Neosporosis and Abortion in Dairy Cattle

 

Neosporosis and Abortion in Dairy Cattle

Mark Anderson, Bradd Barr, Joan Rowe, Karen Sverlow, Andrea Packham and Patricia Conrad

California Veterinary Diagnostic Laboratory and Department of Pathology,
Microbiology and Immunology,
School of Veterinary Medicine,
University of California Davis,
Davis, California, USA, 95617-1770.
E-mail to the Group at manderso@cvdls.ucdavis.edu

Take Home Messages

Fetal infections by the protozoa parasite, Neospora sp., is a newly recognized cause of abortion and congenital infection in cattle. This infection is the most common cause of abortion seen in many dairies throughout the world. Diagnosis of the infection is assisted by examinations of aborted fetuses and serologic testing of cows. At the present time there is no treatment or prevention for the infection and the life cycle of the parasite is not known. Cattle can be chronically infected with Neospora and can pass the infection onto their offspring during pregnancy.

Introduction

Neospora is a newly recognized genus that was first identified as a Toxoplasma-like protozoa in dogs with encephalomyelitis and myositis (19) and later shown to be the same Neospora caninum parasite isolated from a litter of puppies in the United States (18, 31, 34, 36). The genus designation Neospora has since been applied to a similar protozoal parasite identified in livestock (11, 66). In 1991, the first bovine Neospora isolates were obtained from aborted fetuses and have been maintained in continuous cell culture (24). Whether the canine isolate (Neospora caninum) and bovine isolate represent identical species is not certain. For the present, the infection in livestock is most appropriately referred to as a Neospora species. Neospora have morphologic similarities to Toxoplasma gondii, but can be antigenically differentiated by immunohistochemistry (14). By light microscopy, Neospora can only be differentiated from Toxoplasma in the tissue cyst stage when Neospora often have a thicker cyst wall. There are distinct ultrastructural differences between Toxoplasma gondii and Neospora (14,49).

Infection due to Neospora has been reported in various species of livestock, including cattle (1-5, 10, 11, 14, 26, 27, 32, 35, 37, 40, 42, 45, 52-56, 59, 64, 65, 66, 67, 74, 75, 76), sheep (33,38), goats (13,30), horses (41), and deer (74). Although only recently recognized, bovine neosporosis has emerged as an important reproductive disease. Since its first association with an abortion storm in 1987 in a dairy in New Mexico (66), there have been reports of Neospora abortions in California and the Midwest which have confirmed this infection as a significant cause of abortion, particularly among dairy cattle (1,5,11,53,64). Retrospective studies in California suggest that the parasite has been endemic since at least 1985 (6). In California, 18 to 19% of all aborted bovine fetuses submitted to the California Veterinary Diagnostic Laboratory System (CVDLS) are diagnosed with this infection (5,11). In dairy cattle submissions from California, the proportion of Neospora abortion is even higher, 24.4% (6).

Bovine neosporosis has a worldwide distribution and has been diagnosed in the United States (48), Canada (*,17,48,52), Mexico (1), Britain (26,27,58,59), Netherlands (75), Denmark (2), Australia (20,32,54), Israel (42), New Zealand (67), South Africa (43), Japan (*,56), and Zimbabwe(*). Within the United States 32 states have reported cases including Alabama (48), Arizona (*,53,76), California (3-11), Colorado (*,64), Georgia (48), Idaho (*), Illinois (*), Indiana (35), Iowa (53, 76), Kansas (*), Maryland (*,40), Massachusetts (76), Michigan (*), Minnesota (*, 53,76), Missouri (*,76), Montana (*), Nebraska (48,76), New Hampshire (*), New Mexico (66), New York (*), North Dakota (*), Ohio (*), Oklahoma (*), South Dakota (53,76), Texas (*,53,76), Utah (*), Vermont (*,76), Virginia (*,76), Washington (63,65), West Virginia (*), and Wisconsin (*,53,76).

Pathogenesis

The pathogenic potential of bovine Neospora sp. has been confirmed by experimental infection of pregnant cattle resulting in fetal death and in the birth of an in utero exposed or congenitally infected calf (15). Neospora caninum from dogs also has been experimentally inoculated into pregnant cattle and sheep resulting in transplacental fetal infection in a cow and abortion in sheep (38,39).

