Identification of a Highly Antigenic Region of Subtilisin-Like Serine Protease 1 for Serodiagnosis of Neospora caninum Infection

Neospora caninum is an apicomplexan parasite that causes abortion in cattle; hence, accurate diagnosis of this pathogen is important to the cattle farming industry. Our previous proteomics and immunoscreening analyses revealed that the N. caninum subtilisin-like serine protease 1 (NcSUB1) has potential as a serodiagnostic tool for Neospora. Consequently, we expressed two fragments containing five NcSUB1 tandem repeat copies covering amino acids (aa) 524 to 843 (NcSUB1t) and 555 to 679 (NcSUB1tr) to identify the antigenic regions. The serodiagnostic performances of NcSUB1t and NcSUB1tr were compared with that of N54, which contains a single copy of the repeats (aa 649 to 784), and with the truncated NcSAG1 (NcSAG1t), which lacks a signal peptide and C-terminal hydrophobic regions, as a positive reference. Serum samples from N. caninum experimentally infected cattle and mice and cattle from a farm with confirmed cases of Neospora abortion were tested by enzyme-linked immunosorbent assay (ELISA) with the four antigens. In the N. caninum experimentally infected cattle, the highest IgG1 antibody titers were detected against NcSUB1t, while specific IgG1 antibodies were detectable from 16 days postinfection (dpi), with levels peaking at 36 dpi for all of the antigens. On the other hand, the levels of anti-NcSUB1 IgG2 antibodies were lower than those of anti-SAG1t IgG2 antibodies. The ELISA with NcSUB1t and NcSUB1tr had good sensitivity (94.59 to 95.95%) and specificity (80 to 100%) with bovine serum field samples compared to NcSAG1t and showed no cross-reactions with sera from Toxoplasma gondii experimentally infected mice. Moreover, IgG antibodies against NcSUB1t were detected during parturition in the NcSAG1t antibody-positive cattle, and NcSUB1t-specific antibody transfer was observed from a mother to her calf. Our results show that the NcSUB1 tandem repeat is potentially useful for serodiagnosis of N. caninum.     

Development of Rapid Immunochromatographic Test with Recombinant NcSAG1 for Detection of Antibodies to Neospora caninum in Cattle

An immunochromatographic test (ICT) with recombinant surface antigen 1 of Neospora caninum (NcSAG1) was developed for the rapid detection of antibodies to N. caninum in cattle. The ICT was used to clearly discriminate between immunofluorescent-antibody test (IFAT)-positive bovine sera and IFAT-negative bovine sera. Serum samples collected from cattle in Yanbian, China, were examined by the ICT. Of the 96 serum samples, 23 (24.0%) were positive by the ICT, and 19 (19.8%) samples were positive by a previously developed enzyme-linked immunosorbent assay (ELISA). Eighteen of 19 ELISA-positive samples were positive according to the ICT. A good agreement was found between the results of the ICT and the ELISA. The results presented here suggest that the ICT with recombinant truncated NcSAG1 fused to glutathione S-transferase is a useful and reliable method for the detection of antibodies to N. caninum in cattle.     

A survey of Neospora caninum-associated bovine abortion in large dairy farms of Mashhad, Iran

Neospora caninum is an apicomplexan protozoon causing abortion in cattle worldwide. The present study was designed to assess the importance of this parasite for causing abortion in dairy farms in the Mashhad area of Iran. Of the aborted bovine fetuses, 151 were collected from dairy farms between 2006 and 2008. First, brain samples were examined by polymerase chain reaction (PCR) for the presence of N. caninum DNA, diagnosis was complemented with immunohistochemistry (IHC) and fetal serology (ELISA). Twenty-two (14.5%) of bovine fetuses were considered to be infected with N. caninum with at least one diagnostic technique being positive. PCR yielded 18 (11.9%) positive out of 151 brain samples. Only 52 brain samples were suitable for IHC examination, and N. caninum organism was detected in six (11.5%) of these 52 brain samples. Fetal fluids (n = 151) were assessed with a N. caninum-ELISA, resulting in 15 (9.9%) seropositive fetal fluids samples. In the present study, a good agreement was observed between PCR and ELISA, and a fair agreement between PCR and IHC. The results indicated that abortion due to N. caninum infection is prevalent among large-size dairy farms in the Mashhad area of Iran, and that different complementary diagnostic techniques should be used to increase the chance to detect N. caninum.     

