Open Access
Volume 29, 2022
Article Number 60
Number of page(s) 15
Published online 21 December 2022

© R. Udonsom et al., published by EDP Sciences, 2022

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Neospora caninum is an apicomplexan protozoan parasite and a primary cause of abortion in cattle throughout the world [16]. Consequently, bovine neosporosis is currently a disease of concern worldwide due to its global distribution and significant economic impact through massive losses in the dairy and meat industries [15, 20, 37]. Currently, no effective drugs or vaccines are available to prevent abortion or transmission caused by N. caninum infection in cattle [20]. Neospora caninum infection is generally latent and asymptomatic in non-pregnant cattle, although persistent infection throughout life is an important feature of bovine neosporosis, resulting in repeated abortions by transplacental (vertical) transmission, the principal route of infection [4, 8]. Prevention and control strategies of neosporosis are dependent on farm management practices and strict hygiene. At present, serological diagnosis is the only option to discriminate between infected and uninfected animals, followed by appropriate treatment management to control bovine neosporosis [19]. Several diagnostic methods for bovine neosporosis are used to detect specific antibodies against N. caninum. The enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody test (IFAT) are the most common techniques used to diagnose N. caninum infections [2, 6]. However, a major problem concerning conventional serological testing is the possibility of low specificity of diagnosis due to cross-reactivity among closely related apicomplexan pathogens, including Toxoplasma gondii, Cryptosporidium parvum and Babesia spp. (B. bovis and B. bigemina) [18].

There are reports of serological cross-reactivity among animals infected with N. caninum and T. gondii. Cross-reactive N. caninum soluble antigens (NLA) were recognised using sera from mice and cats immunised with T. gondii [33]. Of 384 monoclonal antibodies (mAbs), 10 were produced by immunising mice with N. caninum tachyzoites and were found to be cross-reactive between N. caninum and T. gondii. Among these, three antigenic proteins, including protein disulfide isomerase (PDI), heat shock protein 70 (HSP70) and ribosomal protein 1 (RP1), were identified as cross-reactive antigens between both parasites [28]. Similarly, Sohn et al. developed a panel of 46 mAbs using a mouse immunised with a mixed fraction of N. caninum organelles and found that some of these mAbs cross-reacted with T. gondii [41]. Current investigations of the parasite proteome provide comprehensive insights into their biological processes and highlight valuable diagnostic biomarkers, as well as new vaccine targets [44]. It is necessary to identify parasite-specific proteins to develop novel and specific biomarkers to enhance sensitivity and specificity for precise and acceptable diagnosis. The initial proteomics analysis of N. caninum tachyzoite conducted by Lee et al. revealed 31 spots corresponding to 20 different proteins identified from N. caninum tachyzoites by peptide mass fingerprinting and 17 spots corresponding to 11 antigenic proteins identified from N. caninum protein map [27]. Another study identified 64 spots as antigenic proteins on immunoblot profiles using rabbit anti-sera [25]. A comparison of proteomes between N. caninum and T. gondii tachyzoites was also conducted, which revealed the cross-reactive antigens between them [26, 48]. Currently, there are limited proteomics studies on the species-specific antigens or cross-reactivity of N. caninum compared with other apicomplexan parasites in the bovine host. Therefore, this study was conducted to identify the immunoreactive and antigenic proteins of N. caninum tachyzoites using infected bovine sera specific to N. caninum, T. gondii, C. parvum, B. bovis and B. bigemina and healthy host sera by immunoproteomics. MS and bioinformatics analyses were performed to identify and characterise the cross-reactive and species-specific antigens among these parasites. These species-specific immunogenic proteins could be targeted as new biomarkers for N. caninum immunodiagnosis or vaccine development.

Materials and methods

Bovine immune serum samples

Fourteen bovine serum samples infected with the five protozoan parasites N. caninum (N = 2), T. gondii (N = 3), C. parvum (N = 3), B. bovis (N = 4) and B. bigemina (N = 2) and negative sera (N = 4) were used in this study. The details of bovine serum samples are presented in Table 1. This study was approved by the Faculty of Tropical Medicine-Animal Care and Use Committee, Mahidol University (FTM-ACUC 005/2022E).

Table 1

List of known bovine serum samples used this study.

Maintenance and purification of N. caninum

Neospora caninum tachyzoites (Nc-1 strain) were maintained in African green monkey kidney (Vero) cell monolayer with Dulbecco’s Modified Eagle Medium (Cytiva HyClone™) supplemented with 10% foetal bovine serum, L-glutamine (2 mM/mL), penicillin–streptomycin (100 U/mL penicillin and 100 μg/mL streptomycin) and amphotericin B (0.25 μg/mL) in a humidified atmosphere with 5% CO2 at 37 °C. Neospora caninum tachyzoites were harvested by cell scraping of the infected Vero host cell after 3–4 days of inoculation. The tachyzoites were separated from host cells using a 5 μm filter, and the tachyzoite suspension was loaded onto a 30%, 50% and 80% (v/v) osmotic Percoll® (Sigma-Aldrich, Burlington, MA, USA) gradient to purify and eliminate the remaining host cells, followed by centrifugation at 2,000 g for 30 min at 4 °C. The viable tachyzoite band forming between the 50% and 80% osmotic Percoll® gradients were collected and washed three times with phosphate-buffered saline (PBS). The purified N. caninum tachyzoite pellet was stored at −70 °C until use [27].

