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Development and characterization of monoclonal antibodies against Besnoitia besnoiti tachyzoites

Published online by Cambridge University Press:  16 August 2018

P. García-Lunar ,
G. Schares ,
A. Sanz-Fernández ,
A. Jiménez-Meléndez ,
I. García-Soto ,
J. Regidor-Cerrillo Open the ORCID record for J. Regidor-Cerrillo [Opens in a new window] ,
I. Pastor-Fernández ,
A. Hemphill Open the ORCID record for A. Hemphill [Opens in a new window] ,
M. Fernández-Álvarez  and
L. M. Ortega-Mora
...Show all authors
P. García-Lunar
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
G. Schares
Affiliation:
Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Epidemiology, Greifswald-Insel Riems, Germany
A. Sanz-Fernández
Affiliation:
Inmunología y Genética Aplicada, SA (INGENASA), Calle de los Hermanos García Noblejas, 41, 28037, Madrid, Spain
A. Jiménez-Meléndez
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
I. García-Soto
Affiliation:
Inmunología y Genética Aplicada, SA (INGENASA), Calle de los Hermanos García Noblejas, 41, 28037, Madrid, Spain
J. Regidor-Cerrillo
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
I. Pastor-Fernández
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
A. Hemphill
Affiliation:
Institute of Parasitology, Vetsuisse Faculty, University of Bern, Länggass-Strasse 122, CH-3012 Bern, Switzerland
M. Fernández-Álvarez
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
L. M. Ortega-Mora
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
G. Álvarez-García*
Affiliation:
SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain
*
Author for correspondence: G. Álvarez-García, E-mail: [email protected]
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Abstract

This is the first report on the development and characterization of eight monoclonal antibodies (MABs) generated against whole- and membrane-enriched tachyzoite extracts of the apicomplexan parasite Besnoitia besnoiti. Confocal laser scanning immunofluorescence microscopy was used to localize respective epitopes in B. besnoiti tachyzoites along the lytic cycle. A pattern compatible with dense granule staining was observed with MABs 2.A.12, 2.F.3 and 2.G.4, which could be confirmed by immunogold electron microscopy for MABs 2.A.12 and 2.F.3. In particular, MABs 2.F.3 and 2.G.4 were secreted during early invasion, proliferation and egress phases. MABs 3.10.8 and 5.5.11 labelled the tachyzoite surface, whilst MABs 1.17.8, 8.9.2 and 2.G.A recognized the apical tip, which is reminiscent for microneme localization. Besides, the epitopes recognized by the latter two (MABs 8.9.2 and 2.G.A) exhibited a redistribution from the anterior part across the parasite surface towards the posterior end during invasion. Most MABs developed were genus-specific. Indeed, the MABs cross-reacted neither with T. gondii nor with N. caninum tachyzoites. In summary, we have generated MABs that will be useful to study the key processes in the lytic cycle of the parasite and with additional promising diagnostic value. However, the molecular identity of the antigens recognized remains to be elucidated.

