Molecular Identification and Histopathological Findings of Neospora caninum as a Cause of Bovine Abortion in Some Egyptian Dairy Farms

Document Type : Original Article

Authors

1 Biotechnology Research Unit, Animal Reproduction Research Institute, Agricultural Research Center, Giza, Egypt

2 Pathology of Reproduction Research Department, Animal Reproduction Research Institute, Agricultural Research Center, Giza, Egypt

3 Parasitology Department, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt

Abstract

Neospora caninum infection is a major cause of abortion in dairy and beef cattle in many countries. Fast and accurate diagnosis of neosporosis is still the best disease control strategy. Thus, Np6 and Np21 primer sets were employed for the detection of N. caninum in the blood of 139 aborted cows and tissues of 25 foeti during a wave of abortion in five dairy farms in Egypt. The results revealed a 47.5% overall prevalence of N. caninum infection in aborted cows and a 64% overall prevalence in foeti. Two farms were PCR negative for the infection, and the other farms presented 45.7%, 87.0% and 90.9% molecular prevalence. The majority of PCR-positive foeti were aborted in the fifth month of pregnancy. The most common PCR-positive fetal tissue was the brain, followed by the heart, liver, stomach contents, and then the lung. The histopathological lesions in 5-month-old aborted foeti were microgliosis in the brain meninges and submeningially and myocarditis in heart tissue. At 7 months of abortion, multifocal necrosis, mononuclear cell infiltration, and neuron degeneration were observed in the brain. Heart tissue showed hemorrhage and necrotic changes. A tissue cyst was observed in the heart of 5-month aborted foeti and the brain of 7-month-old foeti. The sequenced amplicons from aborted cows and foeti were 100% identical to each other. As far as we are aware, our investigation is the first to sequence isolates of N. caninum from cattle hosts. The sequences were submitted to GenBank with the accession numbers OR939832.1 and PP708713.1. Multiple sequence alignments revealed variation between the study isolates and other published isolates from different regions and hosts. Phylogeny revealed clustering of our sequences with sequences of isolates from South Korea, China, and Italy. The sequences were distinct from sequences previously isolated from camels in Egypt.

