Reproductive Problems Due to Neospora Caninum in Dairy Cows from Greece

Neospora caninum (Sarcocystidae) is a protozoan parasite which is the etiological agent of neosporosis [1-3]. This apicomplexan parasite was initially recognized in 1984 from dogs in Norway [4] and was described as a new genus and species in 1988 [5]. In the N. caninum life cycle, dogs and other related canids are, beside their role as intermediate hosts, the only definitive hosts that shed through their feces the oocysts for a variable period of time into the environment [6-9]. N. caninum has been reported in a large number of intermediate hosts worldwide, such as ruminants, horses, rabbits, mice, deer, badgers, polecats, ferrets and mink [10-12], while it is not considered as a zoonotic agent although antibodies against N. caninum have been reported in human tissues [13]. Neosporosis has emerged as a serious disease only in cattle and dogs. Regarding cattle, neosporosis is recognized as one of the most important causes of cow reproductive and infertility problems and abortions worldwide while it can also cause increased mortality in newborn calves [8,10]. When a cow becomes infected from dog feces, the infection spreads via tahyzoites to other tissues of the body and to the placenta in cases of pregnancy, where damage to the placenta or vertical transmission through the placenta to the fetus can occur. Vertical transmission of N. caninum is considered the principal route of infection in cattle [10] while a cow may pass the infection to multiple offspring [14]. Abortion may be a result of both the primary damage and the immune mediated inflammatory response of the cow [15,16]. Calves that are infected in utero may be born weak, underweight and with neurological symptoms such as ataxia, decreased reflexes and exophthalmia [3,8]. Neosporosis has a serious economic impact within a flock especially if the prevalence is high. This results from direct costs such as the value of fetuses and indirect costs, including veterinarian support, costs associated with rebreeding, possible loss of milk yield, and replacement costs if aborted cows are culled [17,18]. The aim of this study was to describe the rate of N. caninum infection among cows that presented reproductive problems in Greece.

During 2018-2021, blood samples were collected in Greece from 931 dairy cows that presented reproductive problems, including abortions (n=384) and decreased reproductive performance (n=547). All samples were examined by an indirect Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of IgG antibodies against N. caninum (IDVet, Montpellier, France). In addition, tissue samples from aborted fetuses were collected from cows that were found positive for N. caninum antibodies and were examined by nested polymerase chain reaction (PCR) for N. caninum. The presence of the NC-5 gene was evaluated using the primers and conditions described by Corbellini [19].

Antibodies against N. caninum were found in 200 (21.5%) of the 931 samples tested (Table 1). Eighty-seven of the positive samples originated from cows that had aborted and 113 from cows with decreased fertility. Among the cows that presented abortion, 48 had aborted in the first lactating period, 10 in the second and 29 in early gestation (32-70 days). Nested PCR for N. caninum in seven brain samples, which were collected from the aborted fetuses of an equal number of positive for N. caninum cows, recorded a positive result. Brain samples were not collected from the fetuses of all the cows that had aborted either due to early abortion (32-70 days) and subsequent insufficient development of the brain (n=29) or due to advanced autolysis of the fetus (n=51).

Table 1: Characteristics of the farms included in the study.

Abortions and neonatal mortality are a major problem in livestock. Neosporosis is recognized as one of the most important causes of reproductive problems and abortion in cattle worldwide [8,10,20,21]. Abortions and neonatal mortality can cause severe financial losses, especially when the disease is endemic or epidemic. In this study a large amount of reproductive problem cases was investigated using the combination of two techniques (ELISA and PCR). The results recorded in our research showed the spread of Neosporosis among 21.5% of the dairy cows that had such problems. Previous studies have shown prevalence of N. caninum infection among cows presenting abortion that varies from 12 to 42% [22-25]. Abortions due to N. caninum have been described to occur starting in month three of gestation and until delivery [10,21,26] in an epidemic or endemic manner [27]. Of note, our study records several cases occurring between the first 32 to 70 days of gestation. In addition, the present study records that most miscarriages due to neosporosis occurred during the first lactation period, which indicates that the infections were recent and that preventive measures should be implemented on the infected farms. N. caninum can also cause fetal viability disorders or neurological birth defects in newborn calves [28,29] and calves younger than 2 months of age [30]. The N. caninum-infected young calves may present neurologic signs, low birth weight [6], difficulties to rise and stand, flexed or hyperextended hind and/or forelimbs, and in some cases exophthalmia or asymmetrical appearance of the eyes. However, most of the calves born congenitally infected remain clinically healthy [31-33]. The aforementioned situations lead to additional financial losses for the breeders and, thus, the employment of preventive measures is necessary [32]. In cattle, the transplacental transmission is the most frequent route of infection, being observed in up to 93.7% of cases [10]. Dogs play an important role in the horizontal transmission and maintenance of N. caninum infection in dairy cattle [6-8] and subsequently keeping dogs away from infected breeding is recommended [34].