Keywords

B. besnoiticonfocal laser scanning microscopymonoclonal antibodytachyzoitetransmission electron microscopyWestern blot
Type
Research Article
Information
Parasitology , Volume 146 , Issue 2 , February 2019 , pp. 187 - 196
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Copyright
Copyright © Cambridge University Press 2018 

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References

Aguado-Martínez, A, Álvarez-García, G, Schares, G, Risco-Castillo, V, Fernández-García, A, Marugán-Hernández, V and Ortega-Mora, LM (2010) Characterisation of NcGRA7 and NcSAG4 proteins: immunolocalisation and their role in the host cell invasion by Neospora caninum tachyzoites. Acta Parasitologica 55, 304–312.Google Scholar
Alexander, DL, Mital, J, Ward, GE, Bradley, P and Boothroyd, JC (2005) Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles. PLoS Pathogens 1, e17.Google Scholar
Álvarez-García, G, Frey, CF, Mora, LM and Schares, G (2013) A century of bovine besnoitiosis: an unknown disease re-emerging in Europe. Trends in Parasitology 29, 407–415.Google Scholar
Álvarez-García, G, García-Lunar, P, Gutiérrez-Expósito, D, Shkap, V and Ortega-Mora, LM (2014) Dynamics of Besnoitia besnoiti infection in cattle. Parasitology 141, 1419–1435.Google Scholar
Bartels, CJ, Arnaiz-Seco, JI, Ruiz-Santa-Quitera, A, Bjorkman, C, Frossling, J, von, BD, Conraths, FJ, Schares, G, van, MC, Wouda, W and Ortega-Mora, LM (2006) Supranational comparison of Neospora caninum seroprevalences in cattle in Germany, The Netherlands, Spain and Sweden. Veterinary Parasitology 137, 17–27.Google Scholar
Baszler, TV, Knowles, DP, Dubey, JP, Gay, JM, Mathison, BA and McElwain, TF (1996) Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay. Journal of Clinical Microbiology 34, 1423–1428.Google Scholar
Baszler, TV, Adams, S, Vander-Schalie, J, Mathison, BA and Kostovic, M (2001) Validation of a commercially available monoclonal antibody-based competitive-inhibition enzyme-linked immunosorbent assay for detection of serum antibodies to Neospora caninum in cattle. Journal of Clinical Microbiology 39, 3851–3857.Google Scholar
Black, MW and Boothroyd, JC (2000) Lytic cycle of Toxoplasma gondii. Microbiology and Molecular Biology Reviews: MMBR 64, 607–623.Google Scholar
Blader, IJ, Coleman, BI, Chen, C and Gubbels, M (2015) Lytic cycle of Toxoplasma gondii: 15 years later. Annual Review of Microbiology 69, 463–485.Google Scholar
Cortes, H, Leitao, A, Gottstein, B and Hemphill, A (2014) A review on bovine besnoitiosis: a disease with economic impact in herd health management, caused by Besnoitia besnoiti (Franco and Borges, 1916). Parasitology 141, 1406–1417.Google Scholar
Dubey, JP, Hattel, AL, Lindsay, DS and Topper, MJ (1988) Neonatal Neospora caninum infection in dogs: isolation of the causative agent and experimental transmission. Journal of the American Veterinary Medical Association 193, 1259–1263.Google Scholar
Dubey, JP, Shkap, V, Pipano, E, Fish, L and Fritz, DL (2003) Ultrastructure of Besnoitia besnoiti tissue cysts and bradyzoites. The Journal of Eukaryotic Microbiology 50, 240–244.Google Scholar
Dubey, JP, Sreekumar, C, Rosenthal, BM, Vianna, MC, Nylund, M, Nikander, S and Oksanen, A (2004) Redescription of Besnoitia tarandi (Protozoa: Apicomplexa) from the reindeer (Rangifer tarandus). International Journal for Parasitology 34, 1273–1287.Google Scholar
Dunn, JD, Ravindran, S, Kim, SK and Boothroyd, JC (2008) The Toxoplasma gondii dense granule protein GRA7 is phosphorylated upon invasion and forms an unexpected association with the rhoptry proteins ROP2 and ROP4. Infection and Immunity 76, 5853–5861.Google Scholar
Eiras, C, Arnaiz, I, Álvarez-García, G, Ortega-Mora, LM, Sanjuanl, ML, Yus, E and Dieguez, FJ (2011) Neospora caninum seroprevalence in dairy and beef cattle from the northwest region of Spain, Galicia. Preventive Veterinary Medicine 98, 128–132.Google Scholar