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Main Subjects

References

AHMED, N. E., AL–AKABWAY, L. M., RAMADAN, M. Y., EL-GAWAD, A., MOHAMED, S., and MOUSTAFA, M., 2017. Serological and PCR-sequencing assays for diagnosis of Toxoplasma gondii and Neospora caninum infecting camels in Egypt. Benha Veterinary Medical Journal, 33 (2), pp.200-210. https://doi.org/10.21608/bvmj.2017.30466
AL-QASSAB, S. E., REICHEL, M. P., and ELLIS, J., 2010. On the biological and genetic diversity in Neospora caninum. Diversity 2, 27. https://doi.org/10.3390/d2030411
ALMERÍA, S., ARAUJO, R., TUO, W., LOPEZ-GATIUS, F., DUBEY, J., P., and GASBARRE, L., C., 2010. Fetal death in cows experimentally infected with Neospora caninum at 110 days of gestation. Veterinary Parasitology, 169 (3-4), pp.304-311.  https://doi.org/10.1016/j.vetpar.2009.12.044
ALMERÍA, S., 2013. Neospora caninum and wildlife. International Scholarly Research Notices. https://doi.org/10.5402/2013/947347
ALMERÍA, S., SERRANO-PEREZ, B., DARWICH, L., DOMINGO, M., MUR-NOVALES, R., REGIDOR-CERRILLO, J., CABEZÓN, O., PÉREZ-MAILLO, M., LOPEZ-HELGUERA, I., FERNÁNDEZ-AGUILAR, X., and PUIG-RIBAS, M., 2016. Foetal death in naive heifers inoculated with Neospora caninum isolate Nc-Spain7 at 110 days of pregnancy. Experimental Parasitology, 168, pp.62-69. https://doi.org/10.1016/j.exppara.2016.06.009
AL-KAABI, N. A. B. M., AL-RIKABY, R. S. A., and ALKAABAWI, N. A. M., 2023. Molecular detection and prevalence study of Neospora caninum isolated from blood of aborted cows in babylon province of Iraq. Online Journal of Animal and Feed Research, 13(5), pp. 410-415.  https://doi.org/10.51227/ojafr.2023.57
BJERKÅS, I., MOHN, S. F., and PRESTHUS, J., 1984. Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs.  Z. Parasitenkd. 70, 271–274. https://doi.org/10.1007/BF00942230
BASZLER, T. V., GAY, L. J., LONG, M. T., and MATHISON, B. A., 1999. Detection by PCR of Neospora caninum in fetal tissues from spontaneous bovine abortions. Journal of Clinical Microbiology, 37 (12), pp.4059-4064. https://doi.org/10.1128/jcm.37.12.4059-4064.1999
BUXTON. D., MCALLISTER, M. M., and DUBEY, J. P., 2002. The comparative pathogenesis of neosporosis. Trends in Parasitology, 18 (12), pp.546-552. https://doi.org/10.1016/s1471-4922(02)02414-5
BERECZKY, S., MARTENSSON, A., GIL, J. P., and FARNERT, A., 2005. Rapid DNA extraction from archive blood spots on filter paper for genotyping of Plasmodium falciparum. American Journal of Tropical Medicine and Hygiene, 72(3), pp.249-251. https://doi.org/10.4269/ajtmh.2005.72.249
BANCROFT, J. D., and GAMBLE, M., 2008. Theory and practice of histological techniques (sixth edition). Elsevier Health Sciences. Pages xiii.  https://doi.org/10.1016/b978-0-443-10279-0.50006-3
BAHRAMI, S., HAMIDINEJAT, H., FATEMI-TABATABAEI, S. R., and SARDARIFAR, S., 2018. Effect of natural neosporosis on bull sperm quality. Tropical Animal Health and Production, 50, pp.85-89. https://doi.org/10.1007/s11250-017-1403-8
COLLANTES-FERNÁNDEZ, E., RODRÍGUEZ-BERTOS, A., ARNÁIZ-SECO, I., MORENO, B., ADURIZ, G., and ORTEGA-MORA, L. M., 2006. Influence of the stage of pregnancy on Neospora caninum distribution, parasite loads and lesions in aborted bovine foetuses. Theriogenology, 65 (3), pp. 629-641. https://doi.org/10.1016/j.theriogenology.2005.06.003
CALARCO L., and ELLIS, J., 2020. Species diversity and genome evolution of the pathogenic protozoan parasite, Neospora caninum. Infection, Genetics and Evolution 84. 104444. https://doi.org/10.1016/j.meegid.2020.104444
DUBEY, J. P. 2003. Review of Neospora caninum and neosporosis in animals. The Korean Journal of Parasitology, 41(1), p.1. https://doi.org/10.3347/kjp.2003.41.1.1