Preparation of soluble N. caninum proteins

Purified N. caninum tachyzoites were dissolved in a lysis buffer containing 8 M urea, 2 M thiourea, 4% (w/v) CHAPS and 50 mM DTT, and then the N. caninum sample was sonicated at 5.5 W for 2 min (5 s pulse/10 s rest) on ice slurry. The suspension was centrifuged at 14,000 rpm for 30 min at 4 °C, and the resulting supernatant was collected. Protein concentration was estimated by the Bradford assay (BioRad Inc., Hercules, CA, USA) using bovine serum albumin as a standard, and the interfering substance was removed using a 2-D Clean-Up Kit (GE Healthcare Bioscience, Chalfont St Giles, UK), according to the manufacturer’s protocol.

Two-dimensional electrophoresis

ImmobilineTM DryStrip gels (pH 3–10, NL 7 cm; GE Healthcare, Uppsala, Sweden) were rehydrated overnight at 25 °C with N. caninum soluble protein (100 μg/strip), 1% (w/v) bromophenol blue and 0.5% IPG buffer (pH 3–10, NL; GE Healthcare). Isoelectric focusing (IEF) was performed using an Ettan IPG Phor Electrofocusing system (GE Healthcare) under the following running conditions: 0.2 kV/h for the initial 30 min, followed by a gradient of 0.3 kV/h for 30 min, 4.5 kV/h for 90 min and step down and hold at 3.0 kV/h for 35 min. After IEF, the IPG strips were equilibrated in 5 mg/mL dithiothreitol (DTT) for 15 min and 25 mg/mL iodoacetamide for 15 min, and each focused IPG strip was inserted into 12% sodium dodecyl sulphate polyacrylamide gel and sealed with 0.5% agarose gel. Electrophoresis was conducted at 150 V per gel until the bromophenol blue dye reached the lower gel edge. Protein spots were visualised by silver staining, and the immunoreactive spots in these gels (three gels) were excised and pooled for mass spectrometric analysis. Other six gels from two-dimensional gel electrophoresis (2-DE) were used for immunomics analysis.

Immunomics analysis

For immunoblotting studies, the proteins on the 2-DE gels were transblotted onto nitrocellulose membranes (Merck Millipore, Carrigtwohill, Ireland) using the western blot wet/tank transfer (Amersham Bioscience, Amersham, UK) under the running conditions of 20 V, 400 mA at 4 °C, overnight. The blotted membranes were blocked with 5% skimmed milk in PBS containing 0.05% Tween-20 (PBS-T) for 1 h and then probed with pooled bovine sera (diluted 1:400) confirmed to be infected with apicomplexan parasites, including N. caninum (N = 2), T. gondii (N = 3), C. parvum (N = 3), B. bovis (N = 4) and B. bigemina (N = 2) for 2 h. Pooled healthy bovine sera (N = 4) with no history of infection were used as negative controls. After washing with PBS-T, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-bovine immunoglobulin G (Invitrogen, Waltham, MA, USA) at 1:2,000 dilution for 1 h. Ultra TMB-Blotting solution substrate (Thermo Fisher Scientific, Milton Park, UK) was used to visualise the antigen–antibody reactive spots, and then the protein spots specific to N. caninum infection were identified and compared with the immunoreactive spots specific to other apicomplexan parasites. Immunoreactive protein spots were excised from the silver-stained 2-DE gels and subjected to trypsin digestion.

In-gel digestion

Immunoreactive protein spots were manually excised from the silver-stained 2-DE gels. Gel pieces were de-stained at 4 °C overnight with 50% acetonitrile (ACN; Sigma-Aldrich) in 50 mM ammonium bicarbonate (Merck). The disulfide bonds in the proteins were reduced with 4 mM DTT in 50 mM ammonium bicarbonate at 60 °C for 15 min and then alkylated with 250 mM iodoacetamide at room temperature for 30 min in the dark. The reaction was quenched by 4 mM DTT in 50 mM ammonium bicarbonate for 5 min at room temperature, after which the entire solution was removed, and the gel pieces were dehydrated with acetonitrile. The gel pieces were digested with proteomics-grade trypsin (Sigma-Aldrich) in 50 mM ammonium bicarbonate at 37 °C overnight. The digested peptides were extracted by acetonitrile and dried in a vacuum centrifuge.

Mass spectrometry analysis (LC-MS/MS)

Dried tryptic peptides were redissolved in 0.1% formic acid. Each sample was injected and analysed for amino acid sequences using the UltiMate 3000 nano-liquid chromatography (nano-LC) system (Dionex, Camberley, UK). The mass spectra obtained from the mass spectrometry (MS) and tandem mass spectrometry (MS/MS) covered mass ranges of m/z 400–2000 and m/z 50–1500, respectively. A mascot generic file (.mgf) was generated using the data analysis software (Bruker Daltonics, Billerica, MA, USA). Mascot Daemon version 2.3.2 (Matrix Science, London, UK) was used to merge the .mgf files and identified the proteins. The National Center for Biotechnology Information (NCBI) ( was set as the protein sequence database, and ToxoDB Toxoplasma informatics resources ( were also used for protein identification. Peptides with 95% confidence were reported in this study to reduce false-positive results.


