Molecular comparison of the dense granule proteins GRA6 and GRA7 of Neospora hughesi and Neospora caninum

Neospora hughesi is a recently described apicomplexan parasite that has been associated with several cases of equine protozoal myeloencephalitis. The biology of this new parasite is just beginning to be defined. Towards this understanding, we report important differences between the nucleotide and deduced amino acid sequences of the dense granule proteins GRA6 and GRA7 of N. hughesi and Neospora caninum. This information can be used to differentiate the two species and contribute to further understanding of the prevalence and biology of N. hughesi. The newly defined proteins of N. hughesi are referred to as NhGRA6 and NhGRA7 in keeping with the protocol for naming homologous proteins of the Apicomplexa. Genes of the two dense granule proteins of N. hughesi (isolate Nh-A1) and four different isolates of N. caninum were isolated via PCR and their DNA sequences were determined. Computer analysis indicated that the two gene sequences were identical among all four N. caninum isolates. However, the gene for NhGRA6 was found to be 96 nucleotides longer at the 3' end than that of NcGRA6, resulting in a protein product that is 32 amino acids larger than NcGRA6. Two tandem repeat sequences were identified at the 3' end of the NhGRA6 gene. These repeat sequences contributed to the lengthening of the carboxy terminus of NhGRA6 in comparison with that of NcGRA6. The larger size of NhGRA6 was further confirmed by Western blot analysis in which NcGRA6 monospecific antibodies recognised a protein of approximately 42 kDa in N. hughesi whole tachyzoite preparation but a protein of 37 kDa in N. caninum whole tachyzoite preparation. Analysis of GRA7 gene sequences indicated a 6 and 14.8% difference at nucleotide and amino acid sequence level, respectively, between NcGRA7 and NhGRA7. Despite the same number of residues in the deduced amino acid sequences of all the GRA7 proteins, Western blot analysis indicated a difference in the migration pattern of NhGRA7 in comparison with NcGRA7. Results of our study indicate that diagnostic tests based on differences in dense granule sequences and antigenicity may have potential to differentiate between N. hughesi and N. caninum. Such diagnostic tests would be valuable tools to aid in our understanding of the epidemiology of these parasites. Additionally, dense granule proteins are immunogenic and they may have potential as use in recombinant vaccines against neosporosis.     

Differentiation of Neospora hughesi from Neospora caninum based on their immunodominant surface antigen, SAG1 and SRS2

Neospora hughesi is a newly recognised parasite that is closely related to Neospora caninum, and is a cause of equine protozoal myeloencephalitis. We have characterised two N. hughesi immunodominant tachyzoite antigens which exhibit antigenic and molecular differences from the homologous tachyzoite antigens on N. caninum. These antigens on N. hughesi are referred to as NhSAG1 and NhSRS2, using the same mnemonics as used for the N. caninum antigens (NcSAG1 and NcSRS2), and are homologous to Toxoplasma gondii surface antigen 1 (SAG1) and SAG1-related sequence 2 (SRS2). The NcSAG1 and NcSRS2 were antigenically conserved in six different N. caninum isolates from cattle and dogs. The two equine-derived Neospora isolates, one designated as N. hughesi, were similar to each other but different from N. caninum. There was 6% difference in amino acid identity between NcSAG1 and NhSAG1, whereas there was a 9% difference when NcSRS2 and NhSRS2 were compared. The polymorphism of these genes and their corresponding proteins provide additional markers which can be used to distinguish N. caninum from N. hughesi.     

ELISA detection of IgG antibody against a recombinant major surface antigen (Nc-p43) fragment of Neospora caninum in bovine sera

An ELISA was established to measure bovine IgG directed against the recombinant antigenic determinant of Nc-p43, a major surface antigen of Neospora caninum. In a previous study, two thirds of the C-terminal of the molecule was expressed as a 6 x His tagged protein (Ncp43P) for ELISA using 2/3 of the N-terminal of SAG1 from Toxoplasma gondii as a control (TgSAG1A). Among 852 cattle sera collected from stock farms scattered nation-wide, 103 sera (12.1%) were found to react with Ncp43P positively, but no positive reaction was observed with TgSAG1A. This study shows that Ncp43P could be available as an efficient antigen for the diagnosis of neosporosis in cattle. Furthermore, it together with TgSAG1A, could be useful for the differential diagnosis of N. caninum and T. gondii infections in other mammals.     