Fernández-García, A, Risco-Castillo, V, Pedraza-Díaz, S, Aguado-Martínez, A, Álvarez-García, G, Gómez-Bautista, M, Collantes-Fernández, E and Ortega-Mora, LM (2009 a) First isolation of Besnoitia besnoiti from a chronically infected cow in Spain. The Journal of Parasitology 95, 474–476.Google Scholar
Fernández-García, A, Álvarez-García, G, Risco-Castillo, V, Aguado-Martínez, A, Marugán-Hernández, V and Ortega-Mora, LM (2009 b) Pattern of recognition of Besnoitia besnoiti tachyzoite and bradyzoite antigens by naturally infected cattle. Veterinary Parasitology 164, 104–110.Google Scholar
Fernández-García, A, Álvarez-García, G, Marugán-Hernández, V, García-Lunar, P, Aguado-Martínez, A, Risco-Castillo, V and Ortega-Mora, LM (2013) Identification of Besnoitia besnoiti proteins that showed differences in abundance between tachyzoite and bradyzoite stages by difference gel electrophoresis. Parasitology 140, 999–1008.Google Scholar
Frey, CF, Regidor-Cerrillo, J, Marreros, N, García-Lunar, P, Gutiérrez-Expósito, D, Schares, G, Dubey, JP, Gentile, A, Jacquiet, P and Shkap, V (2016) Besnoitia besnoiti lytic cycle in vitro and differences in invasion and intracellular proliferation among isolates. Parasites & Vectors 9, 1.Google Scholar
García-Lunar, P, Ortega-Mora, LM, Schares, G, Gollnick, NS, Jacquiet, P, Grisez, C, Prevot, F, Frey, CF, Gottstein, B and Álvarez-García, G (2013 a). An inter-laboratory comparative study of serological tools employed in the diagnosis of Besnoitia besnoiti infection in Bovines. Transboundary and Emerging Diseases 60, 59–68.Google Scholar
García-Lunar, P, Regidor-Cerrillo, J, Gutiérrez-Expósito, D, Ortega-Mora, L and Álvarez-García, G (2013 b) First 2-DE approach towards characterising the proteome and immunome of Besnoitia besnoiti in the tachyzoite stage. Veterinary Parasitology 195, 24–34.Google Scholar
García-Lunar, P, Regidor-Cerrillo, J, Ortega-Mora, LM, Gutiérrez-Expósito, D and Álvarez-García, G (2014) Proteomics reveals differences in protein abundance and highly similar antigenic profiles between Besnoitia besnoiti and Besnoitia tarandi. Veterinary Parasitology 205, 434–443.Google Scholar
García-Lunar, P, More, G, Campero, L, Ortega-Mora, LM and Álvarez-García, G (2015) Anti-Neospora caninum and anti-Sarcocystis spp. specific antibodies cross-react with Besnoitia besnoiti and influence the serological diagnosis of bovine besnoitiosis. Veterinary Parasitology 214, 49–54.Google Scholar
Ghalmi, F, China, B, Kaidi, R and Losson, B (2009) First epidemiological study on exposure to Neospora caninum in different canine populations in the Algiers District (Algeria). Parasitology International 58, 444–450.Google Scholar
Gross, U, Bohne, W, Soete, M and Dubremetz, J (1996) Developmental differentiation between tachyzoites and bradyzoites of Toxoplasma gondii. Parasitology Today 12, 30–33.Google Scholar
Gutiérrez-Expósito, D, Ortega-Mora, LM, Gajadhar, AA, García-Lunar, P, Dubey, JP and Álvarez-García, G (2012) Serological evidence of Besnoitia spp. infection in Canadian wild ruminants and strong cross-reaction between Besnoitia besnoiti and Besnoitia tarandi. Veterinary Parasitology 190, 19–28.Google Scholar
Gutiérrez-Expósito, D, Ferre, I, Ortega-Mora, LM and Álvarez-García, G (2017) Advances in the diagnosis of bovine besnoitiosis: current options and applications for control. International Journal for Parasitology 47, 737–751.Google Scholar
Hemphill, A, Vonlaufen, N, Naguleswaran, A, Keller, N, Riesen, M, Guetg, N, Srinivasan, S and Alaeddine, F (2004) Tissue culture and explant approaches to studying and visualizing Neospora caninum and its interactions with the host cell. Microscopy and Microanalysis 10, 602–620.Google Scholar