At present, the only known natural route by which cattle become infected with Neospora is transplacental infection of the fetus from the infected dam. The routes by which cattle may become infected after birth are unknown, but the similarities of Neospora to other apicomplexan coccidia, particularly Toxoplasma, suggests that postnatal infection may be acquired through oral ingestion of coccidial oocysts shed from an unidentified carnivorous definitive host. Exogenous stages of Neospora such as oocysts or sporocysts have not been identified. Tachyzoites and tissue cysts are the life cycle stages of Neospora currently identified.

The life cycle of Neospora is unknown, but attempts are being made to identify the definitive host of bovine Neospora. Dogs, cats, rats and mice have been screened for Neospora coccidia following experimental infection with bovine Neospora sp. (Conrad P and Barr B, unpublished data, 1992).Dogs, cats, and raccoons have been screened for N. caninum coccidia following experimental infections with N. caninum, but no fecal oocysts identified as Neospora have been identified in these species (48). Four species of birds (red tailed hawk, turkey vulture, barn owl, and crow) have been inoculated with Neospora caninum infected mouse tissue with results that suggest that these particular avian species are probably not definitive hosts for neosporosis (9).

A significant method by which Neospora infections may be maintained within the herd is through prenatal infection of fetuses which survive and become replacements that are chronically infected. There is serologic evidence of congenital transmission of neosporosis in California dairies based on the observation that the rate of seropositivity is not associated with the age of the cow, thus indicating that the incidence of new, postnatal infection is low or nil (61).

Congenitally infected calves can have a chronic persistent infection which can be passed on transplacentally to their offspring. In a survey of heifer calves in a known Neospora dairy herd, 25 calves with serologic evidence of congenital exposure were compared with 25 serologically negative cohorts. The two groups were similar until pregnancy and calving. At the time of calving, blood samples were obtained from the heifer and calf from 38 of the 50 heifers. All calves born to heifers that had elevated Neospora titres as calves (evidence of congenital Neospora exposure) also had elevated Neospora titres (20 of 20). Conversely all of the negative heifers had serologically negative calves (18 of 18) (Mark Anderson and Jim Reynolds, unpublished data, 1995). In addition, there was no evidence of seroconversion to Neospora among the negative heifers and the positive heifers retained serologic titres to Neospora. Ten of the calves were purchased and necropsied, seven serologically positive calves from positive heifers, and three serologically negative calves from negative heifers. All seven of the positive calves had histologic lesions in the brain and spinal cord consistent with congenital Neospora infection and Neospora protozoa were identified by immunohistochemistry in five of the seven calves. Neospora was isolated from the tissues from two of these calves. The negative calves had no lesions or other findings suggesting congenital Neospora infection.

These results suggest both that a chronic latent infection can occur with Neospora and, most interestingly, that there is vertical transmission of this disease through generations of cattle.There is a precedent to these observations in that vertical transmission of Toxoplasma gondii, a similar parasite, has been observed in wild rats in the absence of the definitive host, members of the cat family (73).

Clinical Presentation

Although congenital Neospora infections have been diagnosed in most domestic livestock, cattle are the only livestock species in which there is sufficient information available concerning the natural infection to describe its clinical features. There are no signs of clinical illness in cows that abort due to Neospora infection. The aborted fetuses are usually autolysed with no gross lesions and placentas are not retained. Abortions have been diagnosed in both heifers and cows from three months gestation to term (5,11,40). Whether Neospora infection can cause reproductive problems in the first trimester of gestation is unknown.A majority (78%) of Neospora abortions occur between four and six months gestation and this pattern of mid-gestation abortion is distinctive from other diagnosed causes of infectious abortion in dairy cattle which tend to occur later in gestation (6,8).

Although Neospora infections occur in both dairy and beef cattle, most reports attributing significant numbers of abortions to this infection have been associated with dairy cattle, particularly those in drylot dairies (1,3-8,10,11,53,64,66). This apparent disparity between beef and dairy cattle is not thought to represent breed susceptibility as beef cattle are susceptible to experimental infection (15) and both congenital infections and abortions due to Neospora have been documented in beef breeds (*,35,37,40,63). It is possible that the environment of the drylot dairy is more conducive to the spread and transmission of this disease. Cattle in drylot dairies are densely populated and fed a variety of harvested feeds and commodities which are frequently stored on or around the dairy prior to being mixed and fed. These feeding practices offer many opportunities for fecal contamination of individual ration components which would then be mixed and fed, efficiently exposing much of the herd. This pattern of increased exposure and disease associated with intensive management mimics factors affecting the incidence of Toxoplasma abortion in sheep flocks.