Neospora caninum immunoblotting improves serodiagnosisof bovine neosporosis

Neospora caninum ranges among the major causes of infectious abortion in cattle worldwide. The present study was designed to improve the serodiagnostic tools by complementing a conventional ELISA with a highly sensitive and species-specific N. caninum immunoblot. To evaluate this test combination, sera from several groups of cows were tested. The first group, consisting of experimentally infected calves, showed that immunoblot antibody reactivities were detectable 1 to 3 days earlier than those found in ELISA. The first immunodominant bands that appeared were a 29-kDa (NcSAG1) and a 36-kDa (NcSRS2) antigen. Other groups, based upon naturally infected cattle, were used to compare the diagnostic sensitivity of ELISA and immunoblotting. Overall, N. caninum immunoblotting exhibited a higher sensitivity (98%) than ELISA (87%). Conversely, immunoblotting also confirm in two other cases, true transient negativation in some animals. In general, banding patterns and band staining intensity correlated to the semiquantitative ELISA findings. On the other hand, the banding pattern could not be used to discriminate between sera from animals with a recent abortion and those of cows with latent N. caninum infection. We also addressed putative cross-reactions due to infection with Toxoplasma gondii. Sera from animals with a serologically proven T. gondii infection were either clearly negative by Neospora immunoblotting or they yielded a specific immunoblot antibody profile indicating a double infection with N. caninum. Sera from animals with positive findings in both Toxoplasma and Neospora ELISA thus provided dichotomic results in the immunoblot by allowing to confirm or to rule out the specificity of the antibody reaction in Neospora ELISA. Altogether, our findings demonstrate that N. caninum immunoblotting is a very sensitive and specific complementary tool to improve the serology for N. caninum infections in cattle.     

Development of rapid immunochromatographic test with recombinant NcSAG1 for detection of antibodies to Neospora caninum in cattle

An immunochromatographic test (ICT) with recombinant surface antigen 1 of Neospora caninum (NcSAG1) was developed for the rapid detection of antibodies to N. caninum in cattle. The ICT was used to clearly discriminate between immunofluorescent-antibody test (IFAT)-positive bovine sera and IFAT-negative bovine sera. Serum samples collected from cattle in Yanbian, China, were examined by the ICT. Of the 96 serum samples, 23 (24.0%) were positive by the ICT, and 19 (19.8%) samples were positive by a previously developed enzyme-linked immunosorbent assay (ELISA). Eighteen of 19 ELISA-positive samples were positive according to the ICT. A good agreement was found between the results of the ICT and the ELISA. The results presented here suggest that the ICT with recombinant truncated NcSAG1 fused to glutathione S-transferase is a useful and reliable method for the detection of antibodies to N. caninum in cattle.     

Survey of <Emphasis Type="Italic">Neospora caninum</Emphasis> and bovine herpes virus 1 coinfection in cattle

A seroprevalence survey of Neospora caninum and bovine herpesvirus 1 (BHV-1) was conducted in cattle pasturing in an area of the southern Italian Apennines to investigate the coinfection of these two pathogens. Blood samples were collected from 948 pastured cattle raised on 81 farms. Sera were tested for antibodies to N. caninum and to BHV-1 using an ELISA assay and a neutralization test, respectively. Out of the 81 farms sampled, 63 (77.8%) were positive for N. caninum and 80 (98.8%) for BHV-1. Coinfection was found in 62 (76.5%) farms. Out of the 948 bovine sera samples, 303 (32.0%) had antibodies to N. caninum and 735 (77.5%) to BHV-1. The copresence of antibodies to N. caninum and BHV-1 was found in 256 (27.0%) cattle. The logistic regression results indicated that seropositivity for BHV-1 was a risk factor for N. caninum seropositivity and seropositivity for N. caninum was a risk factor for BHV-1 seropositivity.     