Identification of novel proteins in Neospora caninum using an organelle purification and monoclonal antibody approach

Neospora caninum is an important veterinary pathogen that causes abortion in cattle and neuromuscular disease in dogs. Neospora has also generated substantial interest because it is an extremely close relative of the human pathogen Toxoplasma gondii, yet does not appear to infect humans. While for Toxoplasma there are a wide array of molecular tools and reagents available for experimental investigation, relatively few reagents exist for Neospora. To investigate the unique biological features of this parasite and exploit the recent sequencing of its genome, we have used an organelle isolation and monoclonal antibody approach to identify novel organellar proteins and develop a wide array of probes for subcellular localization. We raised a panel of forty-six monoclonal antibodies that detect proteins from the rhoptries, micronemes, dense granules, inner membrane complex, apicoplast, mitochondrion and parasite surface. A subset of the proteins was identified by immunoprecipitation and mass spectrometry and reveal that we have identified and localized many of the key proteins involved in invasion and host interaction in Neospora. In addition, we identified novel secretory proteins not previously studied in any apicomplexan parasite. Thus, this organellar monoclonal antibody approach not only greatly enhances the tools available for Neospora cell biology, but also identifies novel components of the unique biological characteristics of this important veterinary pathogen.     

Antibody reaction of human anti-Toxoplasma gondii positive and negative sera with Neospora caninum antigens

Anti-Neospora caninum antibody was detected in anti-Toxoplasma gondii positive and negative human sera by ELISA, western blot and immunofluorescence assay (IFA). Twelve cases out of 172 (6.7%) Toxoplasma-positive sera cross-reacted with both T. gondii and N. caninum antigens, and one out of 110 Toxoplasma-negative sera reacted with N. caninum antigen by ELISA. By western blot, all 12 sera reacted with T. gondii antigens with various banding patterns but specifically at 30 kDa (SAG1) and 22 kDa (SAG2) bands. With N. caninum antigen, the number of reactive bands was reduced, however a 43 kDa band reacted in three cases in Toxoplasma-positive sera in addition to one in Toxoplasma-negative control sera. All sera of the Toxoplasma-positive group labeled surface membrane of T. gondii, but reacted differently with N. caninum. Fluorescence was detected in surface membrane, subcellular organelles, or both in N. caninum. And one case in the Toxoplasma-negative group also reacted with N. caninum strongly in subcellular organelles. This suggested that the antibody against N. caninum may be present in human sera although the positive rate was very low in this study. The possibility of human infection with N. caninum remains to be evaluated further.     

Qualitative evaluation of selective tests for detection of Neospora hughesi antibodies in serum and cerebrospinal fluid of experimentally infected horses

Neospora hughesi is a newly recognized protozoan pathogen in horses that causes a myeloencephalitis similar to Sarcocystis neurona. There are no validated serologic tests using the gold standard sera that are currently available to detect specific N. hughesi antibodies and, thus, no tests available to detect antemortem exposure or estimate seroprevalence in the horse. The objectives of the present study were to establish a bank of gold standard equine sera through experimental infections with N. hughesi and to assess several serologic tests for the detection of related protozoan antibodies. Seven horses were inoculated with N. hughesi tachyzoites, and 7 horses received uninfected cell culture material. The horses were monitored, and blood and cerebrospinal fluid were collected repeatedly over a 4-mo period. With the sera, 4 different serologic techniques were evaluated. including a whole-parasite lysate enzyme-linked immunosorbent assay (ELISA), a recombinant protein ELISA, a modified direct agglutination test, and an indirect fluorescent antibody test. Qualitative and quantitative evaluation of the results showed that the N. hughesi indirect fluorescent antibody test (IFAT) consistently discriminated between experimentally infected and noninfected horses, using a cutoff of 1:640. Sera from 3 naturally infected horses had titers >1:640. Cerebrospinal fluid in all but I infected horse had very low N. hughesi IFAT titers (<1:160), starting at postinoculation day 30.     

Immune responses to Neospora caninum and prospects for vaccination

Developing an effective vaccine against neosporosis presents several interesting challenges. The parasite is spread efficiently from mother to foetus over several generations, and naturally infected cattle do not appear to develop adequate protective immunity. Modulation of the immune response during pregnancy favours parasite survival and multiplication. However, induction of pro-inflammatory responses that are thought to be protective against Neospora caninum would be detrimental to the pregnancy. So, is vaccination a feasible option to control the disease? This article discusses some of these issues and reports on the progress towards a vaccine for neosporosis.     