Neospora infection has been identified throughout California in more than a third of the dairy herds submitting aborted fetuses to the CVDLS (6,11). The herd prevalence of abortion due to this infection can be quite variable. Rare sporadic cases may occur in some dairies with a nominal abortion rate.However, explosive outbreaks of Neospora abortion may occur. A well-documented example involved a group of 147 drylot dairy heifers in which 27 (18%) aborted during a six week period and all fetuses examined (17) were diagnosed with Neospora infection (Jim Reynolds and Mark Anderson, personal communication, Nov. 1993). In some instances, up to 5% of pregnant cattle have aborted due to neosporosis within one to three months. Annual herd abortion rates up to 30% have been reported in dairies with Neospora abortions and abortions may continue to occur over a period of several years (*,1,64). Over a one year period of time, all aborted fetuses available on 26 selected California dairies were collected and submitted to the CVDLS for diagnosis. A total of 266 abortions were submitted, of which, 113 (42.5%) were confirmed Neospora abortions from 19 (73%) dairies (8). In addition to abortion, fetal mummification has been associated with Neospora outbreaks (*,64). Neospora abortions occur throughout the year, but there is possibly a small increased risk of abortion during the late fall and winter (68).

Bovine fetal Neospora infection does not always produce fetal death resulting in abortion or stillbirth. Fetal infection may result in the birth of live full-term congenitally affected calves (13,14,27,32,35,37,40,57,63). Central nervous system infection and damage in these calves results in highly variable clinical signs which are often limited to limb dysfunctions, ranging from mild proprioceptive defects to complete paralysis. Microscopically there is a multifocal protozoal encephalomyelitis which may be particularly localized in the spinal cord gray matter.

Calves may acquire a Neospora infection during gestation, but be born clinically normal except that they have serologic and even histopathologic evidence of congenital infection. A consistent finding in these calves is a high precolostral antibody titre to Neospora which is useful in detecting in utero infection (13). In a survey of calves on a dairy with a history of Neospora abortions, 67/189 newborn calves (35%) had serologic evidence of in utero Neospora infection, with no evidence of increased morbidity or mortality in these calves (60). The apparent wide variability in clinical presentation of these in utero exposed or congenitally infected calves may be due to multiple factors, including the age and immune development of the fetus at the time of exposure to Neospora, and the distribution of the lesions in the central nervous system.

Cows that abort a Neospora infected fetus can have additional infected fetuses in subsequent pregnancies. The outcome of these subsequent pregnancies is variable, resulting in a congenitally infected calf or possibly another abortion. Barr and colleagues identified five calves born to four cows with Neospora abortions in the previous pregnancy. In all calves there was serologic and histopathologic evidence of congenital infection (13). Repeat abortions can also occur, in a survey of abortions in drylot dairies in California, two confirmed Neospora abortions were identified in four of 41 cows in which information concerning other pregnancies was available (8). It is not known whether these repeat transplacental infections are the result of a release of parasites from tissue cysts in the dam or from reinfection of the dam from the environment. However, in bitches naturally infected with Neospora caninum there is documented evidence of congenital infection in successive litters of puppies (33) and there is experimental evidence of repeat transplacental infection of Neospora caninum in mice that had one infectious exposure (23 ).

Diagnosis

The confirmation of a suspect Neospora infection will require the assistance of a veterinary diagnostic laboratory. The preferred samples in cases of abortion include one or more aborted fetuses submitted with placenta and sera from the dam. The aborted fetus is usually autolysed with serosanguinous fluid accumulation in body cavities. Rarely there are subtle gross lesions, consisting of pale white foci in the skeletal muscles or the heart. Histologic lesions consist of widespread nonsuppurative infiltrates. The most diagnostically significant lesions are found in the brain and consist of scattered foci of nonsuppurative cellular infiltrates with occasional foci of necrosis. Protozoa are not usually seen on routinely stained slides. Other histologic lesions that are consistently found include nonsuppurative epicarditis and/or myocarditis, focal nonsuppurative myositis and nonsuppurative portal hepatitis, frequently with focal hepatic necrosis. The presumptive diagnosis of protozoal infection can usually be made on the basis of histologic lesions. Immunohistochemistry using antibodies to Neospora caninum (45) or the bovine Neospora isolate (14,15) is an effective method to identify Neospora in fetal tissues and establish a definitive diagnosis. Recently, a monoclonal antibody against Neospora caninum has been developed which can also be used to detect infection in aborted fetuses (22). Neospora immunohistochemistry is most successful on sections of fetal brain, although the parasites are also frequently present in the lung, kidney, and skeletal muscle (Mark Anderson, unpublished data, 1994). Immunohistochemistry has been successfully employed to diagnose Neospora infections in mummified fetuses although the autolytic state of these fetuses diminishes the diagnostic accuracy.