Evaluation of bovine abortion associated with <Emphasis Type="Italic">Neospora caninum</Emphasis> by different diagnostic techniques in Mashhad,Iran

Twelve aborted foetuses (gestational ranged from 4–9 months) and dams from dairies cattle farms in (Mashhad) Iran were analysed to investigate the participation of Neospora caninum in abortion. Diagnosis of the infection was determined by histopathology, serology (indirect fluorescent antibody test [IFAT]) and semi-nested polymerase chain reaction (PCR). A total 33% of bovine foetuses were considered to be infected by PCR technique. Microscopic lesions consistent with N. caninum infection in foetal brains were observed in 25% of the samples, whereas 33% were positive using IFAT (with a cut-off titre of 1:20). This study confirms the importance of N. caninum as an important cause of abortion in Iran.     

Identification of vaccine candidate peptides in the NcSRS2 surface protein of Neospora caninum by using CD4+ cytotoxic T lymphocytes and gamma interferon-secreting T lymphocytes of infected holstein cattle

Previously, our laboratory showed that Holstein cattle experimentally infected with Neospora caninum develop parasite-specific CD4+ cytotoxic T lymphocytes (CTL) that lyse infected, autologous target cells through a perforin-granzyme pathway. To identify specific parasite antigens inducing bovine CTL and helper T-lymphocyte responses for vaccine development against bovine neosporosis, the tachyzoite major surface proteins NcSAG1 and NcSRS2 were targeted. In whole tachyzoite antigen-expanded bovine T-lymphocyte lines, recombinant NcSRS2 induced potent memory CD4+- and CD8+-T-lymphocyte activation, as indicated by proliferation and gamma interferon (IFN-gamma) secretion, while recombinant NcSAG1 induced a minimal memory response. Subsequently, T-lymphocyte epitope-bearing peptides of NcSRS2 were mapped by using overlapping peptides covering the entire NcSRS2 sequence. Four experimentally infected cattle with six different major histocompatibility complex (MHC) class II haplotypes were the source of immune cells used to identify NcSRS2 peptides presented by Holstein MHC haplotypes. NcSRS2 peptides were mapped by using IFN-gamma secretion by rNcSRS2-stimulated, short-term T-lymphocyte cell lines, IFN-gamma enzyme-linked immunospot (ELISPOT) assay with peripheral blood mononuclear cells, and 51Cr release cytotoxicity assay of rNcSRS2-stimulated effector cells. Four N. caninum-infected Holstein cattle developed NcSRS2 peptide-specific T lymphocytes detected ex vivo in peripheral blood by IFN-gamma ELISPOT and in vitro by measuring T-lymphocyte IFN-gamma production and cytotoxicity. An immunodominant region of NcSRS2 spanning amino acids 133 to 155 was recognized by CD4+ T lymphocytes from the four cattle. These findings support investigation of subunit N. caninum vaccines incorporating NcSRS2 gene sequences or peptides for induction of NcSRS2 peptide-specific CTL and IFN-gamma-secreting T lymphocytes in cattle with varied MHC genotypes.     

Neospora caninum surface antigen (p40) is a potential diagnostic marker for cattle neosporosis

Neospora caninum is an intracellular protozoan that infects domestic and wild canids as well as many warm-blooded animals as shown by the isolation of viable parasites. The effectiveness of diagnostic tests for detecting specific antibodies against N. caninum is hampered by potential cross-reaction with other Coccidia. So, there is currently an urgent need for a sensitive and specific diagnostic assay for detecting N. caninum in animals. The N. caninum 40-kD surface antigen (p40), similar to NcSAG1 and NcSRS2, was shown to belong to surface antigen super family and thus represents an excellent marker for the diagnosis of neosporosis. In order to test the hypothesis, recombinant Ncp40 (rNcp40) was expressed in Escherichia coli, and an indirect ELISA test was developed using recombinant NCp40 antigen for N. caninum serodiagnosis. The antigen used in this study did not have cross-reactivity with anti-Toxoplasma gondii serum. Anti-p40 antibodies were detected by ELISA in the sera of Yellow cattle and were compared with (IFAT). Optimal sensitivity and specificity (98.2 and 98.6 %) were identified by IFAT. Additionally, 37 positive sera of T. gondii were detected and there was no significant difference with the negative serum of N. caninum. The rNcp40 ELISA developed here provides a specific and sensitive assay for detecting neosporosis in cattle.     

Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by <Emphasis Type="Italic">Neospora caninum</Emphasis> tachyzoites

Neospora caninum negatively impacts bovine reproductive performance around the world. Addressing this problem requires a greater understanding of the parasite’s molecular biology. In this study, monoclonal antibodies against recombinant proteins were successfully developed and employed to characterise two different proteins of N. caninum: the acute phase-associated NcGRA7 and the chronic phase-associated NcSAG4. Immunofluorescence with the anti-rNcGRA7 monoclonal antibody suggested that NcGRA7 trafficks from tachyzoite dense granules to the matrix of the parasitophorous vacuole and parasite’s surroundings. Furthermore, NcGRA7 is also expressed in the bradyzoite stage and localised on the matrix of bradyzoite-positive vacuoles. NcGRA7 appears to be partially involved in the tachyzoite-invasion mechanisms, as an anti-rNcGRA7 monoclonal antibody partially inhibited in vitro tachyzoite-invasion. A monoclonal antibody specific for NcSAG4 confirmed this protein’s bradyzoitespecific expression both by western blot and immunofluorescence. However, some bradyzoite-positive vacuoles only weakly expressed NcSAG4, if it was expressed at all. The specificity of the anti-rNcSAG4 monoclonal antibody was confirmed by the recognition of the NcSAG4 in the membrane surface of Nc-1SAG4^c transgenic tachyzoites, which constitutively expresses NcSAG4. Blocking NcSAG4 of Nc-1SAG4^c tachyzoites with the monoclonal antibody did not affect host cell invasion. However, its implication on the host cell adhesion or host immune evasion should not be discarded.     

Sensitive and Specific Identification of Neospora caninum Infection of Cattle Based on Detection of Serum Antibodies to Recombinant Ncp29

Neosporosis is an economically important disease of dairy cattle caused by the protozoan Neospora caninum. Diagnostic tests for neosporosis are complicated by the potential for cross-reaction of antibodies to antigens that are similar between N. caninum and closely related parasites Toxoplasma gondii and Sarcocystis cruzi. To provide a sensitive and specific assay for detecting antibodies to N. caninum in the serum of infected animals, we have investigated a recombinant form of the antigen known as Ncp29 (rNcp29), which is a major surface protein of the parasite. Ncp29 is encoded by a gene that is homologous to the SAG1 gene previously characterized from T. gondii. An enzyme-linked immunosorbent assay (ELISA) was used to screen animals for the presence of serum antibodies specific to rNcp29. The rNcp29 ELISA readily distinguished between cattle known to be infected with N. caninum (optical density [OD] > 1.2 at 1:500 or greater dilution) and negative controls (OD < 0.5 at 1:500). Additionally, sera from animals that were infected with T. gondii or S. cruzi were negative. The rNcp29 ELISA developed here provides a specific and sensitive assay for detecting neosporosis in cattle.     

Comparative study of protective activities of Neospora caninum bradyzoite antigens, NcBAG1, NcBSR4, NcMAG1, and NcSAG4, in a mouse model of acute parasitic infection

Neospora caninum is an obligate intracellular protozoan parasite that causes severe neuromuscular diseases, repeated abortion, stillbirth, and congenital infection in livestock and companion animals. The development of an effective vaccine against neosporosis in cattle is an important issue due to the significant worldwide economic impact of this disease. We evaluated the immunogenicity of four bradyzoite antigens, NcBAG1 (first described in this study), NcBSR4, NcMAG1, and NcSAG4, using an acute infection mouse model to determine synergistic effects with the tachyzoite antigen as a candidate for vaccine production. Mice were inoculated with the recombinant vaccines (r-)NcBAG1, rNcBSR4, rNcMAG1, rNcSAG4, or phosphate-buffered saline (PBS) (adjuvant control group) in an oil-in-water emulsion with bitter gourd extract, a Th1 immune stimulator, or PBS alone as the infection control group. Mice inoculated with each vaccine developed antigen-specific IgG1 and IgG2a antibodies and isolated splenocytes from mice produced high levels of interferon-γ when infected with the N. caninum tachyzoite. The mice inoculated with rNcBAG1, rNcMAG1, or rNcSAG4 developed slight to moderate clinical symptoms but did not succumb to infection. In contrast, rNcBSR4 and both control groups developed severe disease and some mice required euthanasia. The parasitic burden in the brain tissues of vaccinated mice was assessed by N. caninum-specific real-time PCR at 5 weeks after infection. The parasite load in rNcBAG1-, rNcMAG1-, and rNcSAG4-inoculated mice was significantly lower than that in adjuvant and infection control mice. Therefore, these antigens may be useful for the production of a N. caninum-specific vaccination protocol.     

