Veterinary Parasitology 137 (2006) 222–230 www.elsevier.com/locate/vetpar
Neospora caninum: Serological follow-up in dairy cows during pregnancy Barbara Ha¨sler a,*, Jorge A. Hernandez a,c, Martin Reist a, Heinz Sager b, Christine Steiner-Moret b, Daniela Staubli b, Katharina D.C. Sta¨rk a, Bruno Gottstein b a Swiss Federal Veterinary Office, Schwarzenburgstrasse 155, CH-3003 Bern, Switzerland Institute of Parasitology, University of Bern, La¨nggassstrasse 122, CH-3001 Bern, Switzerland c University of Florida, College of Veterinary Medicine, Department of Large Animal Clinical Sciences, Gainesville, FL 32610-0136, USA b
Received 27 September 2005; received in revised form 19 December 2005; accepted 10 January 2006
Abstract We conducted a longitudinal study to follow-up the anti-Neospora caninum serologic status in 30 initially seropositive and 83 initially seronegative cows during their pregnancy. Study cows were blood-sampled every other month during pregnancy until parturition. Blood serum samples were screened for anti-N. caninum antibodies by ELISA. Cows that seroconverted were retested by immunoblot as a confirmation test. Among 30 seropositive cows, 28 cows remained seropositive during the whole pregnancy, whereas 2 cows transiently tested negative at least once during pregnancy. Among 83 seronegative cows, 82 cows remained seronegative and 1 cow tested positive three times during the sixth, eighth and last month of pregnancy. As only 2 out of 30 seropositive animals and 1 out of 83 animals changed their serologic status during pregnancy, the study results indicate that there is only a minor temporal instability of anti-N. caninum antibody reactivity in adult cattle. # 2006 Elsevier B.V. All rights reserved. Keywords: Anti-N. caninum antibodies; ELISA; Cattle
1. Introduction Neospora caninum is a protozoan parasite which is recognized as an important pathogen associated with abortions in cows (Thilsted and Dubey, 1989; Anderson et al., 1991; Thornton et al., 1991; Duff * Corresponding author. Tel.: +41 31 323 23 96; fax: +41 31 323 95 43. E-mail address:
[email protected] (B. Ha¨sler).
and Otter, 1994; Trees et al., 1994; Boulton et al., 1995; Thurmond et al., 1995; Pare et al., 1997; Jensen et al., 1999; Mainar-Jaime et al., 1999) and occasionally with encephalomyelitis in congenitally infected calves (Dubey et al., 1990; Barr et al., 1991; Bryan et al., 1994). Life cycles and routes of transmission of N. caninum have not been completely elucidated. Because of taxonomic and morphologic similarities with Toxoplasma gondii (Ellis et al., 1994; Holmdahl et al., 1994), N. caninum is believed to have
0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.01.007
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a life cycle similar to that of T. gondii, for which infection can occur in utero or by ingestion of oocysts in the feces of a definitive host (Dubey, 1993). Despite the discovery that dogs can serve as a definitive host for N. caninum (McAllister et al., 1998), congenital infection is generally accepted as the primary mean of transmission and maintenance of N. caninum in cattle (Dubey et al., 1992; Barr et al., 1993; Pare et al., 1996; Anderson et al., 1997; Thurmond and Hietala, 1997; Hietala and Thurmond, 1999). During pregnancy, some fetal infections culminate in abortion, whereas most result in a new generation of chronically infected cattle (Barr et al., 1993; Pare et al., 1997; Thurmond and Hietala, 1997). The economic importance of infection with N. caninum in cattle herds is reportedly attributable to costs associated with abortion (Anderson et al., 1991; Hernandez et al., 2002), increased number of culled cows (Thurmond and Hietala, 1996), and decreased milk production (Thurmond and Hietala, 1997; Hernandez et al., 2001). In herds where congenital infection represents a major route by which infection is maintained within the herd, producers and veterinarians are interested in developing strategies of selective culling and replacement for control and eradication of the disease. A policy of culling N. caninum-infected cattle can reduce the prevalence of infection and, subsequently, abortions due to N. caninum where the endogenous (vertical) transmission is the main mode of infection (French et al., 1999). ELISA is an approved serological test (von Blumroder et al., 2004) that has been used in epidemiological studies to estimate the prevalence of N. caninum infection and to examine the relationship between N. caninum exposure and abortion, milk yield and culling in cattle (Pare et al., 1997; Thurmond and Hietala, 1997; Hernandez et al., 2001, 2002). The reported sensitivity and specificity of the ELISA tests used in these studies were 89–96% and 97–99%, respectively, suggesting that the performance of these tests was adequate for serodiagnosis of N. caninum in cattle. However, results of a previous study in Switzerland (Sager et al., 2001) suggested that the value of serology can be hampered by a temporal instability of anti-N. caninum antibody reactivity status in adult cattle. Similar observations were recently published by Kyaw et al. (2005) who reported