2-DE profile of N. caninum tachyzoite proteins

The 2-DE analysis followed by silver staining revealed approximately 500–600 protein spots, and most protein spots were located between 10 and 130 kDa (Fig. 1). Based on 2-DE immunoblotting, 70 immunoreactive spots were identified that are indicated using circles or ellipse in the figure. Among these, 37 spots (spot numbers 1–37) were recognised by anti-N. caninum serum, and only 20 protein spots marked with arrows (spot numbers 1, 2, 3, 4, 7, 8, 9, 10, 18, 19, 22, 25, 30, 31, 32, 33, 34, 35, 36 and 37) corresponding to 14 different antigenic proteins were specific to N. caninum. Approximately 50 protein spots were cross-reactive with other apicomplexan-infected sera. The antigenic spots were most abundant at molecular masses ranging from 26 to 130 kDa. All the immunoreactive protein spots were excised and identified by LC-MS/MS. Protein identification was performed by MASCOT search engine 2.3 (Matrix Science, Ltd.) using the NCBI N. caninum database. Table 2 shows the data of these spots consisting of identification scores, molecular weight, number of matched peptides, percentage of protein sequence coverage and isoelectric point (pI).

thumbnail Figure 1

Two-dimensional electrophoresis protein patterns of N. caninum (Nc-1) tachyzoites separated using 12% acrylamide and visualised by silver staining. A total of 70 immunoreactive protein spots (spot numbers 1–70) were identified on the 2-DE gel based on immunoblot analysis that were recognised by known bovine sera. The circle or ellipse with arrows indicates 20 specific protein spots that were recognised by N. caninum-positive bovine pooled sera.

Table 2

List of proteins identified on the 2-DE profiles of Neospora caninum (Nc-1) tachyzoites probed with known bovine sera analysed by mass spectrometry (LC-MS/MS).

Detection of immunoreactive spots by 2-DE immunoblotting using immune bovine sera against N. caninum and other apicomplexan parasites

The immunoreactive protein spots recognised in individual bovine sera on the 2-DE immunoblot profiles are shown in Figures 2A2F. A total of 37 immunoreactive protein spots were recognised by anti-N. caninum serum, but only 20 antigenic protein spots (indicated with arrows) corresponding to 14 different proteins were specific to N. caninum (Fig. 2A). The immunoblot analysis also revealed 41 protein spots recognised by anti-T. gondii serum (Fig. 2B), 24 by anti-C. parvum serum (Fig. 2C) and 2 by anti-B. bovis and anti-B. bigemina sera (Fig. 2D and 2E). Furthermore, two protein spots were detected by healthy bovine sera (Fig. 2F). At least 50 protein spots were identified to exhibit cross-reactivity of N. caninum tachyzoite proteins with other apicomplexan parasites. Most protein spots were cross-reactive with the closely related T. gondii and C. parvum. Spot numbers 12, 13, 14, 15, 17, 23, 24 and 29 were recognised by anti-N. caninum sera but demonstrated cross-reactivity with anti-T. gondii and anti-C. parvum sera. Spot numbers 38, 45, 46, 48, 51, 54 and 55 were also cross-reactive against anti-T. gondii and anti-C. parvum sera. Spot number 64 was cross-reactive against anti-T. gondii, anti-C. parvum and anti-B. bovis sera, whereas spot number 28 showed cross-reactivity with anti-T. gondii, anti-C. parvum and anti-B. bigemina sera. All immunogens were classified according to their specific reactivity against N. caninum and other apicomplexan parasite infections in bovine hosts and are listed in Table 3.

thumbnail Figure 2

Immunoblot analysis of 2-DE-separated N. caninum tachyzoite antigens using pooled bovine anti-N. caninum (A), anti-T. gondii (B), anti-C. parvum (C), anti-B. bovis (D) and anti-B. bigemina (E) sera and healthy serum (F). The arrows indicate 20 protein spots corresponding to 14 different antigenic proteins recognised by N. caninum-positive sera, and those without arrows indicate spots cross-reactive with other apicomplexan protozoan-infected sera.

Table 3

List of proteins identified as specific and/or cross-reactive antigens of Neospora caninum and other apicomplexan protozoa in the bovine host.

Functional categorisation of immunoreactive proteins against N. caninum

To further understand the functions of the immunoreactive proteins against N. caninum, the 20 immunoreactive protein spots corresponding to 14 different specific antigens were putatively annotated using GO terms obtained from the ToxoDB Toxoplasma informatics resources ( and previous study reports (Table 4). The functional classification of the 14 different antigenic proteins against N. caninum is shown in Table 4. In the category of biological processes, eight proteins were associated with cell growth and invasion process, including HSP90-like protein, ubiquitin carboxyl-terminal hydrolase, microneme protein 4 (MIC4), inosine-5′-monophosphate dehydrogenase, actin, elongation factor 1-alpha, hypothetical protein NCLIV_005150 and peroxidoxin 2 (also called peroxiredoxin 2). In the molecular function category, four proteins corresponded to ATP, DNA or protein binding. Furthermore, a protein associated with cellular components and an unnamed protein product represented as rhoptry protein (ROP1) with no function data available were identified. Proteins involved in cell proliferation and invasion process were found to be immunogenic.

Table 4

Functional classification of immunoreactive proteins against Neospora caninum.