Isolation and biological characterisation of a new isolate of Neospora caninum from an asymptomatic calf in Brazil

Neospora caninum is a tissue-cyst forming parasite that has been recognized worldwide as a cause of abortion in cattle. Despite the ubiquitous distribution of this parasite and its broad range of hosts, the number of N. caninum isolates obtained to date is limited. In addition, the majority of isolates have been obtained from clinically affected hosts, therefore potentially biasing this population towards more virulent isolates. This report describes the isolation and biological characterisation of a new N. caninum isolate, Nc-Goiás 1, obtained from an asymptomatic, naturally infected calf from Brazil. This new isolate was identified as a member of the N. caninum species by polymerase chain reaction (PCR) using specific primers based on the N. caninum internal transcribed spacer 1 (ITS-1) sequence, and was genetically identified at multiple loci using microsatellite analysis. Finally, a pathogenicity study was conducted in a BALB/c mice model. All Nc-Goiás 1-infected mice survived without exhibiting any clinical signs. Further pathogenic characterisation of this isolate suggested that Nc-Goiás 1 is less virulent than other N. caninum isolates (Nc-Liv and Nc-1) studied in this mouse model. This is the first report of the isolation and biological characterisation of N. caninum from an infected but clinically healthy calf in South America.     

Prevalence of Neospora caninum infection in Sardinian dairy farms (Italy) detected by iscom ELISA on tank bulk milk

Neospora caninum is a heteroxenous cyst-forming coccidian closely related to Toxoplasma gondii and is considered one of the major causes of abortions in cattle worldwide. The present work aims to update the epidemiological trend of N. caninum of dairy cattle in Sardinia island, Western Mediterranean (Italy). For this reason, we used the newest enzyme-linked immunoassay (ELISA) methodology that exploits immune-stimulating complexes (iscoms) principle and allows us to point out the infection in the tank bulk milk too, besides the individual cattle. A total of 624 herds were sampled and tank bulk milk was submitted to iscom ELISA test. The analysis of the tank bulk milk samples revealed a total farm prevalence of 55% for N. caninum in Sardinia. In the provinces of Oristano and Cagliari the prevalences (64 and 65%, respectively) were significantly higher (p<0.01) than in Sassari and Nuoro (41 and 40%, respectively). The iscom Elisa test applied on tank bulk milk seems to be helpful and cost-effective for large epidemiological surveys, for monitoring control strategy plans for N. caninum, and for increasing the bio-safety level in dairy cattle farms.     

Seroprevalence of Neospora caninum infection in dairy cows in Northern provinces,Thailand

Neospora caninum, an obligate intracellular protozoan parasite, is the causative agent of neosporosis, recognized as a major cause of bovine abortion around the world. Thailand is a developing agricultural country located in Southeast Asia. Livestock developments particularly in dairy cows of this country have been hampered by low productivity including milk and slow growth rate due to the impact of many pathogens including N. caninum. Currently, there is no effective method for control of neosporosis since there is less information regarding current status of infections. The objective of this study was to investigate the seroprevalence of neosporosis in dairy cows of the northern part of Thailand. During 2006–2007, the sera of 642 cows from 42 small farm holders with the top three highest consensus of dairy farms in the northern provinces, such as Chiang Rai, Chiang Mai and Lumpang were collected and performed tests. Antibodies to N. caninum were assayed by enzyme-linked immunosorbent assay (ELISA) with recombinant N. caninum surface antigen 1 (NcSAG1) and indirect fluorescent antibody test (IFAT). The overall prevalence of N. caninum infection in this study was 46.9% (301/642) by ELISA and 34.3% (220/642) by IFAT.     

