Scientific
Seroprevalence of Neospora caninum infection following an abortion outbreak in a dairy cattle herd RA ATKINSONa, RW COOKb, LA REDDACLIFFc, J ROTHWELLcd, KW BROADYe, PAW HARPERf and JT ELLISag
Objective To investigate the seroprevalence of Neospora caninum infection in a commercial dairy cattle herd, 15 months after detection of an abortion outbreak. Procedure Sera from the whole herd (n=266) were examined for N caninum antibodies by indirect fluorescent antibody test (IFAT) and immunoblot analysis. Herd records were reviewed to collate serological results with abortion history, proximity to calving, and pedigree data. Results The seroprevalence of N caninum infection was 24% (63/266) for IFAT titre ≥ 160, 29% (78/266) for immunoblot positive (+ve), and 31% (82/266) for IFAT ≥ 160 and/or immunoblot +ve; 94% (59/63) of animals with IFAT ≥ 160 were immunoblot +ve. The association between seropositivity (IFAT ≥ 160 and/or immunoblot +ve) and history of abortion was highly significant (P < 0.001); the seroprevalence was 86% (18/21) in aborting cows, compared with 30% (50/164) in non-aborting animals. The abortion rate for seropositive cows was 26% (18/68) compared with 3% (3/117) for seronegative animals. IFAT titres of infected cows were higher within 2 months of calving than at other times (P < 0.001). The association between seropositivity in dams and daughters was highly significant (P = 0.009). Conclusions The abortions were associated with N caninum infection and there was evidence of reactivation of latent infection close to calving and congenital transmission of infection. Immunodominant antigens identified by immunoblots may prove useful for improved diagnostic tests. Aust Vet J 2000;78:262-266 Key words: Neospora caninum, cattle, abortion, immunoblot, Western blot, indirect fluorescent antibody test, immunodominant antigen.
N
eosporosis is a major cause of bovine abortion in Australia, New Zealand, USA, UK and several other European countries.1-4 The aetiological agent, Neospora caninum, is closely related to the apicomplexan protozoan Toxoplasma gondii but they can be distinguished by their ultrastructural, antigenic and genetic properties.5-9 Bovine and canine isolates of N caninum10-13 are considered to be identical organisms on the basis of their shared antigenic and genetic characteristics.9,13,14 The dog has recently been identified as a aMolecular Parasitology Unit, University of Technology, Sydney, Westbourne
definitive host for N caninum,15 but congenital infection appears to be the major mode of natural transmission in cattle.16-19 Serological tests used to diagnose N caninum infection in cattle include the IFAT,20,21 immunoblot analysis,22 ELISA21,23 and recombinant-ELISA.24,25 In Australia no sero-epidemiological information on bovine neosporosis has been published. The aim of this study was to investigate the seroprevalence of N caninum infection following an abortion outbreak in a commercial dairy cattle herd in New South Wales.
Materials and methods Herd history and initial laboratory investigation The study herd of Friesian cattle was located on the south coast of New South Wales. Breeding was non-seasonal and by artificial insemination, with about 140 cows calving annually. An abortion outbreak was first detected in December 1993 and continued through 1994, with sporadic abortions occurring in subsequent years to December 1998. Specimens (including whole foetuses, selected fresh and formalin-fixed tissues, and maternal blood samples) from 12 cows that aborted from 1993 to 1995, were submitted to EMAI for routine diagnostic testing, which included gross and histological examination, serological testing for leptospirosis and brucellosis, immunoglobin estimation in foetal fluid, bacteriological culture and pestivirus examination. Details of herd history, including abortions, were obtained by interview with the farmer and by examination of computerised herd records. Collection of sera In March 1995, sera were collected from all 266 animals (including heifers and calves) in the herd. Presumptive negative control sera were collected from a herd with no history of Neospora abortion and maintained under quarantine at EMAI. Indirect fluorescent antibody test (IFAT) The test procedure was performed as previously described,20 using teflon-masked, 12-well Neospora caninum NC-1 antigen slides (VMRD Inc Pullman, WA, USA), and fluorescein-conjugated, affinity-purified rabbit anti-bovine IgG (Jackson ImmunoResearch Laboratories, Inc, PA, USA).