Toxoplasma, Cryptosporidium, Babesia and Neospora are important veterinary pathogens that cause diseases in farm animals, resulting in considerable economic losses to the livestock sector [32]. Toxoplasma gondii is the most significant pathogen associated with reproductive problems, especially in small ruminants [14]; C. parvum is one of the most important causes of calf diarrhoea, particularly in neonatal calves [43]; and Babesia spp. cause tick-borne disease with a worldwide economic impact due to severe disease in cattle, among which B. bovis and B. bigemina are the two most important species [7]. Bovine neosporosis is a major cause of abortion in cattle worldwide, which causes serious economic losses to beef and dairy industries [15, 37]. Considering the lack of an effective treatment method or vaccine against neosporosis, there is a need to improve serodiagnostic methods to discriminate N. caninum-infected animals from those infected with other closely related pathogens in the assessment of epidemiology, surveillance and disease management [19].

The proteomics approach can help in the discovery of novel immunogens involved in host immune stimulation and can help in the identification of possible targets for drugs and vaccines [35]. High-resolution 2-DE protein separation combined with immunoblot analysis of antigenic proteins, followed by identification with MS and bioinformatics analysis provides an approach to identify parasite-specific proteins or distinct antigens that represent potential vaccine candidates or targets for serodiagnosis improvement [23, 25]. Although combinations of 2-DE, immunoblotting, and mass spectrometric analysis for analysing N. caninum antigens have been applied, there are limited studies on the identification of N. caninum antigenic proteins in the bovine host, a major victim of neosporosis. Lee et al. detected 102 antigen spots using IPG strips (pH 4–7) on immunoblot profile using serum from rabbit immunised with N. caninum, among which 17 spots corresponding to 11 antigenic proteins were identified as antigens from N. caninum on the 2-DE map [27]. Subsequently, 132, 84, 4 and 40 antigenic protein spots were recognised against bovine IgM, IgE, IgA and IgG, respectively, by immunoproteomics using serum from cow immunised with N. caninum [39]. Comparison of the antigenic proteome between N. caninum KBA-2 and VMDL-1 isolates using serum from cow immunised with N. caninum showed a high similarity pattern on 2-DE separation, and the antigenic spots on immunoblot profiles were also detected at similar locations in terms of pI and molecular weight [38]. In this study, we identified and analysed N. caninum (Nc-1 isolate) tachyzoite antigenic proteins recognised by each of apicomplexan-infected bovine sera, including N. caninum, T. gondii, C. parvum, B. bovis and B. bigemina, and healthy host sera on 2-DE immunoblot profiles. Based on 2-DE immunoblotting, we identified 20 antigenic spots corresponding to 14 specific antigens against N. caninum. Among these, HSP90, hypothetical protein NCLIV_034460, ubiquitin carboxyl-terminal hydrolase, corA-like Mg2 transporter domain-containing protein, microneme 4 (MIC4), inosine-5′-monophosphate dehydrogenase, actin, hypothetical protein NCLIV_004400, rhoptry protein (ROP1), elongation factor 1-alpha, hypothetical protein NCLIV_005150, armadillo/beta-catenin-like repeat-containing protein, peroxidoxin 2 and Gbp1p protein were significantly specifically immunoreactive corresponding to their immunoglobulin reactions against N. caninum.

Most antigenic proteins identified in this study were associated with cell invasion and proliferation processes of the parasite. Among these proteins, HSP90 is a molecule playing a vital role in the biology and virulence of the parasite. In T. gondii, HSP90 plays an important role in bradyzoite differentiation, host cell invasion, growth and virulence [42], whereas in Plasmodium, HSP90 was indicated as a protein regulating parasite growth in human erythrocytes [46]. Another previous report on HSP90 described species-specific antigens against N. caninum using sera from mice immunised with either N. caninum or T. gondii [48]. Elongation factor 1-alpha (EF-1α) is a key element of protein translation and one of the most abundant proteins expressed in eukaryotic cells [24]. Mice vaccinated with recombinant T. gondii EF-1α showed high levels of specific anti-T. gondii antibodies and production of IFN-gamma and interleukin-4, which significantly prolonged the survival time after challenge infection with the T. gondii RH virulent strain [45]. Although our results showed that EF-1α exhibits high immunoreactivity against N. caninum, we also found a strong reactivity pattern with anti-T. gondii on the 2-DE immunoblot profiles. NcMIC4 has been found to be largely upregulated in the N. caninum tachyzoite stage when entering and developing within the host cell, and re-expression of NcMIC4 occurred 30 min after entry into the host cell [22]. MIC1 and MIC4 induce protective immunity against T. gondii by stimulating the production of IL-2, IL-12, IFN-g and IL-10 in immunised mice, indicating that these proteins might become targets for the further development of vaccines [29]. Our study also showed that MIC4 reacted with N. caninum-infected bovine sera, indicating that it may be a promising candidate for diagnosis and vaccine development. However, it is necessary to evaluate the diagnostic and vaccine potential against bovine neosporosis in the near future.