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about seronegativation during pregnancy in N. caninum-infected cows. In the study of Sager et al. (2001), a first serological survey classified 543 (15%) of 3551 cattle as seropositive. In a second survey, cattle were tested again 3–12 months later, and 212 (39%) of the 543 cattle that had tested positive in the first survey were classified as negative in the second survey. Furthermore, 90 (3%) of 3008 cattle that had tested negative in the first survey were classified as positive in the second; mechanisms that could explain the temporal instability of the anti-N. caninum antibody status in adult cattle were not described. It is possible that cattle may have been misclassified as positive or negative to N. caninum because the sensitivity and specificity of the ELISA used was not 100%; cattle with border-line antibody reactions above or below the ELISA cut-off value can be misclassified when tested repeatedly over time; an increase in anti-N. caninum antibody reactivity can develop in naturally infected cows during pregnancy, when reactivation of the parasite and maternal immune response may influence abortion (Pare et al., 1997; Stenlund et al., 1999); an increase in anti-N. caninum antibody reactivity can also develop in non-infected cattle in herds in which N. caninum infection has occurred due to horizontal transmission (Davison et al., 1999). The correct classification of cattle as infected or non-infected with N. caninum is important for a successful control and eradication program. A policy of selective culling of N. caninuminfected cattle could be adversely affected by falsenegative cattle, as such animals would remain in the herd and allow for further spread of the infection by endogenous and exogenous transmission. The objectives of the study reported here were: (1) to follow-up the anti-N. caninum serologic status in dairy cows during pregnancy and (2) to examine the anti-N. caninum serologic status in dairy cows that had an abortion during pregnancy.
2. Materials and methods 2.1. Study population One hundred and forty-three adult cows from 11 dairy farms located in the canton of Bern, Switzerland, were included in the study. All 143 cows were blood
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sampled and ELISA tested in an initial survey and all pregnant cows with complete records and a minimum of three serum samples collected every other month during pregnancy (n = 113) were used to assess objective 1. In addition, blood samples of 10 aborting cows with complete records and insemination, calving and abortion dates were used to investigate objective 2. The herd size ranged from 15 to 52 cows (median, 30 cows). Within-herd seroprevalence of N. caninum ranged from 27 to 93% (median, 40%). The main breeds included Swiss Red-and-White (8 herds), Holstein Frisian (2 herds) and Simmental (1 herd). Ten of the 11 study farms had at least one farm dog with free access to the whole farm. The 11 herds were selected based on their history of N. caninumassociated problems and willingness of herd owners to participate in the study. For the whole study period, abortions occurred only sporadically and temporally distributed over the whole period, thus indicating endogenous transmission. There was no indication for the occurrence of abortion storms (exogenous infection mode), such as found later in another Swiss farm (Sager et al., 2005). 2.2. General approach The approach to the first objective was to follow-up the serologic status of initially N. caninum-seropositive or seronegative cows during pregnancy. Thirty cows initially classified as seropositive and 83 cows classified as seronegative were subsequently blood sampled every other month during pregnancy, until parturition. The approach to the second objective was to follow-up the anti-N. caninum serologic status in seven seronegative and three seropositive cows that experienced an abortion.