The definitive diagnosis of Neospora infection in fullterm calves can be difficult due to the patchy distribution of lesions and low numbers of parasites that may be present (14). The most characteristic lesions are in the spinal cord, consisting of a multifocal nonsuppurative myelitis. In some cases thick-walled tissue cysts may be present within neurons. However, these tissue cysts are extremely rare in many in utero exposed calves, making it difficult to establish a diagnosis on the basis of Neospora immunohistochemistry alone. Neospora serology, utilizing an indirect fluorescent antibody (IFA) test, has proven effective in detecting elevated Neospora antibodies in the serum of congenitally infected or in utero exposed calves (14,25). In addition, the Neospora IFA test may be useful in establishing the diagnosis in aborted fetuses, since infected fetuses six months or more in gestation may have elevated Neospora antibody titres (16). However, just as with Toxoplasma infections, a negative fetal Neospora IFA titre does not rule out the possibility of infection.

The use of Neospora IFA for serodiagnosis of Neospora infection in adult cows requires caution in interpretation because some cows aborting a Neospora infected fetus may not have significantly elevated titres. In one study, 22% of known infected cows that aborted a positive fetus had Neospora IFA titres that were within two dilutions of titres in presumed noninfected cattle (25). In addition, within 2-5 months following abortion, the previously elevated titres in cows aborting a Neospora infected fetus may drop to levels similar to noninfected cattle (25). Laboratories using this or any other serologic test for Neospora must establish appropriate cut off titres using standardized sera and should attempt to confirm their positive results by the identification of parasites in aborted fetuses.

An enzyme-linked immunosorbent assay (ELISA) for detection of Neospora antibodies has been recently developed (62). The ELISA test is being used for routine diagnostic testing at the California Veterinary Diagnostic Laboratory as part of the bovine abortion serologic panel. This new procedure is an improvement in serologic testing for Neospora because the test is rapid, inexpensive and consistent with excellent sensitivity (88.6%) and specificity (96.5%). Cutoff values have been established for the ELISA by which the probability of infection can be estimated in cattle. In addition to its use in individual abortion cases, the test can be used to estimate seroprevalence of Neospora infection within a herd by testing 30 to 50 cows in the herd at least two months postpartum. The test is also useful in investigating the causes of recent abortion storms. The Neospora ELISA values of cows that had aborted can be compared with a group of pregnant herdmates. The number of pregnant cows tested should be twice that of aborting cows tested (70).

Control and Prevention

At present, the only clearly established method of Neospora transmission in herds is through prenatal infections. Cattle that are chronically infected pass the infection transplacentally to their fetus. Although various antimicrobial agents have been tested against Neospora caninum in vitro there is currently no known method whereby an infected cow can be cleared of the infection (46,47,50). Therefore, control of the infection in the herd would need to be focused on reducing the level of latent infection in cows in the herd and to limit introduction of infected cattle into the herd (70).

Culling decisions concerning cows that have a Neospora abortion should be made with the knowledge that repeat abortions or repeat congenital infections might occur in these animals. Currently there is insufficient information concerning the future reproductive performance of these cows by which to establish economic estimates to guide culling.

There are no proven methods available to prevent postnatal infection because there is insufficient information on the biology of this parasite, including the life cycle, on which to base specific recommendations. However, it is prudent to remove all potentially infected tissues, such as aborted fetuses and placentas from the environment, that might serve as a source of infection for susceptible hosts. In addition, fecal contamination of feed and water sources by other animals should be minimized.It is apparent that fecal contamination of the environment or feeds of cattle is extremely common since virtually all cattle are infected with Sarcocystis cruzi through ingestion of coccidia from a canidae definitive host. As is the case with toxoplasmosis, development of an effective Neospora vaccine will be difficult.