Street, Gore Hill, New South Wales 2065 bNSW Agriculture, Regional Veterinary Laboratory, Wollongbar, New South
Wales 2477 cNSW Agriculture, Elizabeth Macarthur Agricultural Institute, Camden, New South Wales 2570 dPresent address: Elanco Animal Health, 112 Wharf Road, West Ryde, NSW 2114 eImmunobiology Unit, University of Technology, Sydney, Westbourne Street, Gore Hill, New South Wales 2065 fNSW Agriculture, Grafton Agricultural Research and Advisory Station, Grafton, New South Wales 2460 gAuthor for correspondence:
[email protected]
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BMU ELISA EMAI IDA IFAT RT SDS-PAGE
BioRad multiscreen unit Enzyme-linked immunosorbent assay Elizabeth Macarthur Agricultural Institute Immunodominant antigen Indirect fluorescent antibody test Room temperature Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
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Scientific Immunoblot analysis Parasite propagation - Neospora caninum (NC-Liverpool, a canine isolate from England, and NC-SweB1, a bovine isolate from Sweden) and Toxoplasma gondii (RH strain) were propagated in vitro following standard procedures.13,26 Every 3 to 6 days cultures were passaged by the addition of a 1/10 dilution of infected monolayer suspension to a fresh flask containing a 24hour culture of Vero cells. Antigen preparation – Cultures were harvested when more than 60% of monolayer cells appeared infected with schizonts and groups of dividing tachyzoites. The resulting suspension was passed several times through 23- and 26-gauge hypodermic needles to aid the disruption of host cells, and then filtered through a 3 µm membrane (Nucleopore). The tachyzoites were counted on a haemocytometer, centrifuged for 10 min at 3000 g and snap-frozen in liquid nitrogen as pellets. When required, pellets were thawed and boiled in SDS-PAGE buffer.27 Insoluble material was separated by centrifugation for 10 min at 10,000 g and the supernatant containing the soluble fraction was aliquoted, and stored at -80°C until required. Gel electrophoresis – Aliquots of antigen were thawed, boiled for 3 to 4 min and applied non-reduced at either 1 µg protein per lane in standard gels or 20 µg protein per slab in preparative gels. Antigen was resolved on 5% stacking and 12% separating gels using a BioRad mini-gel apparatus set at 200 V for 45 min at RT. Western transfer – Antigens separated on SDS-PAGE were transferred onto 0.45 µm PVDF membranes (Amersham) following standard procedures using a Tris/glycine buffer28 and running conditions were standardised to 250 mA constant for 4 h at RT29 for analysis of the herd sera. All other transfers were conducted at RT for 45 min at 50 V. Immunoscreening – Non-specific antibody binding was eliminated by incubation of membranes in Tris-buffered saline supplemented with 5% skim milk powder either overnight at 4°C, or for 1 h at RT. Membranes containing NC-Liverpool antigen were placed in the BMU, and isolated strips were incubated for 45 min at RT with a negative control serum (EMAI), a positive control serum and test sera from the herd, diluted 1/50 or 1/100. Membranes were then washed in Tris-buffered saline-Tween and further incubated for 45 min at RT in a 1/500 dilution of anti-bovine IgG conjugated to alkaline phosphatase (Sigma). Washing was repeated and membranes were placed in the developing solution of nitroblue tetrazoleum and 5-bromo4-chloro-3-indolyl-phosphate (Sigma) for 20 min at RT. Specificity of the immunoblot analysis was tested by placing membranes from a standard gel containing antigens from N caninum (NC-Liverpool and NC-SweB1) and T gondii (RH strain) in the BMU, and incubating isolated strips as above with serum from calves experimentally infected with N caninum (NC-1) and T gondii (RH strain). Analysis of serology results in relation to abortion history, proximity to calving, and dam/daughter pedigree data For the purpose of these analyses, animals were regarded as seropositive if either their IFAT titres were ≥ 160 or their immunoblot results were positive. Cows more than 12 months of age at the time of sampling (March 1995) were classified as either having aborted or not (up to end of 1996). These same cows were classified as being close to calving if they had calved within 2 months either side of the date of sampling; otherwise they were classified as remote (≥ 2 months) from calving. For
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pedigree analysis the serological status of dams, and of daughters, greater than 12 months of age at the time of sampling, were compared for all available dam/daughter pairs, and for a subgroup of these pairs in which the daughters were born before detection of the abortion outbreak in December 1993. The data were analysed using the statistical facilities in EpiInfo 6.1 (2 x 2 tables for abortion and pedigree data, and the means command for proximity to time of calving).
