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Neosporosis in Mexican Dairy Herds: Lesions and Immunohistochemical Detection of Neospora caninum in Fetuses
J. Comp. Path. 2001, Vol. 125, 58–63 doi:10.1053/jcpa.2001.0477, available online at http://www.idealibrary.com on
Neosporosis in Mexican Dairy Herds: Lesions and Immunohistochemical Detection of Neospora caninum in Fetuses E. Morales∗, F. J. Trigo∗, F. Ibarra†, E. Puente‡ and M. Santacruz§ ∗Departamento de Patologı´a and †Departamento de Parasitologı´a, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Auto´noma de Me´xico, 04510, Me´xico, D.F., ‡Pfizer, S.A. de C.V. Insurgentes Sur 1685, 2 Piso, Col. Guadalupe Inn, 01020, Me´xico, D.F. and §Centro Agropecuario Industrial de Tizayuca, S.A. Parque Industrial Tizayuca, Hidalgo, Me´xico Summary Of 211 aborted bovine fetuses collected from Mexican dairy herds between January 1996 and March 1999, 73 showed microscopical lesions consistent with neosporosis. Of these 73 fetuses, 58 (79%) showed lymphocytic myocarditis, 39 (53%) showed microgliosis and multifocal necrosis in the brain, 39 (53%) showed lymphocytic hepatitis, and 19 (26%) showed lymphocytic myositis. Immunohistochemical examination of brain, myocardium and liver from 53 of the same 73 fetuses demonstrated Neospora caninum antigens in 41 (77%), of which 19 (46%) gave positive results in one of the three sites, 15 (37%) in two, and seven (17%) in three. The results indicated the presence of neosporosis in a number of the main dairy farming regions of Mexico. 2001 Harcourt Publishers Ltd
Introduction Neospora caninum, a protozoon first recognized in the USA, causes disease in dogs (Dubey et al., 1988a; Dubey, 1992), dairy cattle (Anderson et al., 1991a,b; Barr et al., 1991a; Nietfeld et al., 1992), beef cattle, sheep, horses, goats and deer (Dubey and Lindsay, 1996). In non-human primates and numerous other hosts, experimental N. caninum infection induces clinical disease (Dubey and Lindsay, 1996). N. caninum was first isolated from dogs (Dubey et al., 1988a,b), and later from bovine aborted fetuses (Conrad et al., 1993). Neosporosis in cattle is characterized by abortion, neonatal paralysis, or the birth of symptomless carrier calves (Anderson et al., 1991a,b; Pare´ et al., 1996). Cattle may be infected transplacentally or as the result of ingesting oocysts produced by dogs, which represent the definitive host (Barr et al., 1993; Pare´ et al., 1996; Anderson et al., 1997; McAllister et al., 1998). Correspondence to: E. Morales 0021–9975/01/050058+06 $35.00
In California, New Zealand and the Netherlands, neosporosis is the most commonly diagnosed cause of bovine abortion (Anderson et al., 1991b; Thornton et al., 1991; Dubey and Lindsay, 1996). Within the American continent, apart from the USA, bovine neosporosis has been identified only in Canada (McIntosh and Haines, 1994), Mexico (Abbitt et al., 1993; Delgado et al., 1995; Morales et al., 1997) and Argentina (Venturini et al., 1995). The diagnostic techniques used consist of (1) histopathological and immunohistochemical examination of tissues from fetuses or newborn animals, and (2) ELISA or immunofluorescence to demonstrate specific antibody in fetal serum and fluids, or in maternal serum (Dubey and Lindsay, 1996). The most significant and distinctive microscopical lesions are lymphocytic encephalomyelitis with multifocal microgliosis and necrosis in the brain and spinal cord. In addition, lymphocytic myocarditis and myositis may be found, as well as lymphocytic periportal hepatitis, with or without foci of hepatocellular necrosis. Occasionally there 2001 Harcourt Publishers Ltd
59
Bovine Neosporosis
is inflammation and necrosis in other organs. Cysts are usually confined to the central nervous system (CNS), with clusters of tachyzoites in muscular tissue, liver and the CNS (Dubey et al., 1989, 1990a, b; Barr et al., 1990, 1991b, 1993). Histopathology and immunohistochemistry, which are reliable diagnostic methods, have been used to identify methods for diagnosis in individual cases, but epidemiological studies based on these techniques are few (Lindsay and Dubey, 1989; Anderson et al., 1990; Barr et al., 1990, 1991b; Wouda et al., 1997) and in Mexico there is little information about this disease. The first report was of six fetuses from a herd of 800 cows in the northeast of