Another immunoreactive protein identified in this study was actin, the protein responsible for several biological processes in apicomplexan parasites, including cell motility, host cell invasion, vesicular transport and apicoplast inheritance [11]. Actin strongly reacted with bovine IgM, IgG and IgE and exhibited immunodominant antigens with bovine IgG on the immunoblot profiles of both N. caninum KBA-2 and VMDL-1 isolates [38, 39]. In addition, it has been reported that there is N. caninum actin in at least nine different isoforms that are functional in cellular processes and might be regulated by mechanisms involving post-translational modifications [3]. Similarly, the armadillo/beta-catenin-like repeat-containing protein has been demonstrated to be crucial in apical rhoptry positioning and consequently aids in host cell invasion in P. falciparum and T. gondii [9, 31]. Ubiquitin plays an important role in protein turnover, cellular signalling and intracellular transport. It is conjugated to the lysine residues of proteins to regulate a large number of cellular processes [40]. A study on the ubiquitylation pathway in apicomplexan parasites suggested that ubiquitin is essential for controlling cellular processes throughout the apicomplexan complex parasitic life cycle [34]. Moreover, peroxidoxins and inosine-5′-monophosphate dehydrogenase exhibited high antigenic activity in our study and in other organisms as well [5, 10, 17], indicating their potential as vaccine candidates and drug targets. Interestingly, we discovered the unnamed protein product ROP1 exhibiting strong immunoreactivity against N. caninum but with no functional data available. Rhoptry proteins of apicomplexan pathogens play a vital role in parasite virulence. ROP5 was found to be critical for the pathogenesis of T. gondii in mice, as deletion of this gene attenuated virulence in the mice [36]. A recent report of N. caninum ROP5 knockout in a plaque assay indicated that N. caninum showed weakened invasion ability and slower intracellular growth, along with loss of virulence, in mice [30]. Similarly, N. caninum ROP2 was identified to play an essential role during host cell invasion processes and exhibits immunoprotective properties that induced host immune responses, indicating its potential as a vaccine candidate [12, 13]. Further study is required to clarify the function of ROP1 protein.

Although several studies have revealed the cross-reaction between N. caninum and T. gondii, there are limited studies describing cross-reactivity using proteomics among apicomplexan parasites in the bovine host. Three proteins, including PDI, HSP70 and RP1, were identified as cross-reactive antigens between N. caninum and T. gondii [28]. Some proteins showed high homology between N. caninum and T. gondii tachyzoites, such as HSP70, tubulin α- and β-chain, PDI, actin and enolase, which were believed to be conserved antigens in both parasites [26]. Zhang et al. also demonstrated that at least 18 protein spots showed cross-reaction between N. caninum and T. gondii using sera from mouse immunised with parasites and further found that some antigens shared high homology with the corresponding antigens of T. gondii [48]. In this study, a large number of N. caninum immunoproteomics profiles cross-reacted with T. gondii and C. parvum (Table 3). In addition, the corA-like Mg2 transporter domain-containing protein, elongation factor 1-alpha and ROP1 were found to be highly specific antigenic proteins against N. caninum, but these antigens were found in different spots recognised by other protozoan-infected sera, which had the same protein accession number. This finding might be attributed to the different forms of post-translational modification or different isoforms of these three proteins. In this study, as we used pooled bovine healthy sera, two protein spots reacted with the healthy sera, which might be due to non-specific binding of the bovine background antibody. Therefore, we deduced these two proteins as cross-reactive antigens. A high degree of cross-reactivity was identified in the antigens among these parasites, especially N. caninum, T. gondii and C. parvum.

Although many parasite antigens were identified in this study, there were several limitations to using this method. Since strong detergent was not added to the 2-DE gel electrophoresis buffer system, very high hydrophobic proteins were not able to dissolve and be detected. It was not possible to separate the proteins with very high or low isoelectric points by 2-DE gel electrophoresis. In addition, protein identification relied on protein visualisation by silver staining; therefore, very low-abundant proteins could not be found. As a result of these obstacles, other cross-reactive proteins might not be resolved and identified using this method.


There is a need for specific biomarkers in veterinary medicine for diagnosis and follow-up treatment. Immunoproteomics is very useful for identifying host immune responses and characteristics of individual antigenic proteins. This study demonstrated the detection of disease-specific proteins using infected bovine sera, which exhibited distinct specific antigens against N. caninum and possible cross-reactive antigens with other apicomplexan parasites, especially T. gondii and C. parvum. Further study is required to evaluate cross-reactive antigens as potential common vaccine candidates or drug targets to control the diseases caused by these parasites in the bovine host. Therefore, we can target these highly specific immunoreactive antigens for further identification and characterisation in immunodiagnosis and vaccine development.

Conflict of interest

The authors declare that they have no conflicts of interest.


We would like to recognise the financial support received from the Thailand Research Fund (TRF), the National Research Council of Thailand (NRCT), the Thailand Science Research and Innovation (TSRI) through the Royal Golden Jubilee PhD Program (RGJ-PhD) (Grant No. PHD/0067/2561) and Faculty of Tropical Medicine, Mahidol University. We would like to thank the native English-speaking staff of the Office of Research Services, Faculty of Tropical Medicine, Mahidol University for helping us in editing our manuscript.