2.4. ELISA procedure Serum samples (n = 143) were tested for detection of anti-N. caninum antibodies by using a commercial ELISA test CHEKIT1 at the laboratory of the Institute of Parasitology, University of Bern, Switzerland, following the manufacturers’ instructions (Bommeli Diagnostics, Bern, www.bommeli.com). This commercial test used microtiter plates that were coated with crude antigen lysates of whole tachyzoites. The test and control sera were diluted 1:10 and incubated for 60 min at 37 C. After incubation unbound material was removed from the wells by washing. A peroxidase-labelled anti-bovine IgG conjugate was added and incubated for 60 min at 37 C. Unbound conjugate was removed by washing, and a chromogen-containing substrate was added to the wells. The degree of color developed (absorbance measured at 404 nm, reference wavelength 492 nm) was directly proportional to the amount of anti-N. caninum antibodies present in the sample. Serum samples were tested in triplicates. The A404 nm of triplicates was averaged and the values of both the positive control and the samples were corrected by subtracting the A404 nm of the negative control, as presented below: Positive control : Apositive Anegative Samples : Asample Anegative If the coefficient of variation was 0.10, the most divergent value was ignored and the remaining two values were averaged. If there were no outliers identifiable, all three values were averaged. The results are expressed as percentages using the following formula: Sample value
2.3. Collection of blood samples ¼ Blood samples were collected from study cows by coccygeal venipuncture and transported to the Institute of Parasitology at the University of Bern. In the laboratory, samples were centrifuged within 24 h and serum was harvested and dispensed into plastic vials. Serum aliquots were properly identified and stored frozen at 30 C until testing. Blood samples were collected during May 2003–October 2004.
Asample Anegative Apositive Anegative 100 ðantibody reactivity expressed in %Þ
Cows with serum samples with a value 40 AR% were classified as seropositive. The reported sensitivity and specificity are 97.5% (94.1–100%) and 95.1% (90.5–99.8%), respectively (Bommeli Diagnostics, Bern, www.bommeli.com).
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2.5. Immunoblotting Immunoblotting was used to re-test the sera of the cows that – as tested by ELISA – seroconverted during pregnancy. Immunoblots and respective test interpretation were carried out exactly as described by Schares et al. (1998). 2.6. Pregnancy In the 11 study herds, the herd veterinarian carried out pregnancy diagnosis of cows by palpating per rectum the uterus and its contents approximately 42– 49 days after insemination.
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cows that remained seropositive or seronegative, respectively, during pregnancy was the same, was tested by using the McNemar’s chi-square test. The Friedman test was used to assess the effect of pregnancy phase (first trimester, second trimester and third trimester) on antibody reactivities in seropositive cows. Follow-up between group comparisons were done using the Wilcoxon signed-rank test. Bonferroni corrections were used to correct for multiple testing. For all statistical analyses, a P-value equal to or less than 0.05 was considered significant. Statistical software used were NCSS 2001 (NCSS, Kaysville, UT, USA) and S-PLUS 6.1 Professional (Insightful Corporation, Seattle, USA).