Mexico (Abbitt et al., 1993), and subsequently fetuses with distinctive neosporosis lesions were reported from diagnostic laboratories in Torreon Coahuila (Delgado et al., 1995) and Mexico City (Morales et al., 1997). In view of the meagre data on bovine neosporosis in Mexico, this report presents epidemiological information on the disease, based on histopathological and immunohistochemical examination of tissues from aborted fetuses. Materials and Methods Animals Aborted fetuses (n=211) from the main dairy production states of Hidalgo, Coahuila, Queretaro, Guanajuato, Durango, Aguascalientes, Jalisco, Estado de Mexico, Tamaulipas, Tlaxcala and Chihuahua were submitted to the Faculty of Veterinary Medicine and Zootechnics for diagnosis between January 1996 and March 1999. Histopathology Samples of brain, myocardium, diaphragmatic muscle, liver, lung, kidney and spleen were fixed in 10% neutral buffered formalin and processed by routine methods. Sections (5 m) were cut and stained with haematoxylin and eosin (HE), and selected sections of brain were also stained by the periodic acid-Schiff (PAS) method. Immunohistochemistry Of 73 fetuses with distinctive lesions of neosporosis, 53 (73%) were subjected to immunohistochemical examination of the brain, myocardium and liver (i.e., the organs most severely affected) to identify the parasite. Serial sections (3 m) were mounted on poly-L-lysine-coated slides, heated at 56°C, dewaxed in xylene, and rehydrated through graded
ethanols. Protease (Sigma Chemical Company, St Louis, Missouri, USA) was applied for 10 min at 37°C, followed by washing for 5 min with running deionized water. Endogenous peroxidase was quenched by immersion in 3% H2O2. Slides were blocked by the application of normal rabbit sera (Dako Corporation, Carpinteria, California, USA) for 10 min. The sections were then treated for 30 min at 37°C with goat anti-N. caninum primary antibody (Veterinary Medical Research and Development, Pullman, Washington, USA), diluted 1 in 3000. A secondary biotinylated antibody (Dako) diluted 1 in 600 was applied for 20 min at 37°C, followed by an avidin–biotin peroxidase complex (ABC) (Dako) for 20 min at 37°C. To develop the immunological reaction, the slides were treated for 5 min with a diaminobenzidine solution (DAB) (Zymed Laboratories, South San Francisco, California, USA). The sections were then counterstained with haematoxylin. They were considered positive when structures consistent with the morphology of the parasite were immunolabelled by this method. As a negative control, antiN. caninum antibody was replaced by normal goat serum (Dako) and positive controls consisted of sections of canine brain containing clusters of N. caninum tachyzoites (supplied by Dr Deborah Haines, University of Saskatchewan, Saskatoon, Canada). Results Histopathology Of the 211 fetuses examined, only 104 (49%) had detectable lesions. Of these, 73 had lesions consistent with neosporosis, the remainder showing changes suggestive of bacterial or viral infection. The 73 suspected neospora cases came from Hidalgo (38 cases), Coahuila (29), Aguascalientes (2), Queretaro (1), Guanajuato (1), Estado de Mexico (1) and Tlaxcala (1). The neospora lesions were classified as mild, severe or moderate. In mild cases, foci composed of only a few inflammatory cells were present in the myocardium or liver, or a few foci of microgliosis, with or without necrosis, were seen in the brain. In severe cases, there was an abundance of inflammatory cells in the myocardium or liver, or foci of microgliosis, with or without necrosis. Moderate cases fell between the two extremes of mild and severe. The most significant and consistent lesions of neosporosis were those in the myocardium, brain, liver and diaphragmatic muscle. The type and
60
E. Morales et al. Table 1 Type and severity of lesions consistent with neosporosis in 73 bovine fetuses Severity of lesions
Mild Moderate Severe
Number (and %) of the 73 fetuses showing microgliosis and necrosis
myocarditis
hepatitis
myositis
28 (38) 9 (12) 2 (3)
26 (36) 21 (29) 11 (15)
26 (36) 11 (15) 2 (3)
11 (15) 6 (8) 2 (3)