  1. Abdelbaky HH, Nishimura M, Shimoda N, Hiasa J, Fereig RM, Tokimitsu H, Inokuma H, Nishikawa Y. 2020. Evaluation of Neospora caninum serodiagnostic antigens for bovine neosporosis. Parasitology International, 75, 102045. [CrossRef] [PubMed] [Google Scholar]
  2. Anderson ML, Andrianarivo AG, Conrad PA. 2000. Neosporosis in cattle. Animal Reproduction Science, 60–61, 417–431. [CrossRef] [PubMed] [Google Scholar]
  3. Baroni L, Pollo-Oliveira L, Heck AJ, Altelaar AM, Yatsuda AP. 2019. Actin from the apicomplexan Neospora caninum (NcACT) has different isoforms in 2D electrophoresis. Parasitology, 146(1), 33–41. [CrossRef] [PubMed] [Google Scholar]
  4. Bartels CJ, Huinink I, Beiboer ML, van Schaik G, Wouda W, Dijkstra T, Stegeman A. 2007. Quantification of vertical and horizontal transmission of Neospora caninum infection in Dutch dairy herds. Veterinary Parasitology, 148(2), 83–92. [CrossRef] [PubMed] [Google Scholar]
  5. Bayih AG, Daifalla NS, Gedamu L. 2014. DNA-protein immunization using Leishmania peroxidoxin-1 induces a strong CD4 + T cell response and partially protects mice from cutaneous leishmaniasis: role of fusion murine granulocyte-macrophage colony-stimulating factor DNA adjuvant. PLoS Neglected Tropical Diseases, 8(12), e3391. [CrossRef] [PubMed] [Google Scholar]
  6. Bjorkman C, Uggla A. 1999. Serological diagnosis of Neospora caninum infection. International Journal for Parasitology, 29(10), 1497–1507. [CrossRef] [PubMed] [Google Scholar]
  7. Bock R, Jackson L, de Vos A, Jorgensen W. 2004. Babesiosis of cattle. Parasitology, 129(Suppl), S247–S269. [CrossRef] [PubMed] [Google Scholar]
  8. Buxton D, McAllister MM, Dubey JP. 2002. The comparative pathogenesis of neosporosis. Trends in Parasitology, 18(12), 546–552. [CrossRef] [PubMed] [Google Scholar]
  9. Cabrera A, Herrmann S, Warszta D, Santos JM, John Peter AT, Kono M, Debrouver S, Jacobs T, Spielmann T, Ungermann C, Soldati-Favre D, Gilberger TW. 2012. Dissection of minimal sequence requirements for rhoptry membrane targeting in the malaria parasite. Traffic, 13(10), 1335–1350. [CrossRef] [PubMed] [Google Scholar]
  10. Cao S, Aboge GO, Terkawi MA, Zhou M, Luo Y, Yu L, Li Y, Goo Y, Kamyingkird K, Masatani T, Suzuki H, Igarashi I, Nishikawa Y, Xuan X. 2013. Cloning, characterization and validation of inosine 5’-monophosphate dehydrogenase of Babesia gibsoni as molecular drug target. Parasitology International, 62(2), 87–94. [CrossRef] [PubMed] [Google Scholar]
  11. Das S, Stortz JF, Meissner M, Periz J. 2021. The multiple functions of actin in apicomplexan parasites. Cellular Microbiology, 23(11), e13345. [PubMed] [Google Scholar]
  12. Debache K, Alaeddine F, Guionaud C, Monney T, Muller J, Strohbusch M, Leib SL, Grandgirard D, Hemphill A. 2009. Vaccination with recombinant NcROP2 combined with recombinant NcMIC1 and NcMIC3 reduces cerebral infection and vertical transmission in mice experimentally infected with Neospora caninum tachyzoites. International Journal for Parasitology, 39(12), 1373–1384. [CrossRef] [PubMed] [Google Scholar]
  13. Debache K, Guionaud C, Alaeddine F, Mevissen M, Hemphill A. 2008. Vaccination of mice with recombinant NcROP2 antigen reduces mortality and cerebral infection in mice infected with Neospora caninum tachyzoites. International Journal for Parasitology, 38(12), 1455–1463. [CrossRef] [PubMed] [Google Scholar]
  14. Dubey JP. 2009. Toxoplasmosis in sheep-the last 20 years. Veterinary Parasitology, 163(1–2), 1–14. [CrossRef] [PubMed] [Google Scholar]
  15. Dubey JP, Schares G. 2011. Neosporosis in animals-the last five years. Veterinary Parasitology, 180(1–2), 90–108. [CrossRef] [PubMed] [Google Scholar]
  16. Dubey JP, Schares G, Ortega-Mora LM. 2007. Epidemiology and control of neosporosis and Neospora caninum. Clinical Microbiology Reviews, 20(2), 323–367. [CrossRef] [PubMed] [Google Scholar]
  17. Fereig RM, Kuroda Y, Terkawi MA, Mahmoud ME, Nishikawa Y. 2017. Immunization with Toxoplasma gondii peroxiredoxin 1 induces protective immunity against toxoplasmosis in mice. PLoS One, 12(4), e0176324. [CrossRef] [PubMed] [Google Scholar]
  18. Gondim LF, Mineo JR, Schares G. 2017. Importance of serological cross-reactivity among Toxoplasma gondii, Hammondia spp., Neospora spp., Sarcocystis spp. and Besnoitia besnoiti. Parasitology, 144(7), 851–868. [CrossRef] [PubMed] [Google Scholar]
  19. Guido S, Katzer F, Nanjiani I, Milne E, Innes EA. 2016. Serology-based diagnostics for the control of bovine neosporosis. Trends in Parasitology, 32(2), 131–143. [CrossRef] [PubMed] [Google Scholar]
  20. Horcajo P, Regidor-Cerrillo J, Aguado-Martínez A, Hemphill A, Ortega-Mora L. 2016. Vaccines for bovine neosporosis: current status and key aspects for development. Parasite Immunology, 38(12), 709–723. [CrossRef] [PubMed] [Google Scholar]
  21. Ichikawa-Seki M, Fereig RM, Masatani T, Kinami A, Takahashi Y, Kida K, Nishikawa Y. 2019. Development of CpGP15 recombinant antigen of Cryptosporidium parvum for detection of the specific antibodies in cattle. Parasitology International, 69, 8–12. [CrossRef] [PubMed] [Google Scholar]
  22. Keller N, Riesen M, Naguleswaran A, Vonlaufen N, Stettler R, Leepin A, Wastling JM, Hemphill A. 2004. Identification and characterization of a Neospora caninum microneme-associated protein (NcMIC4) that exhibits unique lactose-binding properties. Infection and Immunity, 72(8), 4791–4800. [CrossRef] [PubMed] [Google Scholar]
  23. Klade CS. 2002. Proteomics approaches towards antigen discovery and vaccine development. Current Opinion in Molecular Therapeutics, 4(3), 216–223. [PubMed] [Google Scholar]
  24. Kristensen R, Mona T, Kosiak B, Holst-Jensen A. 2005. Phylogeny and toxigenic potential is correlated in Fusarium species as revealed by partial translation elongation factor 1 alpha gene sequences. Mycological Research, 109(2), 173–186. [CrossRef] [PubMed] [Google Scholar]