Additional Readings

WCDS-1995:
Reproductive Health Programs
Achieving a Sound Reproductive Health Management Program
Improving Heat Detection in Tie-stall and Free-Stall Environments
An Economic Approach to Reproductive Programs in Dairy Herds

Applied Dairy Science Course - University of Alberta:
Managing Reproduction in Dairy Cattle

Alberta Dairy Management Fact Sheet:
Getting Cows Bred - 1. Preparing cows for high conception rates

References

1. Abbitt B., T.M. Craig, L.P. Jones, R.L. Huey and A.K. Eugster. 1993. Protozoal abortion in a herd of cattle concurrently infected with Hammondia pardalis. J Am Vet Med Assoc 203:444.
2. Agerholm J.S. and B.C. Barr. 1994. Bovine abortions associated with Neospora in Denmark. Acta Veterinaria Scandinavica 35(4):461-464.
3. Anderson M.L., B.C. Barr, P.A. Conrad, M. Thurmond, J. Picanso and J.P. Dubey.. 1991. Bovine Protozoal Abortions in California. The Bovine Practitioner 26:102.
4. Anderson, M.L., P.C. Blanchard, B.C. Barr and R.L. Hoffman. 1990. A survey of causes of Abortion in San Joaquin Valley, California. J Vet Diagn Invest 2:283.
5. Anderson, M.L., P.C. Blanchard, B.C. Barr, J.P. Dubey, R.L. Hoffman and P.A. Conrad. 1991. Neospora-like protozoan infection as a major cause of abortion in California dairy cattle. J Am Vet Med Assoc 198:241.
6. Anderson, M.L., J. Picanso, M. Thurmond, P.C. Blanchard, A.W. Layton, C. Palmer, J. Case, B.C. Barr, J.P. Dubey and P.A. Conrad. 1992. Epidemiological investigations of bovine protozoal abortion in California dairy herds. Proc XVII World Buiatrics Cong and XXV Am Assoc Bovine Pract Conf 2:74.
7. Anderson, M.L., B.C. Barr and P.A. Conrad. 1994. Protozoal causes of reproductive failure in domestic ruminants. Veterinary Clinics of North America, Food Animal Practice, 10(3):439-461.
8. Anderson, M.L., C. Palmer, M. Thurmond, J.P. Picanso, P.C. Blanchard, R.E. Breitmeyer, A.W. Layton, M. McAllister, B.M. Daft, H. Kinde, D.H. Read, J.P. Dubey, P.A. Conrad and B.C. Barr. 1995. Evaluation of abortions in cattle attributable to neosporosis in selected dairy herds in California. J Am Vet Med Assoc 207(9):1206-1210.
9. Baker, D.G., T.Y. Morishita, D.L. Brooks and J.P. Dubey. 1995. Experimental oral inoculations in birds to evaluate potential definitive hosts of Neospora caninum. J. Parasitol. 81(5):783-785.
10. Barr, B.C., M.L. Anderson, P.C. Blanchard, B.M. Daft, K. Kinde and P.A. Conrad. 1990. Bovine fetal encephalitis and myocarditis associated with protozoal infection. Vet Pathol 27:354.
11. Barr, B.C., M.L. Anderson, J.P. Dubey and P.A. Conrad. 1991. Neospora-like protozoal infections associated with bovine abortions. Vet Pathol 28:116.
12. Barr, B.C., M.L. Anderson, L.W. Woods, J.P. Dubey and P.A. Conrad. 1992. Neospora-like protozoal infections associated with abortions in goats. J Vet Diagn Invest 4:365.
13. Barr, B.C., P.A. Conrad, R. Brietmeyer, K. Sverlow, M.L. Anderson, J. Reynolds, A.E. Chauvet, J.P. Dubey and A.A. Ardans. 1993. Congenital Neospora infection in calves born from cows that had previously aborted Neospora-infected fetuses: Four cases (1990-1992). J Am Vet Med Assoc 202:113.
14. Barr, B.C., P.A. Conrad, J.P. Dubey and M.L. Anderson. 1991. Neospora-like encephalomyelitis in a calf: pathology, ultrastructure, and immunoreactivity. J Vet Diag Invest 3:39.