Prevalence of antibodies to Neospora caninum and Sarcocystis neurona in sera of domestic cats from Brazil

Parasite Biology, Epidemiology and Systematics Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Building 1001, Beltsville, Maryland 20705-2350 Antibodies to Neospora caninum and Sarcocystis neurona were determined in serum samples of 502 domestic cats from Brazil using direct agglutination tests with the respective antigens. Antibodies to S. neurona were not found in 1:50 dilution of any serum in the S. neurona agglutination test. suggesting that domestic cats from S?o Paulo city were not exposed to S. neurona sporocysts from opossums. Antibodies to N. caninum were found in 60 (11.9%) of 502 cats with titers of 1:40 in 36 cats, 1:80 in 18 cats, 1:160 in 5 cats, and 1:800 in 1 cat using the Neospora agglutination test (NAT). Antibodies to N. caninum were confirmed by Western blotting in the sera of 10 cats with NAT titers of 1:80 to 1:800; this finding suggests that at least 10 cats had N. caninum-specific antibodies confirmed by 2 tests. This is the first documentation of natural exposure of cats to N. caninum.     

Mathematical models of Neospora caninum infection in dairy cattle: transmission and options for control

The transmission and control of Neospora caninum infection in dairy cattle was examined using deterministic and stochastic models. Parameter estimates were derived from recent studies conducted in the UK and from the published literature. Three routes of transmission were considered: maternal vertical transmission with a high probability (0.95), horizontal transmission from infected cattle within the herd, and horizontal transmission from an independent external source. Putative infection via pooled colostrum was used as an example of within-herd horizontal transmission, and the recent finding that the dog is a definitive host of N. caninum supported the inclusion of an external independent source of infection. The predicted amount of horizontal transmission required to maintain infection at levels commonly observed in field studies in the UK and elsewhere, was consistent with that observed in studies of post-natal seroconversion (0.85-9.0 per 100 cow-years). A stochastic version of the model was used to simulate the spread of infection in herds of 100 cattle, with a mean infection prevalence similar to that observed in UK studies (around 20%). The distributions of infected and uninfected cattle corresponded closely to Normal distributions, with S.D.s of 6.3 and 7.0, respectively. Control measures were considered by altering birth, death and horizontal transmission parameters. A policy of annual culling of infected cattle very rapidly reduced the prevalence of infection, and was shown to be the most effective method of control in the short term. Not breeding replacements from infected cattle was also effective in the short term, particularly in herds with a higher turnover of cattle. However, the long-term effectiveness of these measures depended on the amount and source of horizontal infection. If the level of within-herd transmission was above a critical threshold, then a combination of reducing within-herd, and blocking external sources of transmission was required to permanently eliminate infection.     

Identification of antigenic proteins from Neospora caninum recognized by bovine immunoglobulins M, E, A and G using immunoproteomics

Antigenic proteins of Neospora caninum (N. caninum) against bovine immunoglobulins M, E, A, and G were investigated by using immunoproteomics. Proteins of N. caninum (KBA-2) tachyzoite lysates separated by two-dimensional gel electrophoresis were transferred to polyvinylidene difluoride (PVDF) membranes, probed with different bovine immunoglobulin class and classified. Antigenic spots recognized were also identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. 132, 84, 4, and 40 antigenic protein spots were recognized on N. caninum immunoblot profiles against bovine IgM, IgE, IgA, and IgG, respectively. Of these protein spots, the antigenic proteins recognized by either IgM, IgE, and IgG, or IgM and IgG were HSP70, pyruvate kinase, actin, NCDG-1, tubulin alpha-chain, and putative ribosomal protein S2. On the other hand, IgM, IgE, and IgA reacted with NTPase, HSP60, tubulin beta-chain, putative protein disulfide isomerase, enolase, lactate dehydrogenase, serine-threonine phosphatase, 14-3-3 protein homologue, and GRA2 protein. Most of the antigenic proteins identified were associated with the process of invasion, proliferation, and egression of apicomplexans. In our study, HSP70, actin, NTPase, HSP60, pyruvate kinase, enolase, putative ribosomal protein S2, NCDG-1, and GRA2 proteins were found to be immunodominant proteins, which may contribute to the development of diagnostic markers and vaccine.     