Epidemiology of neosporosis in dairy cattle in Galicia (NW Spain)

This comprehensive study of neosporosis in dairy cattle in Galicia (NW Spain) included: (1) a comparative study of three serological techniques for detection of Neospora caninum antibodies (direct agglutination, enzyme-linked immunosorbent assay and indirect immunofluorescence); (2) a cross-sectional serological survey in which 276 herds and 5,196 animals were tested; (3) a study of N. caninum antibody dynamics; (4) the isolation of viable tachyzoites of N. caninum. Data were analysed to determine the risk factors associated with the infection. A total of 219 herds (79.3%) and 816 heads of cattle (15.7%) were found to be seropositive. Seropositivity was higher on farms with dogs than on farms without dogs, and there was a negative correlation between the size of the herds and seroprevalence. Co-infection with Toxoplasma gondii increased the risk of seropositivity. Cows infected with N. caninum were 5.3 times more likely to abort than non-infected cows. The dynamics study showed an increase in anti-N. caninum antibody titres during the third trimester of pregnancy. Viable tachyzoites were isolated from brain samples. These results indicate that the economic impact of N. caninum is high in Galicia, and therefore, the inclusion of control measures for neosporosis in the official control health programmes is strongly recommended.     

Specific IgG4 response directed against the 45-kDa glycoprotein in trichinellosis: a re-evaluation of patients 15 years after infection

The aim of this study was to evaluate the humoral immune response in late human trichinellosis with particular attention to the presence of IgG4 antibodies directed against the Trichinella-45-kDa glycoprotein (gp). This study re-evaluates subjects 15 years after they were involved in a trichinellosis outbreak that occurred in Central Italy following the consumption of raw boar meat infected with Trichinella britovi. The results show that ELISA tests using the E/S antigen identified five IgM- and eight IgG-positive patients and no IgA-positive patients. Tests using immunoblot (IB) with E/S antigens identified three IgM-, five IgA-, seven- IgG1- and three IgG4-positive sera. When the purified 45-kDa gp was used as an antigen, the IB revealed that six of the ten sera tested were positive for IgG4. Sera were also evaluated with a commercial kit, revealing that 11 of 12 patients had a highly sensitive reactivity against Trichinella proteins (64 and 44–43 kDa). In conclusion, humoral immune response against Trichinella is still present in these patients 15 years after the initial infection, including an IgG4 response directed to the 45-kDa gp.     

Delivery of Neospora caninum surface protein, NcSRS2 (Nc-p43), to mouse using recombinant vaccinia virus

In order to develop a vaccine against Neospora caninum in dogs and cattle, we constructed a recombinant vaccinia virus expressing the N. caninum surface protein, NcSRS2 (Nc-p43). Monoclonal antibodies to NcSRS2 and anti-N. caninum tachyzoite mouse serum recognized the NcSRS2 expressed by the recombinant vaccinia virus. In addition, recombinant NcSRS2 was transported to the cell surface. Mice infected with the recombinant virus predominantly produced IgG1 antibody (Ab) to N. caninum, rather than producing IgG2a Ab. Moreover, splenocytes from mice infected with the recombinant virus proliferated in the presence of the N. caninum antigen. Mice immunized with the recombinant virus gave rise to humoral and cellular immune responses to N. caninum tachyzoites. This study showed that a recombinant vaccinia virus expressing NcSRS2 might be useful for the production of a live vaccine against N. caninum infection. Received: 24 March 2000 / Accepted: 18 May 2000     

Precolostral serology in calves born from Neospora-seropositive mothers

The present study was designed to exploratively determine (a) how many healthy calves, born from seropositive mothers, were also precolostrally seropositive; (b) how many precolostrally negative calves became postcolostrally positive; and (c) in these calves, how the IgG1/IgG2 situation developed pre- and postcolostrally. All calves were born from mothers that were determined to be seropositive in a conventional Neospora caninum-ELISA and by immunoblotting. When the diagnostic performance of the conventional ELISA was compared with that of immunoblotting and an IgG1/IgG2-ELISA in the calves, the latter two exhibited a higher sensitivity: From a total of 15 precolostral calf sera, 7 were positive in the conventional ELISA (diagnostic sensitivity 47%) compared to 15 that were positive by immunoblotting (diagnostic sensitivity 100%) and 12 that were positive by the IgG1/IgG2-ELISA (diagnostic sensitivity 80%). With regard to IgG1/IgG2 findings in the dams, IgG2 appeared as the dominant subclass in the humoral immune response of adult cattle, while in calves, IgG1 appeared as the main prenatally/precolostrally reactive antibody isotype. Provided that precolostral seropositivity reflects postnatal persistent infection with N. caninum, we then postulate that, basically, all of our study calves born form N. caninum-seropositive mothers were prenatally infected with the parasite, and may, thus, all become members of the next transmitting generation.