15. Barr, B.C., J.D. Rowe, K.W. Sverlow, B.H. BonDurant, A.A. Ardans, M.N. Oliver and P.A. Conrad. Experimental reproduction of bovine fetal Neospora infection and death with a bovine Neospora isolate. J Vet Diagn Invest 6:207-215.
16. Barr, B.C., M.L. Anderson, K.W. Sverlow and P.A. Conrad. 1995. Diagnosis of fetal Neospora infection with the Neospora indirect fluorescent antibody test (IFAT). Vet Rec (in press).
17. Bildfell, R, J. Davidson and J.P. Dubey. 1994. Neospora-induced protozoal bovine abortion in Prince Edward Island. Canadian Veterinary Journal, 35(2):122.
18. Bjerkas, I. and J.P. Dubey. 1991. Evidence that Neospora caninum is identical to the Toxoplasma-like parasite of Norwegian dogs. Acta Vet Scand 32:407.
19. Bjerkas, I., S.F. Mohn and J. Presthus. 1984. Unidentified cystforming sporozoan causing encephalomyelitis and myositis in dogs. Z. Parasitenkd 70:271.
20. Boulton, J.G., P.A. Gill, R.W. Cook, G.C. Fraser, P.A. Harper and J.P. Dubey. 1995. Bovine Neospora abortion in northeastern New South Wales. Australian Veterinary Journal 72(3):119-120.
21. Buxton, D and J. Finlayson. 1986. Experimental infection of pregnant sheep with Toxoplasma gondii: pathological and immunological observations on the placenta and foetus. J Comp Path 96:319.
22. Cole, R.A., D.S. Lindsay, J.P. Dubey and B.L. Blagburn. 1993. Detection of Neospora caninum in tissue section using a murine monoclonal antibody. J Vet Diagn Invest 5:579.
23. Cole, R.A., D.S. Lindsay, B.L. Blagburn, D.C. Sorjonen and J.P. Dubey. 1995. Vertical transmission of Neospora caninum in mice. J. Parasitol.,81(5) 730-732.
24. Conrad, P.A., B.C. Barr, K.W. Sverlow, M.L. Anderson, B.M. Daft, H. Kinde, J.P. Dubey, L. Munson and A.A. Ardans. 1993. In vitro isolation and characterization of a Neospora sp. from aborted bovine fetuses. Parasitology 106:239.
25. Conrad, P.A., K.W. Sverlow, M.L. Anderson, J. Rowe, B. BonDurant, G. Tuter, R.E. Brietmeyer, C. Palmer, M. Thurmond, A.A. Ardans and B.C. Barr. 1993. Detection of serum antibody responses in cattle with natural or experimental Neospora infections. J Vet Diagn Invest 5:572.
26. Dannatt, L., F. Guy and A.J. Trees. 1995. Abortion due to Neospora species in a dairy herd. Vet Rec 137:566-567.
27. Dubey, J.P. 1989. Congenital neosporosis in a calf. Vet Rec 125:486.
28. Dubey, J.P. 1992. A review of Neospora caninum and Neospora-like Infection in animals. J Protozool Res 2:40.
29. Dubey, J.P. 1993. Toxoplasma, Neospora, Sarcocystis, and other tissue cyst-forming coccidia of humans and animals. In Kreier JP:Parasitic Protozoa, ed 2. New York, Academic Press Inc, p1.
30. Dubey, J.P., H.M. Acland and A.N. Hamir. 1992. Neospora caninum (Apicomplexa) in a still born goat. J Parasitol 78:532.
31. Dubey, J.P., J.L. Carpenter, C.A. Speer, M.J. Topperrk and A. Uggla. 1988. Newly recognized fatal protozoan disease of dogs. J Am Vet Med Assoc 192:1269.
32. Dubey, J.P., W.J. Hartley and D.S. Lindsay. 1990. Congenital Neospora caninum infection in a calf with spinal cord anomaly. J Am Vet Med Assoc 197:1043.
33. Dubey, J.P., W.J. Hartley, D.S. Lindsay and M.J. Topper. 1990. Fatal congenital Neospora caninum infection in a lamb. J. Parasitol 76:127.
34. Dubey, J.P., A.L. Hattel, D.S. Lindsay and M.J. Topper. 1988. Neonatal Neospora caninum infection in dogs: Isolation of the causative agent and experimental transmission. J Am Vet Med Assoc 193:1259.
35. Dubey, J.P., E.B. Janovitz and A.J. Skowronek. 1992. Clinical neosporosis in a 4-week-old Hereford calf. Vet Parasitol 43:137.