2.7. Abortion 3. Results In the study herds, abortion was determined in cows that had grossly detectable signs of abortion or a return to service after being confirmed pregnant. Seven aborted fetuses (three from seropositive cows and four from seronegative cows, seropositivity/negativity having been assessed at the beginning of the study) were collected and submitted for bacteriological, virological, parasitological and pathohistological examinations at the Institute of Pathology, University of Bern, Switzerland. 2.8. Polymerase chain reaction (PCR) DNA was extracted from the brain of aborted fetuses and subsequently processed for molecular diagnosis by a N. caninum specific PCR, exactly as described by Muller et al. (1996), using the UDG-system and appropriate controls to avoid carry-over contamination and to detect putative inhibition processes. 2.9. Statistical analyses In the present study, seronegative cows that ‘‘positively seroconverted’’ were determined by the occurrence of at least one seropositive result (N. caninum ELISA reaction 40%) during pregnancy. Seropositive cows that ‘‘negatively seroconverted’’ were those animals that tested negative in the N. caninum-ELISA at least once during pregnancy. Among cows classified as persistently seropositive or seronegative, the null hypothesis that the number of
3.1. Follow-up of anti-N. caninum antibody reactivities in dairy cows during pregnancy 3.1.1. ELISA testing One hundred and thirteen cows (30 seropositive and 83 seronegative cows as tested at the beginning of the study) were blood-sampled successfully three to five times (median, four samples) during pregnancy until calving. Among 30 seropositive cows, 26 cows remained seropositive thereafter (Table 1). Four cows had at least one negative serum sample (cutoff = 40%) during pregnancy (Table 1; Fig. 1). Cow 139 and cow 312 tested negative in all five pregnancy samples, whereas cow 60 and cow 202 tested negative only once during their fifth and ninth month of pregnancy, respectively. Among 83 seronegative cows, 82 cows remained persistently seronegative thereafter and one cow (cow 332) tested positive three times during the sixth, eighth and last month of pregnancy. Table 1 Test results of dairy cows investigated for anti-N. caninum antibodies at the beginning of the study and during pregnancy Sampling during pregnancy
Initial sampling Positive Negative Total
Total
Positive
Negative
26 1
4 82
30 83
27
86
113
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Fig. 1. Dynamics of anti-N. caninum antibody reactivity in four seropositive cows nos. (60, 139, 202, 312) and one seronegative cow (#332) that seroconverted during pregnancy. () = cut-off point; (*) cow #60; (&) cow #139; (~) cow #202; (^) cow #312; () cow #332.
3.1.2. Immunoblotting The sera of the cows that – by ELISA – exhibited seroconversion during pregnancy, namely cows 60, 139, 202, 312 and 332 were re-tested by immunoblotting. The positive or negative ELISA results in cows 139, 312 and 332 were confirmed by immunoblot. The two individual negative ELISA results of cows 60 and 202, however, were positive in the immunoblotting. 3.1.3. Statistics section The proportions of cows classified as seropositive before (30/113 = 26.5%) or during pregnancy (28/
Fig. 2. Box plots of anti-N. caninum antibody sample values of 30 seropositive cows in the first, second and last third of pregnancy. Statistically significant difference marked by A and B. Outliers are symbolized by (*).
113 = 24.8%) were not significantly different (P = 0.56). The box plots of the N. caninum sample values of 30 seropositive cows in the first, second and last third of pregnancy are presented in Fig. 2. The influence of the variable ‘‘pregnancy trimester’’ on the ELISA values was highly significant (P = 0.0002, Friedman test). The Wilcoxon test showed that during the third trimester, the values were significantly higher than during the first (P = 0.001) and during the second (P = 0.006) trimester. No significant difference in values was detected between the first and second trimester (P = 0.15).
Table 2 Aborting cows were serologically tested by N. caninum-ELISA and temporal changes of the serostatus (seroconversion) were assessed Cow (#)
Age of fetus (days)
N. caninum-PCR
N. caninum-serology
Seroconversion (ELISA)
5 44 76 110 134 243 166 58 150 162-1c 162-2c
106 49 101 137 279 62 173 165 174 191 200
nd nd nd + nd + + +
b + + + +
+a
Seven of the aborted fetuses were tested for the presence of N. caninum-DNA in the brain by PCR. nd, not done. a Cow #243 started seronegative and subsequently exhibited elevated ELISA-values for N. caninum 8 months after abortion (in the seventh month of a subsequent pregnancy). Immunoblotting revealed seropositivity for all samples of this animal. b The sera from this cow were additionally tested by immunoblotting, which also yielded negative results. Thus, the negative serologic status was confirmed. c Cow #162 had two N. caninum-PCR positive abortions.
