as judged

by high association between seropositivity in dams

and their daughters (Bjorkman et al., 1999; Schares

et al., 2002).

The exact mechanism for the parasite transmission

from a dam to her fetus and what decides the outcome

of pregnancy in an infected cow are not known (Innes

et al., 2002). In the present investigation, two of the

eight cows that were kept throughout pregnancy gave

birth to seropositive calves, one of which was stillborn.

These calves both had high antibody levels and avidity

values below 35, whereas the cows that had been

inoculated more than 17 weeks earlier, had avidities

>50. The lower avidity values in the calves might reflect

that they were only recently infected, i.e. that therewas a

significant time span between when the dams were

infected and when they had transmitted the parasite to

C. Bjorkman et al. / Veterinary Parasitology 128 (2005) 195–200 199

the foetuses. Alternatively, the immature immune

system in the foetus may result in a slower maturation

of the avidity of the IgG antibodies.

Recently, the avidity iscom ELISA has been used to

investigate infection patterns in cattle herds experien-

cing abortionoutbreaks (Jenkins et al., 2000;McAllister

et al., 2000). The aborting cows had lower avidity than

the normally calving cows suggesting a recent point

source exposure to the parasite in these herds. However,

each avidity test must be carefully evaluated before it is

used to establish whether aborting cows are recently or

chronically infected. The reason is that even though the

abortion per se does not seem to affect the avidity

results as measured by the iscom ELISA, a decrease in

avidity at abortion was found by an avidity test based

on tachyzoite extract (Sager et al., 2003). Perhaps this

reflects a difference in binding pattern between

antibodies directed to different epitopes.

The N. caninum iscom avidity ELISA have

previously been applied in naturally N. caninum

infected cattle herds to investigate when the infection

has been acquired and to elucidate the infection

pattern in the herds (Jenkins et al., 2000; McAllister

et al., 2000; Dijkstra et al., 2002; Bjorkman et al.,

2003). Avidity testing provides robust epidemiologi-

cal information when used to analyse groups or herds,

but interpretation of the avidity results for any single

animal is less certain because of individual variation.

The results in the present study validate the ability of

the test to assess the duration of infection following

ingestion of N. caninum oocysts.

Acknowledgements

The authors thank Liying Gao for technical

assistance. The study was financially supported in-

part by the Swedish Research Council for Environ-

ment, Agricultural Sciences and Spatial Planning and

the Swedish Farmers’ Foundation for Agricultural

Research and in-part by United States Department of

Agriculture (USDA-NRICGP 2000–01997).

References

Anderson, M.L., Reynolds, J.P., Rowe, J.D., Sverlow, K.W., Pack-

ham, 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, 1169–1172.

Bjorkman, C., Johansson, O., Stenlund, S., Holmdahl, J., Uggla, A.,

1996. Neospora species infection in a herd of dairy cattle. J. Am.

Vet. Med. Assoc. 208, 1441–1444.

Bjorkman, C., Holmdahl, O.J.M., Uggla, A., 1997. An indirect

enzyme-linked immunoassay (ELISA) for demonstration of

antibodies to Neospora caninum in serum and milk of cattle.

Vet. Parasitol. 68, 251–260.

Bjorkman, C., Naslund, K., Stenlund, S., Maley, S.W., Buxton, D.,

Uggla, A., 1999. An IgG avidity ELISA to discriminate between

recent and chronic Neospora caninum infection. J. Vet. Diagn.

Invest. 11, 41–44.

Bjorkman, C., Uggla, A., 1999. Serological diagnosis of Neospora

caninum infection. Int. J. Parasitol. 29, 1497–1507.

Bjorkman, C., McAllister, M.M., Frossling, J., Naslund, K., Leung,

F., Uggla, A., 2003. Application of the Neospora caninum IgG

avidity ELISA in assessment of chronic reproductive losses

following an outbreak of neosporosis in a herd of beef cattle.

J. Vet. Diagn. Invest. 15, 3–7.

Davison, H.C., Otter, A., Trees, A.J., 1999a. Significance of Neos-

pora caninum in British dairy cattle determined by estimation of

seroprevalence in normally calving cattle and aborting cattle.

Int. J. Parasitol. 29, 1189–1194.

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, 1683–1689.

Davison, H.C., Guy, C.S., McGarry, J.W., Guy, F., Williams, D.J.L.,

Kelly, D.F., Trees, A.J., 2001. Experimental studies on the

transmission of Neospora caninum between cattle. Res. Vet.

Sci. 70, 163–168.

Dijkstra, T., Barkema, H.W., Bjorkman, C., Wouda, W., 2002. A

high rate of seroconversion for Neospora caninum in a dairy herd

without and obvious increased incidence of abortions. Vet.

Parasitol. 109, 203–211.