36. Dubey, J.P., A. Koestner and R.C. Piper. 1990. Repeated transplacental transmission of Neospora caninum in dogs. J Am Vet Med Assoc 197:857.
37. Dubey, J.P., C.W. Leathers and D.S. Lindsay. 1989. Neospora caninum-like protozoan associated with fatal myelitis in newborn calves. J Parasitol 75:146.
38. Dubey, J.P. and D.S. Lindsay. 1990. Neospora caninum induced abortion in sheep. J Vet Diagn Invest 2:230.
39. Dubey, J.P., D.S. Lindsay, M.L. Anderson, S.W. Davis and S.K. Shen. 1992. Induced transplacental transmission of Neospora caninum in cattle. J Am Vet Med Assoc 201:709.
40. Dubey, J.P., S. Miller, D.S. Lindsay and M.J. Topper. 1990. Neospora caninum-associated myocarditis and encephalitis in an aborted calf. J Vet Diagn Invest 2:66.
41. Dubey, J.P. and M.L. Porterfield. 1990. Neospora caninum (Apicomplexa) in an aborted equine fetus. J Parasitol 76:732.
42. Harmelin, A., S. Perl, A. Nyska, B. Yakobson, N. Shpigel, U. Orgad and J.P. Dubey. 1995. Neosporosis-associated bovine abortion in Israel. Vet Rec, 136(3):80.
43. Jardine, J.E. and R.D. Last. 1993. Neospora caninum in aborted twin calves. Journal of South Africa Veterinary Association 64:101.
44. Jerret, I.V., S. McOrist, J. Waddington, J.W. Browning, J.C. Malecki and I.P. McCausland. 1984. Diagnostic studies of the fetus, placenta and maternal blood from 265 bovine abortions. Cornell Vet 74:8.
45. Lindsay, D.S. and J.P. Dubey. 1989. Immunohistochemical diagnosis of Neospora caninum in tissue sections. Am J Vet Res 50:1981-1983.
46. Lindsay, D.S. and J.P. Dubey. 1989. Evaluation of anti-coccidial drugs' inhibition of Neospora caninum development in cell cultures. J. Parasitol., 75(6):990-992.
47. Lindsay, D.S. and J.P. Dubey. Effects of sulfadiazine and amprolium on Neospora caninum (protozoa:apicomplexa) infections in mice. J. Parasitol., 76(2):177-179.
48. Lindsay, D.S., J.P. Dubey, R.A. Cole, L.P. Nuehring and B.L. Blagburn. 1993. Neospora-induced protozoal abortions in cattle. Comp Cont Ed Pract Vet 15:882.
49. Lindsay, D.S., C.A. Speer, M.A. Toivio-Kinnucan, J.P. Dubey and B.L. Blagburn. 1993. Use of infected culture cells to compare ultrastructural features of Neospora caninum from dogs and Toxoplasma gondii. Am J vet Res 54:103.
50. Lindsay, D.S., N.S. Rippey, R.A. Cole, L.C. Parsons, J.P. Dubey, R.R. Tidwell and B.L. Blagburn. 1994. Examination of the activities of 43 chemotherapeutic agents against Neospora caninum tachyzoites in cultured cells. Am J Vet Res, 55(7):976-981.
51. McCausland, I.P., R.T. Badman, S. Hides and K.J. Slee. 1984. Multiple apparent Sarcocystis abortion in four bovine herds. Cornell Vet 74: 146.
52.McIntosh, D.W. and D.M. Haines. 1994. Neospora infection in an aborted fetus in British Columbia. Canadian Veterinary Journal, 35(2):114-115.
53. Nietfeld, J.C., J.P. Dubey, M.L. Anderson, M.C. Libal, M.J. Yaeger and R.D. Neiger. 1992. Neospora-like protozoan infection as a cause of abortion in dairy cattle. J Vet Diagn Invest 4:223.
54. Obendorf, D. and R. Mason. 1990. Neospora caninum infection detected in a bovine aborted foetus. Aust Sac Vet Path Report 28:36.
55. Obendorf, D.L., N. Murray, G. Veldhuis, B.L. Munday and J.P. Dubey. 1995. Abortion caused by neosporosis in cattle. Australian Veterinary Journal, 72(3):117-118.
56. Ogino, H., E. Watanabe, S. Watanabe, H. Agawa, M. Narita, M. Haritani and K. Kawashima. 1992. Neosporosis in the aborted fetus and newborn calf. J Comp Path 107:231.