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3.2. Abortions Ten out of 143 cows had an abortion during the study period. Two herds recorded one abortion each, two herds recorded two abortions each and one herd had five abortions (one cow in this herd aborted twice). Fetal survival time was 49, 62, 101, 106, 137, 173 and 279 days in seven seronegative cows and 165, 174, 191 and 200 days in three seropositive cows (Table 2). N. caninum-DNA was detected by PCR in the brains of four out of seven fetuses submitted to the laboratory; cow 150 and cow 162 (aborted twice) were classified as positive and cow 166 was classified as negative to anti-N. caninum antibodies. No other infectious agent was identified in the remaining three fetuses (Table 2). 3.3. Follow-up of anti-N. caninum antibody reactivities in aborting cows Five of seven seronegative cows remained seronegative before, during and after abortion (Table 2). One seronegative cow (#243) seroconverted 8 months after abortion (ELISA value, 42%). Finally, another seronegative cow (#166) remained persistently seronegative during pregnancy, but N. caninum-DNA was detected by PCR in the brain of the aborted fetus (Table 2). Among three seropositive cows that aborted, two cows remained seropositive, without relevant changes in antibody reactivities. Cow 162 aborted twice (Table 2). There was an increase in the ELISA A404 nm value from 59% (52 days prior to the first abortion) to 91% (10 days after the first abortion). No increase in the ELISA A404 nm value was observed between the second conception and the second abortion.
4. Discussion The present study was prompted by results of a previous study in Switzerland (Sager et al., 2001) suggesting that the value of serology can be hampered by a temporal instability of anti-N. caninum antibody reactivity in cattle in terms of switching from seropositive to seronegative and vice-versa. We used 30 out of 38 (79%) pregnant, seropositive cows and 83 out of 105 (79%) pregnant, seronegative cows with
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three or more blood samples collected during pregnancy to detect possible changes in the reactivity pattern of anti-N. caninum serum antibodies during pregnancy. ELISA testing demonstrated that among 30 seropositive cows, 26 cows remained seropositive thereafter and 4 cows transiently converted to seronegative. Two cows were positive in the initial sample but tested negative in all five samples collected during pregnancy. These results were qualitatively confirmed by immunoblot examination, which was introduced in the lab after starting the study and which had proved to be superior in terms of sensitivity and specificity (Schares et al., 1998). The third seropositive cow tested negative once during her fifth month of pregnancy, while the fourth cow tested negative once during her last month of pregnancy. Immunoblot findings did not confirm these two negative ELISA results. Anticipating a higher specificity and sensitivity of immunoblotting, we concluded that the onetime negative serum of each cow during pregnancy occurred as false negative results; the reported sensitivity of the ELISA test is 97% (95% confidence interval, 94–100%; Bommeli Diagnostics, Bern, www.bommeli.com). In a previous study, 47 out of 1075 (4%) seropositive cows had tested negative for anti-N. caninum-antibodies in subsequent samples; false positive results or maternal antibodies were identified as possible reasons for misclassification (Dijkstra et al., 2003). Finally, in another study, 250 seropositive cows remained seropositive to anti-N. caninum antibodies when sampled and tested again 6– 12 months after the first sample (Lopez-Gatius et al., 2004). An explanation for the high rate of seroconversion (39%) among seropositive cows observed in a previous study (Sager et al., 2001) might be that cattle with border-line antibody reactivities closely above or below the ELISA cut-off value could have been misclassified. Among 83 seronegative cows, 82 cows remained seronegative thereafter and 1 cow tested positive three times during the sixth, eighth and last month of pregnancy. The positive results of the ELISA testing were confirmed by immunoblot procedure, suggesting that this seroconversion was real. The probability that horizontal transmission was responsible for infection and seroconversion is low, as all other nine seronegative cows in the same herd did not