Neosporosis is a very important parasitic disease which can cause infertility, abortions, neonatal mortality and other clinical symptoms in dairy cattle. Frequent preventive laboratory tests are needed to determine the causes of infertility, miscarriages and birth of calves with characteristic clinic symptoms. Preventive measures and special management are needed in infected flocks with N. caninum.

This research has been co‐financed by the European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation, under the call RESEARCH– CREATE–INNOVATE (Acronym: the summer cow, project code: T1EDK-01078).

1. Donahoe SL, Lindsay SA, Krockenberger M, Phalen D, Slapeta J (2015) A review of neosporosis and pathologic findings of Neospora caninum infection in wildlife. Int J Parasitol Parasites Wildl 24(2): 216-238.
2. Dubey JP, Jenkins MC, Kwok OCH, Ferreira LR, Choudhary S, et al. (2013) Congenital transmission of Neospora caninum in white-tailed deer (Odocoileus virginianus). Vet Parasitol 196(3-4): 519-522.
3. Dubey JP, Lindsay DS (1996) A review of Neospora caninum and neosporosis. Vet Parasitol 67(1-2): 1-59.
4. Bjerkås I, Mohn SF, Presthus J (1984) Unidentified cyst-forming sporozoon causing encephalomyelitis and myositis in dogs. Z Parasitenkd 70(2): 271-274.
5. Dubey JP, Hattel AL, Lindsay DS, Topper MJ (1988) Neonatal Neospora caninum infection in dogs: isolation of the causative agent and experimental transmission. J Am Vet Med Assoc 193(10): 1259-1263.
6. Dubey JP, Schares G (2011) Neosporosis in animals the last five years. Veterinary Parasitology 180(1-2): 90-108.
7. King JS, Šlapeta J, Jenkins DJ, Qassab SE, Ellis JT, et al. (2010) Australian dingoes are definitive hosts of Neospora caninum. Int J Parasitology 40(8): 945-950.
8. Reichel MP, Alejandra AA, Gondim LFP, Ellis JT (2013) What is the global economic impact of Neospora caninum in cattle-the billion-dollar question. Int J Parasitol 43(2): 133-142.
9. Dubey J, Barr B, Barta J, Bjerkås I, Björkman C, et al. (2002) Redescription of Neospora caninum and its differentiation from related coccidia. Int J Parasitol 32(8): 929-946.
10. Dubey JP, Schares G, Ortega M (2007) Epidemiology and control of neosporosis and caninum. Clin Microbiol Rev 20(2): 323-367.
11. Gondim LFP, McAllister MM, Pitt WC, Zemlicka DE (2004) Coyotes (Canis latrans) are definitive hosts of Neospora caninum. Int J Parasitol 34(2): 159-161.
12. Haddad JPA, Dohoo IR, VanLeewen JA (2005) A review of Neospora caninum in dairy and beef cattle a Canadian perspective. Can Vet J 46(3): 230-243.
13. Lobato J, Silva DAO, Mineo TWP, Amaral JDHF, Segundo GRS, et al. (2006) Detection of immunoglobulin G antibodies to Neospora caninum in humans: high seropositivity rates in patients who are infected by human immunodeficiency virus or have neurological disorders. Clin Vaccine Immunol 13(1) 84-89.
14. Corbellini LG, Pescador FF, Wunder E, Steffen D, Smith D, et al. (2006) Diagnostic survey of bovine abortion with special reference to Neospora caninum infection: importance, repeated abortion and concurrent infection in aborted fetuses in Southern Brazil. Vet J 172(1): 114-112.
15. Maley SW, Buxton D, Rae AG, Wright SE, Schock A, et al. (2003) The pathogenesis of neosporosis in pregnant cattle: inoculation at mid-gestation. J Comp Pathol 129(2-3): 186-195.
16. Hernandez J, Risco C, Donovan A (2001) Association between exposure to Neospora caninum and milk production in dairy cows. J Am Vet Med Assoc 219(5): 632-635.