  25. Lee EG, Kim JH, Shin YS, Shin GW, Kim YH, Kim GS, Kim DY, Jung TS, Suh MD. 2004. Two-dimensional gel electrophoresis and immunoblot analysis of Neospora caninum tachyzoites. Journal of Veterinary Science, 5(2), 139–145. [CrossRef] [PubMed] [Google Scholar]
  26. Lee EG, Kim JH, Shin YS, Shin GW, Kim YR, Palaksha KJ, Kim DY, Yamane I, Kim YH, Kim GS, Suh MD, Jung TS. 2005. Application of proteomics for comparison of proteome of Neospora caninum and Toxoplasma gondii tachyzoites. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 815(1–2), 305–314. [CrossRef] [Google Scholar]
  27. Lee EG, Kim JH, Shin YS, Shin GW, Suh MD, Kim DY, Kim YH, Kim GS, Jung TS. 2003. Establishment of a two-dimensional electrophoresis map for Neospora caninum tachyzoites by proteomics. Proteomics, 3(12), 2339–2350. [CrossRef] [PubMed] [Google Scholar]
  28. Liao M, Xuan X, Huang X, Shirafuji H, Fukumoto S, Hirata H, Suzuki H, Fujisaki K. 2005. Identification and characterization of cross-reactive antigens from Neospora caninum and Toxoplasma gondii . Parasitology, 130(Pt 5), 481–488. [CrossRef] [PubMed] [Google Scholar]
  29. Lourenco EV, Bernardes ES, Silva NM, Mineo JR, Panunto-Castelo A, Roque-Barreira MC. 2006. Immunization with MIC1 and MIC4 induces protective immunity against Toxoplasma gondii. Microbes and Infection, 8(5), 1244–1251. [CrossRef] [PubMed] [Google Scholar]
  30. Ma L, Liu J, Li M, Fu Y, Zhang X, Liu Q. 2017. Rhoptry protein 5 (ROP5) is a key virulence factor in Neospora caninum. Frontiers in Microbiology, 8, 370. [PubMed] [Google Scholar]
  31. Mueller C, Klages N, Jacot D, Santos JM, Cabrera A, Gilberger TW, Dubremetz JF, Soldati-Favre D. 2013. The Toxoplasma protein ARO mediates the apical positioning of rhoptry organelles, a prerequisite for host cell invasion. Cell Host & Microbe, 13(3), 289–301. [CrossRef] [PubMed] [Google Scholar]
  32. Muller J, Hemphill A. 2013. In vitro culture systems for the study of apicomplexan parasites in farm animals. International Journal for Parasitology, 43(2), 115–124. [CrossRef] [PubMed] [Google Scholar]
  33. Nishikawa Y, Claveria FG, Fujisaki K, Nagasawa H. 2002. Studies on serological cross-reaction of Neospora caninum with Toxoplasma gondii and Hammondia heydorni. Journal of Veterinary Medical Science, 64(2), 161–164. [CrossRef] [PubMed] [Google Scholar]
  34. Ponts N, Yang J, Chung DW, Prudhomme J, Girke T, Horrocks P, Le Roch KG. 2008. Deciphering the ubiquitin-mediated pathway in apicomplexan parasites: a potential strategy to interfere with parasite virulence. PLoS One, 3(6), e2386. [CrossRef] [PubMed] [Google Scholar]
  35. Reamtong O. 2013. Mass spectrometry-based parasitic proteomics. Journal of Tropical Medicine and Parasitology, 36, 40–48. [Google Scholar]
  36. Reese ML, Boothroyd JC. 2011. A conserved non-canonical motif in the pseudoactive site of the ROP5 pseudokinase domain mediates its effect on Toxoplasma virulence. Journal of Biological Chemistry, 286(33), 29366–29375. [CrossRef] [Google Scholar]
  37. Reichel MP, Alejandra Ayanegui-Alcerreca M, Gondim LF, Ellis JT. 2013. What is the global economic impact of Neospora caninum in cattle – the billion dollar question. International Journal for Parasitology, 43(2), 133–142. [CrossRef] [PubMed] [Google Scholar]
  38. Shin Y-S, Shin G-W, Kim Y-R, Lee E-Y, Yang H-H, Palaksha K, Youn H-J, Kim J-H, Kim D-Y, Marsh A. 2005. Comparison of proteome and antigenic proteome between two Neospora caninum isolates. Veterinary Parasitology, 134(1–2), 41–52. [CrossRef] [PubMed] [Google Scholar]
  39. Shin YS, Lee EG, Shin GW, Kim YR, Lee EY, Kim JH, Jang H, Gershwin LJ, Kim DY, Kim YH, Kim GS, Suh MD, Jung TS. 2004. Identification of antigenic proteins from Neospora caninum recognized by bovine immunoglobulins M, E, A and G using immunoproteomics. Proteomics, 4(11), 3600–3609. [CrossRef] [PubMed] [Google Scholar]
  40. Silmon de Monerri NC, Yakubu RR, Chen AL, Bradley PJ, Nieves E, Weiss LM, Kim K. 2015. The ubiquitin proteome of Toxoplasma gondii reveals roles for protein ubiquitination in cell-cycle transitions. Cell Host & Microbe, 18(5), 621–633. [CrossRef] [PubMed] [Google Scholar]
  41. Sohn CS, Cheng TT, Drummond ML, Peng ED, Vermont SJ, Xia D, Cheng SJ, Wastling JM, Bradley PJ. 2011. Identification of novel proteins in Neospora caninum using an organelle purification and monoclonal antibody approach. PLoS One, 6(4), e18383. [CrossRef] [PubMed] [Google Scholar]
  42. Sun H, Zhuo X, Zhao X, Yang Y, Chen X, Yao C, Du A. 2017. The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth. Parasite, 24, 22. [CrossRef] [EDP Sciences] [PubMed] [Google Scholar]
  43. Thomson S, Hamilton CA, Hope JC, Katzer F, Mabbott NA, Morrison LJ, Innes EA. 2017. Bovine cryptosporidiosis: impact, host-parasite interaction and control strategies. Veterinary Research, 48(1), 42. [CrossRef] [PubMed] [Google Scholar]
  44. Toledo R, Bernal MD, Marcilla A. 2011. Proteomics of foodborne trematodes. Journal of Proteomics, 74(9), 1485–1503. [CrossRef] [PubMed] [Google Scholar]
  45. Wang S, Zhang Z, Wang Y, Gadahi JA, Xu L, Yan R, Song X, Li X. 2017. Toxoplasma gondii elongation factor 1-alpha (TgEF-1alpha) is a novel vaccine candidate antigen against toxoplasmosis. Frontiers in Microbiology, 8, 168. [PubMed] [Google Scholar]
  46. Wang T, Maser P, Picard D. 2016. Inhibition of Plasmodium falciparum Hsp90 contributes to the antimalarial activities of aminoalcohol-carbazoles. Journal of Medicinal Chemistry, 59(13), 6344–6352. [CrossRef] [PubMed] [Google Scholar]
  47. Wiengcharoen J, Thompson RA, Nakthong C, Rattanakorn P, Sukthana Y. 2011. Transplacental transmission in cattle: is Toxoplasma gondii less potent than Neospora caninum? Parasitology Research, 108(5), 1235–1241. [CrossRef] [PubMed] [Google Scholar]
  48. Zhang H, Lee E-g, Yu L, Kawano S, Huang P, Liao M, Kawase O, Zhang G, Zhou J, Fujisaki K. 2011. Identification of the cross-reactive and species-specific antigens between Neospora caninum and Toxoplasma gondii tachyzoites by a proteomics approach. Parasitology Research, 109(3), 899–911. [CrossRef] [PubMed] [Google Scholar]