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seroconvert, and there was no evidence that an abortion storm occurred in the cow herd during the study period. The mentioned cow gave birth to a healthy calf. It is known that acquisition of infection during pregnancy is not necessary for abortion to occur (Pare et al., 1997). In our study, seven initially seronegative cows and three initially seropositive cows had an abortion. Five seronegative cows remained seronegative before and after abortion, suggesting that pathogens other than N. caninum or other reasons may have been implicated as cause of abortion. Another seronegative cow seroconverted 8 months after abortion. Evidence of a N. caninum abortion would have provided additional information about the infection status of the cow; however, the fetus (age, 62 days) was not submitted to the laboratory for respective diagnosis. Thus, abortion could not be associated with N. caninum infection. However, a retrospective testing with immunoblotting revealed that the cow was truly seropositive by this test. Finally, another seronegative cow (seronegativity now confirmed by immunoblotting) that aborted during the last month of pregnancy remained seronegative, but the brain of the fetus tested positive for N. caninum DNA by respective PCR. False positive PCR reactions are very unlikely due to the extreme precautions used in our laboratory, e.g. use of appropriate controls, UDG-system and others to avoid carry-over contamination; the whole procedure was described by Muller et al. (1996). Consequently, this cow was apparently harboring and transmitting the parasite without detectable anti-N. caninum serum antibodies, even after abortion. This observation is in line with earlier experiences where seronegative cows aborted N. caninum-PCR-positive fetuses (Sager et al., 2001). In this study, sera from eight cows with N. caninum-abortion were tested in different laboratories by ELISA and immunoblotting, and the absence of detectable anti-N. caninum-specific antibodies was confirmed in all laboratories. Among the three seropositive cows that aborted, two cows had high and one cow had low anti-N. caninum ELISA values during gestation. Pare et al. (1997) have reported that considering only cows seropositive during pregnancy, no difference in abortion risk was found for cows with low daily S/ P values compared to cows with high S/P values. Cows with high S/P values at 180 and 210 days of gestation,
however, had a lower risk of abortion than cows with low S/P values at those times. A significant increase in anti-N. caninum-specific ELISA values was observed in the last trimester of pregnancy. Field and experimental studies of others have shown that most cattle that transmitted the infection to their progeny had a marked rise in N. caninum-specific antibody reactions during pregnancy (Conrad et al., 1993; Pare et al., 1997; Stenlund et al., 1999; Guy et al., 2001). In contrast, cows that did not vertically transmit, did not have a marked increase in antibodies (Guy et al., 2001). The dams in our study did not abort, but most probably transmitted the parasite. The final proof is lacking as we could not collect diagnostic specimen from the newborn calves. In summary, the N. caninum serologic status in dairy cows only changed in 1 out of 83 seronegative cows with a subsequent positive testing during pregnancy, and in 2 out of 30 seropositive cows with a subsequent negative testing at least once during pregnancy. Veterinarians involved in N. caninum control and eradication efforts in cattle herds need to be aware that misclassification of cattle as infected or non-infected with N. caninum using ELISA protocols is possible, as the sensitivity and specificity of available N. caninum ELISA tests is not 100%. Retesting of cattle with border-line antibody concentrations by immunoblotting can reduce the risk of misclassification and is thus strongly suggested.
Acknowledgements We are very grateful to the farmers for the good compliance in the study. We are indebted to Mrs. U. Ma¨usli and Mr. P. Stuenzi for their technical support. The study was financially supported by the Swiss Federal Veterinary Office and the Swiss Federal Office of Science and Education (grant nos. BBW 00.0498 and BBW C01.0122 in the frame of COST 854).
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