DUBEY, J. P., KNICKMAN, E., and GREENE, C. E., 2005. Neonatal Neospora caninum infections in dogs. Acta Parasitologica 50(2), pp.176-179.
DUBEY, J. P. and SCHARES, G., 2006. Diagnosis of bovine neosporosis. Veterinary Parasitology, 140(1-2), pp. 1-34. https://doi.org/10.1016/j.vetpar.2006.03.035
DUBEY, J. P., BUXTON, D., and WOUDA, W., 2006. Pathogenesis of bovine neosporosis. Journal of Comparative Pathology, 134 (4), pp. 267-289. https://doi.org/10.1016/j.jcpa.2005.11.004
DUBEY, J. P., SCHARES, G., and ORTEGA-MORA, L., 2007a. Epidemiology and control of neosporosis and Neospora caninum. Clinical Microbiology Reviews, 20(2), pp.323-367. https://doi.org/10.1128/cmr.00031-06
DUBEY, J. P., and SCHARES, G., 2011. Neosporosis in animals—the last five years. Veterinary Parasitology, 180 (1-2), pp. 90-108. https://doi.org/10.1016/j.vetpar.2011.05.031
DA COSTA, L. S., WITHOEFT, J. A., BILICKI, J. V., MELO, I. C., SNAK, A., DAS NEVES, G. B., MILETTI, L. C., DE MOURA, A. B., and CASAGRANDE, R. A., 2022. Neospora caninum-associated abortions in cattle from Southern Brazil: anatomopathological and molecular characterization. Veterinary Parasitology: Regional Studies and Reports, 36, p.100802. https://doi.org/10.1016/j.vprsr.2022.100802
EAGLESOME, M. D., and GARCIA, M. M., 1997. Disease risks to animal health from artificial insemination with bovine semen. Revue Scientifique et Technique (International Office of Epizootics), 16 (1), pp. 215-225. https://doi.org/10.20506/rst.16.1.1017
FELSENSTEIN, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-791. https://doi.org/10.1111/j.1558-5646.1985.tb00420.x
GHANEM, M. E., SUZUKI, T., AKITA, M., and NISHIBORI, M., 2009. Neospora caninum and complex vertebral malformation as possible causes of bovine fetal mummification. The Canadian Veterinary Journal, 50(4), p.389. https://doi.org/10.1016/j.anireprosci.2007.05.006
GABER, A., HEGAZY, Y. M., OREIBY, A. F., and AL-GAABARY, M. H., 2021. Neosporosis: a neglected abortifacient disease in Egypt, seroprevalence and farmers’ knowledge, attitudes and practices. Journal of the Hellenic Veterinary Medical Society, 72 (3), pp. 3109-3116. https://doi.org/10.12681/jhvms.28500
INNES, E. A., WRIGHT, S. E., MALEY, S., RAE, A., SCHOCK, A., KIRVAR, E., BARTLEY, P., HAMILTON, C., CAREY, I. M., BUXTON D., 2001. Protection against vertical transmission in bovine neosporosis. International Journal for Parasitology, 31 (13), pp.1523-1534. https://doi.org/10.1016/s0020-7519(01)00284-3
JAPA, O., NUANGMEK, A., PRAKHAMMIN, K., and FLYNN, R. J., 2019. Prevalence of vertically transmitted Neospora caninum amongst beef cattle in Phayao, Thailand. Parasitology International, 70, pp. 98-101. https://doi.org/10.1016/j.parint.2019.02.008
KUMAR, S., STECHER, G., and TAMURA, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7), pp.1870-1874. https://doi.org/10.1093/molbev/msw054
MORALES, E., TRIGO, F. J., IBARRA, F., PUENTE, E., and SANTACRUZ, M., 2001. Neosporosis in Mexican dairy herds: lesions and immunohistochemical detection of Neospora caninum in foeti. Journal of Comparative Pathology, 125 (1), pp.58-6 https://doi.org/10.1053/jcpa.2001.0477 
MOORE, D. P., REGIDOR-CERRILLO, J., MORRELL, E., POSO, M. A., CANO, D. B., LEUNDA, M. R, LINSCHINKY, L., ODEON, A. C., ODRIOZOLA, E., ORTEGA-MORA, L. M., and CAMPERO, C. M., 2008. The role of Neospora caninum and Toxoplasma gondii in spontaneous bovine abortion in Argentina. Veterinary Parasitology, 156(3-4), pp.163-167. https://doi.org/10.1016/j.vetpar.2008.06.020 