17. Ansari LM, Rowshan GA, Jesmani H, Masoudian M, Badkoobeh M (2017) Association of Neospora caninum with reproductive performance in dairy cows: A prospective study from Iran. Vet res Forum 8(2): 109-114.
18. Hughes JM, Williams RH, Morley EK, Cook DA, Terry RS, et al. (2006) The prevalence of Neospora caninum and co-infection with Toxoplasma gondii by PCR analysis in naturally occurring mammal populations. Parasitology 132(1): 29-36.
19. Corbellini LG, Pescador FF, Wunder E, Steffen D, Smith D, et al. (2006) Diagnostic survey of bovine abortion with special reference to Neospora caninum infection: importance, repeated abortion and concurrent infection in aborted fetuses in Southern Brazil Vet J 172(1): 114-112.
20. Kim JH, Lee JK, Lee BC, Park BK, Yoo HS, et al. (2002) Diagnostic survey of bovine abortion in Korea: with special emphasis on Neospora caninum. J Vet Med Sci 64(12): 1123-1127.
21. Piagentini M, Moya ACF, Prestes NC, Sartor IF (2012) Neospora caninum infection dynamics in dairy cattle. Parasitol Res 111(2): 717-721.
22. Xu MJ, Liu QY, Fu JH, Nisbet AJ, Shi DS, et al. (2012) Seroprevalence of toxoplasma gondii and Neospora caninum infection in dairy cows in dairy cows in subtropical southern China. Parasitology 139(11): 1425-1428.
23. Hall CA, Reichel MP, Ellis JT (2005) Neospora abortions in dairy cattle: diagnosis, mode of transmission and control. Vet Parasitol 128(3-4): 231-241.
24. Jenkins M, Baszler T, Bjorkman C, Schares G, Williams D (2002) Diagnosis and seroepidemiology of Neospora caninum-associated bovine abortion. Int J Parasitol 32(5): 631-636.
25. Reiterová K, Špilovská S, Antolová D, Dubinský P (2009) Neospora caninum, potential cause of abortions in dairy cows: The current serological follow-up in Slovakia. Vet Parasitol 159(1): 1-6.
26. Wouda W, Bartels CJM, Moen AR (1999) Characteristics of Neospora caninum associated abortion storms in dairy herds in the Netherlands (1995-1997). Theriogenology 52(2): 233-245.
27. Lassen B, Orro T, Aleksejev A, Raaperi K, Järvis T, Viltrop A (2012) Neospora caninum in Estonian dairy herds in relation to herd size, reproduction parameters, bovine virus diarrhoea virus, and bovine herpes virus. Vet Parasitol 190(1-2): 43-50.
28. Malaguti JMA, Cabral AD, Abdalla RP, Salgueiro YO, Galleti NTC, et al. (2012) Neospora caninum as causative agent of bovine encephalitis in Brazil. Rev Bras Parasitol Vet 21: 48-54.
29. Dubey JP (2003) Review of Neospora caninum and neosporosis in animals. Korean J Parasitol 41(1): 1-16.
30. Pan Y, Jansen GB, Duffield TF, Hietala S, Kelton, et al. (2004) Genetic susceptibility to Neospora caninum infection in Holstein cattle in Ontario. J Dairy Sci 87(11): 3967-3975.
31. Bielanski A, Phipps TB, Robinson J (2002) Effect of Neospora caninum on in vitro development of preimplantation stage bovine embryos and adherence to the zona pellucida. Veterinary Record 150: 316-318.
32. Dubey JP (1999) Neosporosis in cattle: biology and economic impact. J Am Vet Med Assoc 214(8): 1160-1163.
33. Schares G, Peters M, Wurm R, Bärwald A, Conraths F (1998) The efficiency of vertical transmission of Neospora caninum in dairy cattle analysed by serological techniques. Vet Parasitol 80(2): 87-98.
34. Thurmond MC, Hietala SK (1997) Effect of congenitally acquired Neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle. Am J Vet Res 58(12): 1381-1385.