Jiménez-Meléndez, A, Ojo, KK, Wallace, AM, Smith, TR, Hemphill, A, Balmer, V, Regidor-Cerrillo, J, Ortega-Mora, LM, Hehl, AB, Fan, E, Maly, DJ, Van Voorhis, WC and Álvarez-García, G (2017) In vitro efficacy of bumped kinase inhibitors against Besnoitia besnoiti tachyzoites. International Journal for Parasitology 47, 811–821.Google Scholar
Langenmayer, M, Gollnick, N, Scharr, J, Schares, G, Herrmann, D, Majzoub-Altweck, M and Hermanns, W (2015) Besnoitia besnoiti infection in cattle and mice: ultrastructural pathology in acute and chronic besnoitiosis. Parasitology Research 114, 955–963.Google Scholar
Leineweber, M, Spekker-Bosker, K, Ince, V, Schares, G, Hemphill, A, Eller, SK and Däubener, W (2017) First characterization of the Neospora caninum dense granule protein GRA9. BioMed Research International 2017, PMID:29259983. doi: 10.1155/2017/6746437.Google Scholar
Li, W, Liu, J, Wang, J, Fu, Y, Nan, H and Liu, Q (2015) Identification and characterization of a microneme protein (NcMIC6) in Neospora caninum. Parasitology Research 114, 2893–2902.Google Scholar
Lodoen, MB, Gerke, C and Boothroyd, JC (2010) A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection. Cellular Microbiology 12, 55–66.Google Scholar
Lunde, MN and Jacobs, L (1963) Toxoplasma hemagglutination and dye test antibodies in experimentally infected rats. The Journal of Parasitology 49, 932–936.Google Scholar
Marcelino, E, Martins, TM, Morais, JB, Nolasco, S, Cortes, H, Hemphill, A, Leitao, A and Novo, C (2011) Besnoitia besnoiti protein disulfide isomerase (BbPDI): molecular characterization, expression and in silico modelling. Experimental Parasitology 129, 164–174.Google Scholar
Marotel, M (1912) Discussion of paper by Besnoit e Robin. Bull.et Mem.de la Soc.des Sciences Vet.de Lyon et de la Soc.de Med.Vet.de Lyon e du Sud-Est 15, 196–217.Google Scholar
More, G, Basso, W, Bacigalupe, D, Venturini, MC and Venturini, L (2008) Diagnosis of Sarcocystis cruzi, Neospora caninum, and Toxoplasma gondii infections in cattle. Parasitology Research 102, 671–675.Google Scholar
Naguleswaran, A, Cannas, A, Keller, N, Vonlaufen, N, Schares, G, Conraths, FJ, Bjorkman, C and Hemphill, A (2001) Neospora caninum microneme protein NcMIC3: secretion, subcellular localization, and functional involvement in host cell interaction. Infection and Immunity 69, 6483–6494.Google Scholar
Naguleswaran, A, Alaeddine, F, Guionaud, C, Vonlaufen, N, Sonda, S, Jenoe, P, Mevissen, M and Hemphill, A (2005) Neospora caninum protein disulfide isomerase is involved in tachyzoite-host cell interaction. International Journal for Parasitology 35, 1459–1472.Google Scholar
Njagi, ON, Entzeroth, R, Nyaga, PN and Musoke, AJ (2004) Monoclonal antibodies identify two neutralization-sensitive epitopes in Besnoitia besnoiti endocytes. Parasitology Research 94, 247–253.Google Scholar
Pastor-Fernández, I, Regidor-Cerrillo, J, Álvarez-García, G, Marugán-Hernández, V, García-Lunar, P, Hemphill, A and Ortega-Mora, LM (2016 a) The tandemly repeated NTPase (NTPDase) from Neospora caninum is a canonical dense granule protein whose RNA expression, protein secretion and phosphorylation coincides with the tachyzoite egress. Parasites & Vectors 9, 352.Google Scholar
Pastor-Fernández, I, Regidor-Cerrillo, J, Jiménez-Ruiz, E, Álvarez-García, G, Marugán-Hernández, V, Hemphill, A and Ortega-Mora, LM (2016 b) Characterization of the Neospora caninum NcROP40 and NcROP2Fam-1 rhoptry proteins during the tachyzoite lytic cycle. Parasitology 143, 97–113.Google Scholar
Risco-Castillo, V, Fernández-García, A and Ortega-Mora, LM (2004) Comparative analysis of stress agents in a simplified in vitro system of Neospora caninum bradyzoite production. Journal of Parasitology 90, 466–470.Google Scholar