MELENDEZ, P., ILHA, M., WOLDEMESKEL, M., GRAHAM, J., COARSEY, M., BAUGHMAN, D., WHITTINGTON, L., and NAIKARE, H., 2021. An outbreak of Neospora caninum abortion in a dairy herd from the State of Georgia, United States. Veterinary Medicine and Science, 7 (1), pp. 141-147. https://doi.org/10.1002/vms3.346
METWALLY, S., HAMADA, R., SOBHY, K., FREY, C. F., and FEREIG, R. M., 2023. Seroprevalence and risk factors analysis of Neospora caninum and Toxoplasma gondii in cattle of Beheira, Egypt. Frontiers in Veterinary Science, 10, p.1122092. https://doi.org/10.3389/fvets.2023.1122092
NEMATOLLAHI, A., MOGHADDAM, G. H., JAAFARI, R., HELAN, J. A., and NOROUZI, M., 2013. Study on outbreak of Neospora caninum-associated abortion in dairy cows in Tabriz (Northwest Iran) by serological, molecular and histopathologic methods. Asian Pacific Journal of Tropical Medicine, 6(12), pp.942-946. https://doi.org/10.1016/s1995-7645(13)60168-6
OKEOMA, C. M., WILLIAMSON, N. B., POMROY, W. E., STOWELL, K. M., and GILLESPIE, L., 2004. The use of PCR to detect Neospora caninum DNA in the blood of naturally infected cows. Veterinary Parasitology, 122 (4), pp. 307-315. https://doi.org/10.1016/j.vetpar.2004.06.001
PESCADOR, C. A., CORBELLINI, L. G., OLIVEIRA, E. C., RAYMUNDO, D. L., and DRIEMEIER, D., 2007. Histopathological and immunohistochemical aspects of Neospora caninum diagnosis in bovine aborted foeti. Veterinary Parasitology, 150(1-2), pp.159-163.. https://doi.org/10.1016/j.vetpar.2007.08.028
PEREIRA, G. R., VOGEL, F. S. F., BOHRER, R. C., DA NÓBREGA, J. E., ILHA, G. F., DA ROSA, P. R A., GLANZNER, W. G., CAMILLO, G., BRAUNIG, P., DE OLIVEIRA, J. F. C., and GONÇALVES, P. B. D., 2014. Neospora caninum DNA detection by TaqMan real-time PCR assay in experimentally infected pregnant heifers. Veterinary Parasitology, 199 (3-4), pp. 129-135. https://doi.org/10.1016/j.vetpar.2013.10.018
 REITT, K., HILBE, M., VOEGTLIN, A., CORBOZ, L., HAESSIG, M., and POSPISCHIL, A., 2007. Aetiology of bovine abortion in Switzerland from 1986 to 1995–a retrospective study with emphasis on detection of Neospora caninum and Toxoplasma gondii by PCR. Journal of Veterinary Medicine Series A, 54(1), pp.15-22. https://doi.org/10.1111/j.1439-0442.2007.00913.x
RAZMI, G, R., MALEKI, M., FARZANEH, N., TALEBKHAN, G. M., and FALLAH, A. H., 2007. First report of Neospora caninum-associated bovine abortion in Mashhad area, Iran. Parasitology Research, 100, pp.755-757. https://doi.org/10.1007/s00436-006-0325-6
RAZMI, G. R., ZAREA, H., NASERI., Z., 2010. A survey of Neospora caninum-associated bovine abortion in large dairy farms of Mashhad, Iran. Parasitology Research, 106, pp.1419-1423. https://doi.org/10.1007/s00436-010-1820-3
ROJO-MONTEJO, S., COLLANTES-FERNÁNDEZ, E., REGIDOR-CERRILLO, J., RODRÍGUEZ-BERTOS, A., PRENAFETA, A., GOMEZ-BAUTISTA, M., and ORTEGA-MORA, L. M., 2011. Influence of adjuvant and antigen dose on protection induced by an inactivated whole vaccine against Neospora caninum infection in mice. Veterinary Parasitology, 175(3-4), pp. 220-229. https://doi.org/10.1016/j.vetpar.2010.10.028
REGIDOR-CERRILLO, J., ARRANZ-SOLÍS, D., BENAVIDES, J., GÓMEZ-BAUTISTA, M., CASTRO-HERMIDA, J. A., MEZO M., PÉREZ, V., ORTEGA-MORA, L. M., GONZÁLEZ-and WARLETA, M., 2014. Neospora caninum infection during early pregnancy in cattle: how the isolate influences infection dynamics, clinical outcome and peripheral and local immune responses. Veterinary Research, 45,pp.1-15. https://doi.org/10.1186/1297-9716-45-10
SINGER-SAM J. 1989. Use of Chelex to improve the PCR signal from a small number of cells. In Forum for PCR Users (Vol. 3).
SALEHI, N., HADADZADEH, H., ASHRAFI, H. J., SHAYAN, P., and SADREBAZZAZ, A., 2009. Molecular and pathological study of bovine aborted foeti and placenta from Neospora caninum infected dairy cattle. Iranian Journal for Parasitology: 4 (3), pp.40-51.