Schistosomiasis: Evaluation of the indirect fluorescent antibody,complement fixation,and slide flocculation tests in screening for schistosomiasis

Foreign Service personnel undergo pertinent parasitologic examinations upon return from foreign duty posts. Under this program, 2800 sera have been evaluated for schistosomiasis in this laboratory. The majority of individuals tested were considered to have limited exposure to schistosomiasis, although a few indigenous people from endemic areas also were screened. Nonindigenous populations usually gave stronger serological reactions than did indigenous populations. A comparison was made between those having protozoan and helminthic infections and those that were negative parasitologically. A number of subjects with tissue-phase helminths were evaluated and consistently gave strong reactions in the indirect fluorescent antibody (IFA) tests. On the other hand, there was no characteristic pattern observed in individuals with low serum titers. The IFA test proved to be highly sensitive and sufficiently specific for screening, provided that low background reactions were disregarded (i.e., when +/? and 1+ reactions were ignored at low serum dilutions). Thus, the IFA test was the method of choice for screening. Recourse to the complement fixation (CF) and slide flocculation (SF) tests, however, was necessary for definitive diagnosis. In view of the differences in the antigens and the serodiagnostic technics used in this survey, absolute correlation of test results could not be expected. Nevertheless, the three procedures (IFA, CF, and SF) showed excellent correlation in proven cases of schistosomiasis.     

Detection of typhoid fever by Widal and indirect fluorescent antibody (IFA) tests. A comparative study

Widal test is a conventional method for the detection of typhoid fever. However, it takes 18-24 hours to complete the test. In the present study indirect fluorescent antibody test has been compared with the Widal test using single serum specimens and was found to be rapid, sensitive and specific. Serum specimens from 41 culture proven cases of typhoid fever, 14 clinically suspected cases and 22 normal individuals were collected. Whereas Widal test detected 63.41% positive cases, IFA test detected 87.80% from among culturally proven typhoid cases. Among the clinically suspected cases of typhoid fever, IFA test detected 85.71% (28.57 + 57.14%) while Widal test detected only 57.13% (35.71 + 21.42%) positive cases out of above 14 cases.     

Detection of Vibrio parahaemolyticus in Penaeus chinensis using an indirect fluorescent antibody staining procedure

An indirect fluorescent antibody technique (iFAT) incorporating fluorescein-isothiocyanate conjugated anti-rabbit globulin goat serum, and rhodamine-isothiocyanate conjugated bovine serum albumin as background stain has been developed for the detection of Vibrio parahaemolyticus.     

The potency determination of human varicella-zoster immunoglobulin by enzyme-linked immunosorbent assay, complement-fixation test and indirect fluorescent antibody tests

Traditionally, plasma for the production of the human varicella-zoster immunoglobulin (VZIG) has been selected on the basis of the complement-fixing antibody (CFA) titre. Since immune individuals may lack CFA to varicella-zoster virus (VZV), non-CFA may be of importance in protection. In a search for a simple and reliable method for potency determination, 24 VZIG preparations were quantified by enzyme-linked immunosorbent assay (ELISA), the complement-fixation test (CFT), the indirect fluorescent antibody test to acetone-fixed (IF) and viable (FAMA) VZV-infected cells, respectively. The antibody titres obtained by the various methods were compared. Arranged in order of decreasing agreement, the correlation coefficients (r) of the regression equations between the variables were 0.62 for CFT and FAMA, 0.50 for CFT and ELISA and 0.26 for CFT and IF in a log2 plot. There was complete agreement between the titres obtained by the commercially available Enzygnost Varicella/Zoster kits (Behring Institute, Marburg, F.R. Germany) and the ELISA microtitre plates produced at our institute (r = 1). The regression equation lines for ELISA/CFT and FAMA/CFT titres tended to be parallel to each other, while the line for IF/CFT titres had a less steep slope. Similar titration curves were obtained for VZIGs fractionated by two different methods. Furthermore, the titration curves of serum pools from varicella and zoster convalescents, respectively, had a similar shape below delta OD = 0.4. Generally, a steeper slope was observed above delta OD = 0.4. As antibody detectable by ELISA seems to correlate with protection and the method is sensitive, specific, reproduceable, simple to carry out and easily automated, it may be suitable for the potency determination of VZIGs.