Risco-Castillo, V, Fernández-García, A, Zaballos, A, Aguado-Martínez, A, Hemphill, A, Rodríguez-Bertos, A, Álvarez-García, G and Ortega-Mora, LM (2007) Molecular characterisation of BSR4, a novel bradyzoite-specific gene from Neospora caninum. International Journal for Parasitology 37, 887–896.Google Scholar
Schares, G, Conraths, FJ and Reichel, MP (1999 a). Bovine neosporosis: comparison of serological methods using outbreak sera from a dairy herd in New Zealand. International Journal for Parasitology 29, 1659–1667.Google Scholar
Schares, G, Dubremetz, JF, Dubey, JP, Barwald, A, Loyens, A and Conraths, FJ (1999 b) Neospora caninum: identification of 19-, 38-, and 40-kDa surface antigens and a 33-kDa dense granule antigen using monoclonal antibodies. Experimental Parasitology 92, 109–119.Google Scholar
Schares, G, Rauser, M, Söndgen, P, Rehberg, P, Bärwald, A, Dubey, J, Edelhofer, R and Conraths, F (2000) Use of purified tachyzoite surface antigen p38 in an ELISA to diagnose bovine neosporosis. International Journal for Parasitology 30, 1123–1130.Google Scholar
Schares, G, Basso, W, Majzoub, M, Rostaher, A, Scharr, JC, Langenmayer, MC, Selmair, J, Dubey, JP, Cortes, HC, Conraths, FJ and Gollnick, NS (2010) Comparative evaluation of immunofluorescent antibody and new immunoblot tests for the specific detection of antibodies against Besnoitia besnoiti tachyzoites and bradyzoites in bovine sera. Veterinary Parasitology 171, 32–40.Google Scholar
Schares, G, Langenmayer, MC, Scharr, JC, Minke, L, Maksimov, P, Maksimov, A, Schares, S, Barwald, A, Basso, W, Dubey, JP, Conraths, FJ and Gollnick, NS (2013) Novel tools for the diagnosis and differentiation of acute and chronic bovine besnoitiosis. International Journal for Parasitology 43, 143–154.Google Scholar
Schares, G, Venepally, P and Lorenzi, HA (2017) Draft genome sequence and annotation of the apicomplexan parasite Besnoitia besnoiti. Genome Announcements 5, e01200–17.Google Scholar
Shkap, V, Pipano, E and Zwernemann, B (1995) Activity of a monoclonal antibody against Besnoitia besnoiti endozoites. Veterinary Research 26, 328–334.Google Scholar
Sohn, CS, Cheng, TT, Drummond, ML, Peng, ED, Vermont, SJ, Xia, D, Cheng, SJ, Wastling, JM and Bradley, PJ (2011) Identification of novel proteins in Neospora caninum using an organelle purification and monoclonal antibody approach. PLoS ONE 6, e18383.Google Scholar
Soldati, D, Dubremetz, JF and Lebrun, M (2001) Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii. International Journal for Parasitology 31, 1293–1302.Google Scholar
Tenter, AM, Barta, JR, Beveridge, I, Duszynski, DW, Mehlhorn, H, Morrison, DA, Thompson, RC and Conrad, PA (2002) The conceptual basis for a new classification of the coccidia. International Journal for Parasitology 32, 595–616.Google Scholar
Uzeda, R, Schares, G, Ortega-Mora, L, Madruga, C, Aguado-Martinez, A, Corbellini, L, Driemeier, D and Gondim, LFP (2013) Combination of monoclonal antibodies improves immunohistochemical diagnosis of Neospora caninum. Veterinary Parasitology 197, 477–486.Google Scholar
Vonlaufen, N, Muller, N, Keller, N, Naguleswaran, A, Bohne, W, McAllister, MM, Bjorkman, C, Muller, E, Caldelari, R and Hemphill, A (2002) Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures. International Journal for Parasitology 32, 1253–1265.Google Scholar
Weiss, LM, Ma, YF, Halonen, S, McAllister, MM and Zhang, YW (1999) The in vitro development of Neospora caninum bradyzoites. International Journal for Parasitology 29, 1713–1723.Google Scholar
Wouda, W, Brinkhof, J, van Maanen, C, de Gee, AL and Moen, AR (1998) Serodiagnosis of neosporosis in individual cows and dairy herds: a comparative study of three enzyme-linked immunosorbent assays. Clinical and Diagnostic Laboratory Immunology 5, 711–716.Google Scholar