ŞUTEU, O., TITILINCU, A., MODRÝ, D., MIHALCA, A., MIRCEAN, V., and COZMA, V., 2010. First identification of Neospora caninum by PCR in aborted bovine foetuses in Romania. Parasitology Research, 106, pp.719-722. https://doi.org/10.1007/s00436-009-1684-6
SINNOTT, F. A., MONTE, L. G., COLLARES, T. F., SILVEIRA, R. M., and BORSUK, S., 2017. Review on the immunological and molecular diagnosis of neosporosis (years 2011–2016). Veterinary Parasitology, 239, pp. 19-25. https://doi.org/10.1016/j.vetpar.2017.04.008
SINGH, U. A., KUMARI, M., and IYENGAR, S., 2018. Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin. Biological Procedures Online, 20, pp. 1-8 https://doi.org/10.1186/s12575-018-0077-6.
SELIM, A., ALSHAMMARI, A., GATTAN, H. S., MARZOK, M, SALEM, M., and AL-JABR, O. A., 2023. Neospora caninum infection in dairy cattle in Egypt: a serosurvey and associated risk factors. Scientific Reports, 13(1), p.15489. https://doi.org/10.1038/s41598-023-42538-8
SILVA, R. C. D., DE LIMA, S. C., DOS SANTOS REIS, W. P. M., DE MAGALHÃES, J. J. F., MAGALHÃES, R. N. D. O., RATHI, B., KOHL, A., BEZERRA, M. A. C., and PENA, L., 2023. Comparison of DNA extraction methods for COVID-19 host genetics studies. Plos One, 18 (10), p.e 0287551. https://doi.org/10.1371/journal.pone.0287551
TAMURA, K., and NEI, M., 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution, 10 (3), pp. 512-526. https://doi.org/10.1093/oxfordjournals.molbev.a040023
VAN MAANEN, C., WOUDA, W., SCHARES, G., VON BLUMRÖDER, D., CONRATHS, F. J., NORTON, R, WILLIAMS, D. J., ESTEBAN-REDONDO, I., INNES, E. A., MATTSSON, J. G., and BJÖRKMAN, C., 2004. An interlaboratory comparison of immunohistochemistry and PCR methods for detection of Neospora caninum in bovine foetal tissues. Veterinary Parasitology, 126 (4), pp.351-364. https://doi.org/10.1016/j.vetpar.2004.08.016
WOUDA, W., MOEN, A. R., VISSER, I. J. R., and VAN KNAPEN, F., 1997. Bovine fetal neosporosis: a comparison of epizootic and sporadic abortion cases and different age classes with regard to lesion severity and immunohistochemical identification of organisms in brain, heart, and liver. Journal of Veterinary Diagnostic Investigation, 9 (2), pp.180-185.https://doi.org/10.1177/104063879700900212
WALSH, P. S., METZGER, D. A., and HIGUCHI, R., 2013. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques, 54 (3), pp.134-139. https://doi.org/10.2144/000114018
YAMAGE, M., FLECHTNER, O., and GOTTSTEIN, B., 1996. Neospora caninum: specific oligonucleotide primers for the detection of brain" cyst" DNA of experimentally infected nude mice by the polymerase chain reaction (PCR). The Journal of Parasitology, pp. 272-279. https://doi.org/10.2307/3284160
YAO, L., YANG, N., LIU, Q., WANG, M., ZHANG, W., QIAN, W. F., HU, Y. F., and DING, J., 2009. Detection of Neospora caninum in aborted bovine foeti and dam blood samples by nested PCR and ELISA and seroprevalence in Beijing and Tianjin, China. Parasitology, 136 (11), pp. 1251-1256. https://doi.org/10.1017/s0031182009990813
ZHANG, W., DENG, C., LIU, Q., LIU, J., WANG, M., TIAN, K. G., YU, X. L., and HU, D. M., 2007. First identification of Neospora caninum infection in aborted bovine foetuses in China. Veterinary Parasitology, 149 (1-2), pp. 72-76. https://doi.org/10.1016/j.vetpar.2007.07.013
 
Journal of Applied Veterinary Sciences
Volume 10, Issue 1
January 2025
Pages 46-56

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  • Receive Date: 11 October 2024
  • Revise Date: 11 November 2024
  • Accept Date: 20 November 2024

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