Cite this article as: Udonsom R, Reamtong O, Adisakwattana P, Popruk S, Jirapattharasate C, Nishikawa Y, Inpankaew T, Toompong J, Kotepui M & Mahittikorn A. 2022. Immunoproteomics to identify species-specific antigens in Neospora caninum recognised by infected bovine sera. Parasite 29, 60.

All Tables

Table 1

List of known bovine serum samples used this study.

Table 2

List of proteins identified on the 2-DE profiles of Neospora caninum (Nc-1) tachyzoites probed with known bovine sera analysed by mass spectrometry (LC-MS/MS).

Table 3

List of proteins identified as specific and/or cross-reactive antigens of Neospora caninum and other apicomplexan protozoa in the bovine host.

Table 4

Functional classification of immunoreactive proteins against Neospora caninum.

All Figures

thumbnail Figure 1

Two-dimensional electrophoresis protein patterns of N. caninum (Nc-1) tachyzoites separated using 12% acrylamide and visualised by silver staining. A total of 70 immunoreactive protein spots (spot numbers 1–70) were identified on the 2-DE gel based on immunoblot analysis that were recognised by known bovine sera. The circle or ellipse with arrows indicates 20 specific protein spots that were recognised by N. caninum-positive bovine pooled sera.

In the text
thumbnail Figure 2

Immunoblot analysis of 2-DE-separated N. caninum tachyzoite antigens using pooled bovine anti-N. caninum (A), anti-T. gondii (B), anti-C. parvum (C), anti-B. bovis (D) and anti-B. bigemina (E) sera and healthy serum (F). The arrows indicate 20 protein spots corresponding to 14 different antigenic proteins recognised by N. caninum-positive sera, and those without arrows indicate spots cross-reactive with other apicomplexan protozoan-infected sera.

In the text

Current usage metrics show cumulative count of Article Views (full-text article views including HTML views, PDF and ePub downloads, according to the available data) and Abstracts Views on Vision4Press platform.

Data correspond to usage on the plateform after 2015. The current usage metrics is available 48-96 hours after online publication and is updated daily on week days.

Initial download of the metrics may take a while.