Results Neosporosis was the only cause of abortion identified in the study herd. Diagnosis was based on the detection of characteristic histological lesions of multifocal necrogranulomatous encephalitis, usually associated with a nonsuppurative myocarditis,2 in eight of 12 aborted foetuses examined, and by the exclusion of other causes of bovine abortion. Comparison of the IFAT and immunoblot serological results (Table 1) suggested that an IFAT titre of 160 was a reasonable cut-off value for the presence of Neospora-specific antibody; 94% (59/63) of animals with IFAT titres ≥ 160 were immunoblot +ve, In contrast, for IFAT titres 80, 40 and < 40, the percentage immunoblot +ve decreased to 11%, 15% and 7%, respectively. The seroprevalence of Neospora infection was 24% (63/266) for IFAT ≥ 160, 29% (78/266) for immunoblot +ve, and 31% (82/266) for IFAT ≥ 160 and/or immunoblot +ve. Immunodominant antigens of Neospora caninum and Toxoplasma gondii The immunoblot antigen profiles of N caninum (NCLiverpool and NC-SweB1) and T gondii (RH) screened with sera from calves experimentally infected with either N caninum (NC-1) or T gondii (RH) and with the negative control serum, are shown in Figure 1. Identical IDAs of canine and bovine Neospora isolates were detected by anti-NC-1 serum at positions corresponding to apparent molecular weights of 45, 37, 30-36, 29/30 and 16/17 kDa. The IDAs of T gondii were detected only by anti-RH serum, at 36 kDa and at 28-32 kDa. The failure of anti-NC-1 serum to react with T gondii antigens and of antiRH serum to react with NC-Liverpool or NC-SweB1 antigens demonstrated that there was no cross-reactivity between N caninum and T gondii in this assay. The negative control serum did not react with any of the antigens. A representative immunoblot of Neospora-specific IDAs detected by herd sera of
Table 1. Neospora seroreactivity of dairy cattle by indirect fluorescent antibody test (IFAT) and immunoblot. IFAT titre
≥ 1280
Immunoblot positive reactions Total reactions % (no. positive/no. tested)
Excluding weak reactions % (no. positive/no. tested)
100 (14/14)
100 (14/14)
640
93 (13/14)
93 (13/14)
320
100 (18/18)
100 (18/18)
160
82 (14/17)
59 (10/17)
80
11 (4/35)
3 (1/35)
40
15 (7/48)
2 (1/48)
< 40
7 (8/120)
3 (4/120)
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different IFAT titres is shown in Figure 2. The Neospora-specific IDAs were consistently detected by sera with IFAT titres ≥ 160, but at lower titres the immunoblot banding pattern was incomplete and inconsistent between samples. Sera with the highest IFAT titres had the strongest reactivity with Neospora-specific IDAs. Association between Neospora seropositivity and abortion history Among 185 cows that could be classified as aborting or nonaborting, the association between Neospora seropositivity (IFAT ≥ 160 and/or immunoblot +ve) and history of abortion was highly significant (Yates corrected chi square P < 0.001); the seroprevalence of Neospora infection was 86% (18/21) in aborting cows, compared with 30% (50/164) in non-aborting animals. The abortion rate for seropositive cows was 26% (18/68) compared with only 3% (3/117) for seronegative cows. Association between Neospora IFAT titre and proximity to calving in Neospora infected cows There were 51 cows older than 12 months of age, with IFAT titres ≥ 160, and the association of higher titres with proximity to calving was highly significant (P < 0.001) (Table 2). Association between Neospora seropositivity in dams and daughters Among 86 dam/daughter pairs identified in the study, the association between seropositivity in dams and their daughters was highly significant (Yates corrected chi square P = 0.009); 53% (20/38) of seropositive and 23% (11/48) of seronegative daughters, had seropositive dams. This association of dam/daughter seropositivity remained significant (P = 0.023) when only the 72 dam/daughter pairs born before the observed abortion outbreak were considered; 48% (16/33) of seropositive and 20% (8/39) of seronegative daughters had seropositive dams.
Figure 1. Immunoblot analyses of N caninum (NC-Liverpool, lanes 1-3; NC-SweB1, lanes 4-6) and T gondii (RH, lanes 7-9) with bovine anti- N caninum (lanes 1, 4 and 7), anti-T gondii (lanes 2, 5 and 8) and negative control (lanes 3, 6 and 9) sera. Protein molecular weights in kilodaltons (kDa) are shown on the left.There is no cross reactivity between N caninum and T gondii.
Figure 2. Immunoblot analyses of N caninum (NC-Liverpool) with 19 sera from the study herd. Protein molecular weights in kilodaltons (kDa) are shown on the left. Lane (IFAT titre): a (80); d,o (160); b,c,m,p,q,r,s (320); f,n (640); e,g,h,i,j,k,l (≥ 1280); t (negative control serum). Immunoblot reactivity increases with IFAT titre.
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Scientific Table 2. Neospora IFAT titres and proximity to calving. IFAT titre
Number of cows Months from calving
≥ 1280
≥2