57. O'Toole, J.M. and M. Jeffrey. 1987. Congenital sporozoan encephalomyelitis in a calf. Vet Rec 121:563.
58. O'Toole, D., M. Jeffrey, D. Challoner, R. Maybey and V. Welch. 1993. Ovine myeloencephalitis-leukomyelomalacia associated with a Sarcocystis-like protozoan. J Vet Diagn Invest 5:212.
59. Otter, A., I.B. Griffiths and M. Jeffrey. 1993. Bovine Neospora caninum abortion in the U.K. (letter) Vet Rec 133:375.
60. Pare', J., S.K. Hietala and M.C. Thurmond. 1993. Precolostral titers to the Neospora-like agent in dairy calves and effect of congenital infection on preweaning health. (Abstract) Conference of Research Workers in Animal Diseases, Chicago, 52, November 1993.
61. Pare', J., M.C. Thurmond and S.K. Hietala. 1994. Congenital Neospora infection in dairy cattle. Vet Rec 134:531-532.
62. Pare', J., S.K. Hietala and M.C. Thurmond. 1995. An enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of Neospora infection in cattle. J Vet Diagn Invest 7:352-359.
63. Parish, S.M., L. Maag-Miller, T.E. Basser, J.P. Weidner, T. McElwain, D.P. Knowles and C.W. Leathers. 1987. Myelitis associated with protozoal infection in newborn calves. J Am Vet Med Assoc 191:1599.
64. Rogers, D.G., M.S. McCullough, W.S. Shain and M.L. Anderson. 1993. Endemic protozoal abortions in a dairy cow herd. Agri-practice 14:16.
65. Shivaprasad, H.L., R. Ely and J.P. Dubey. 1989. A Neospora-like protozoan found in an aborted bovine placenta. Vet Parasitol 34:145.
66. Thilsted, J.P. and J.P. Dubey. 1989. Neosporosis-like abortions in a herd of dairy cattle. J Vet Diagn Invest 1:205.
67. Thornton, R.N., E.J. Thompson and J.P. Dubey. 1991. Neospora abortion in New Zealand cattle. New Zealand Veterinary Journal 39:129.
68. Thurmond, M.C., M.L. Anderson and J. Picanso. 1991. Estimation seasonality of bovine protozoal abortion with a variable and unknown population at risk. (Abstract) 72nd Annual Meeting of the Conference of Research Workers in Animal Diseases, Chicago, November 11-12.
69. Thurmond, M.C., M.L. Anderson and P.C. Blanchard. 1995. Secular and seasonal trends of Neospora abortion in California dairy cows. J of Parasitology, 81(3):364-367.
70. Thurmond, M.C. and S.K. Hietala. 1995. Stratigies to control Neospora infection in cattle. Bovine Practitioner, 29:60-63.
71. Trees, A.J., F. Guy, J.C. Low, L. Roberts, D. Buxton and J.P. Dubey. 1994. Serological evidence implicating Neospora species as a cause of abortion in British cattle. Vet Rec 134:405-407.
72. Vickers, M. and H.V. Brooks. 1983. Suspected Sarcocystis infection in an aborted bovine foetus. NZ Vet J 31:166.
73. Webster, J.P. 1994. Prevalence and transmission of Toxoplasma gondii in wild brown rats, Rattus norvegicus. Parasitology, 108:407-411.
74. Woods, L.W., M.L. Anderson, P.K. Swift and K.W. Sverlow. 1994. Systemic neosporosis in a California black-tailed deer (Odocoileus hemionus columbianus). J Vet Diagn Invest 6:508-510.
75. Wouda, W., I.J.R. Visser and F. Van Knapen. 1992. Bovine protozoal abortion (letter) Vet Rec 130(13):279.
76. Yaeger, M.J., S. Shawd-Wessels and P. Leslie-Steen. 1994. Neospora abortion storm in a Midwestern dairy. J Vet Diagn Invest Oct 6(4):506-8.

* Anderson, M.L., B.C. Barr and P.A. Conrad: Unpublished Neospora immunohistochemistry results from submissions to California Veterinary Diagnostic Laboratory, 1995.