Dubey, J.P., 1999. Neosporosis in cattle: biology and economic

impact. J. Am. Vet. Med. Assoc. 214, 1160–1163.

Dubey, J.P., 2003. Neosporosis in cattle. J. Parasitol. 89, S42–

S56.

Frossling, J., Bonnett, B., Lindberg, A., Bjorkman, C., 2003. Vali-

dation of a Neospora caninum iscom ELISA without a gold

standard. Prev. Vet. Med. 57, 141–153.

Gondim, L.F.P., Laski, P., Gao, L., McAllister, M.M., 2004a.

Variation of the internal transcribed spacer 1 sequence within

individual strains and among different strains of Neospora

caninum. J. Parasitol. 90, 119–122.

Gondim, L.F.P., McAllister, M.M., Anderson-Sprecher, R.C., Bjork-

man, C., Lock, T.F., Firkins, L.D., Gao, L., Fisher, W.R.T.,

2004b. Transplacental transmission and abortion in cows admi-

nistered Neospora caninum oocysts. J. Parasitol. 90, 1394–1400.

Gondim, L.F.P., McAllister, M.M., Mateus-Pinilla, N.E., Pitt, W.C.,

Mech, L.D., Nelson, M.E., 2004c. Transmission of Neospora

caninum between wild and domestic animals. J. Parasitol. 90,

1361–1365.

Gondim, L.F.P., McAllister, M.M., Pitt, W.C., Zemlicka, D.E.,

2004d. Coyotes (Canis latrans) are definitive hosts of Neospora

caninum. Int. J. Parasitol. 32, 159–161.

C. Bjorkman et al. / Veterinary Parasitology 128 (2005) 195–200200

Guy, C.S., Williams, D.J.L., Kelly, D.F., McGarry, J.W., Guy, F.,

Bjorkman, C., Smith, R.F., Trees, A.J., 2001. Neospora caninum

in persistently infected, pregnant cows: spontaneous transpla-

cental infection is associated with an acute rise in maternal

antibody. Vet. Rec. 149, 443–449.

Innes, E.A., Andrianarivo, A.G., Bjorkman, C., Williams, D.J.L.,

Conrad, P.A., 2002. Immune responses to Neospora caninum

and prospects for vaccination. Trends Parasitol. 18, 497–

504.

Jenkins, M.C., Caver, J.A., Bjorkman, C., Anderson, T.C., Romand,

S., Vinyard, B., Uggla, A., Thulliez, P., Dubey, J.P., 2000.

Serological investigation of an outbreak of Neospora cani-

num-associated abortion in a dairy herd in southeastern United

States. Vet. Parasitol. 94, 17–26.

Maley, S.W., Buxton, D., Thomson, K.M., Schriefer, C.E.S., Innes,

E.A., 2001. Serological analysis of calves experimentally

infected with Neospora caninum: a 1-year study. Vet. Parasitol.

96, 1–9.

McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R.,

Wills, R.A., McGuire, A.M., 1998. Dogs are definitive

hosts of Neospora caninum. Int. J. Parasitol. 28, 1473–

1478.

McAllister, M.M., Bjorkman, 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. 217, 881–887.

Pare, J., Thurmond, M.C., Hietala, S.K., 1996. Congenital Neospora

caninum infection in dairy cattle and associated calfhood mor-

tality. Can. J. Vet. Res. 60, 133–139.

Sager, H., Gloor, M., Bjorkman, C., Kritzner, S., Gottstein, B., 2003.

Assessment of antibody avidity in aborting cattle by a somatic

Neospora caninum tachyzoite antigen IgG avidity ELISA. Vet.

Parasitol. 112, 1–10.

Schares, G., Barwald, A., Staubach, C., Sondgen, P., Rauser, M.,

Schroder, R., Peters, M., Wurm, R., Selhorst, T., Conraths, F.J.,

2002. p38-avidity-ELISA: examination of herds experiencing

epidemic or endemic Neospora caninum-associated bovine

abortion. Vet. Parasitol. 106, 293–305.

Stenlund, S., Kindahl, H., Magnusson, U., Uggla, A., Bjorkman, C.,

1999. Serum antibody profile and reproductive performance

during two consecutive pregnancies of cows naturally infected

with Neospora caninum. Vet. Parasitol. 85, 227–234.

Thurmond, M.C., Hietala, S.H., 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.

Trees, A.J., McAllister, M.M., Guy, C.S., McGarry, J.W., Smith,

R.F., Williams, D.J.L., 2002. Neospora caninum: oocyst chal-

lenge of pregnant cows. Vet. Parasitol. 109, 147–154.

Uggla, A., Stenlund, S., Holmdahl, O.J.M., Jakubek, E.-B., Thebo,

P., Kindahl, H., Bjorkman, C., 1998. Oral Neospora caninum

inoculation of neonatal calves. Int. J. Parasitol. 28, 1467–1472.