severity of these lesions are summarized in Table 1. Fifty-eight (79%) of the 73 fetuses displayed lymphocytic myocarditis (multifocal or diffuse), of variable severity, the mild form being the most common, followed by moderate, and then severe. In none of these cases were tachyzoites visible in muscle fibres with HE staining. Focal necrotic lesions and foci of microgliosis were observed in cerebral tissue from 39 fetuses (53%), in some cases the necrosis being surrounded by microglial cells and lymphocytes (Fig. 1). Although these lesions were scattered throughout the CNS, they were found most frequently in the cerebral cortex; they were sometimes multifocal and were commonly near blood vessels. In only one fetus (8 months of gestation) was lymphocytic perivascular infiltration detected, and in only six fetuses (8%) were neospora cysts, of different sizes, found. Each was round or oval in shape and 21×21 m to 49×33 m in size, with a thick PAS-positive wall (Fig. 2). An inflammatory reaction surrounding a tissue cyst, with associated degenerative changes, was seen only once. With HE staining, free tachyzoites were not seen. The severity of CNS lesions was variable, with mild changes occurring most commonly, followed by moderate, and then severe. Thirty-nine fetuses (54%) displayed lymphocytic periportal hepatitis of variable severity, mild lesions being the most common. Randomly distributed focal necrosis was also seen in six of these 39 cases. Parasitic structures were not identified on HE-stained sections of liver. In 19 fetuses (26%), multifocal, generally mild, lymphocytic myositis was seen in diaphragmatic muscle, but no parasitic structures were found. Only in three cases (4%) was a mild pulmonary inflammatory reaction appreciable, consisting of alveolar macrophages with some lymphocytes. The kidney and spleen showed no significant changes. In only 47 of the 73 fetuses could the gestational age be ascertained; it ranged from 3 to 8 months (mean 5·5).
Fig. 1. Fetal brain with a central necrotic focus surrounded by microglial cells. HE. ×400.
Immunohistochemistry Brain, myocardium and liver from 53 of the 73 fetuses with microscopical lesions consistent with neosporosis were examined immunohistochemically. N. caninum antigen was identified in 41 (77%) of the 53 fetuses, immunolabelling being detected in one organ in 19 (46%) of the 41 positive cases, in two organs in 15 (37%), and in all three organs in seven (17%). In the brain, neospora cysts,
61
Bovine Neosporosis
(36%) of the 53 fetuses, sometimes associated with microgliosis and necrosis. In the myocardium, tachyzoites were identified in 24 (45%) of the 53 fetuses, mainly inside myocardial fibres, where they were associated with infiltrates or areas of necrosis, or both. In the liver, tachyzoites, either isolated or in clusters, were identified in 25 (45%) of the 53 fetuses (Fig. 3, Table 2).
Discussion
Fig. 2. Neospora caninum cyst in the brain of an aborted bovine fetus. Note the absence of inflammation. PAS. ×1000.
Fig. 3. Liver with a cluster of immunolabelled Neospora caninum tachyzoites ABC, with antiserum to N. caninum. ×1000.
without associated lesions, were identified in two (4%) of the 53 fetuses. Tachyzoites, either isolated or in clusters, were identified in the brain of 19
From the histopathological results, which were similar to those in other reports (Barr et al., 1990, 1991a; Anderson et al., 1991a; Dubey and Lindsay, 1993; Lindsay et al., 1993), it may be concluded that the tissues most frequently affected, and therefore most useful for diagnostic purposes, are CNS, myocardium, skeletal muscle and liver. Some authors point out that myocarditis tends to be severe but is easily concealed by autolytic changes (Barr et al., 1990; Dubey and Lindsay 1996; Wouda et al., 1997). In the present study, however, myocarditis was commonly mild; some cases displayed autolytic changes. In a study of neospora lesions in the brain of six aborted bovine fetuses, Helman et al. (1998) found most lesions in the cerebrum, followed by the cerebellum and then the medulla. In the present study, the distribution of lesions in the brain was not specifically examined, but most lesions were seen in the cerebral cortex. The neospora cysts, as in other reports (Dubey and Lindsay, 1996), were scarce and only one was associated with an inflammatory reaction; this reaction was possibly due to a break in the cyst wall, with consequent liberation of the contents, as suggested previously (Dubey et al., 1990a; Dubey, 1992). In only one case was there an appreciable perivascular lymphocytic infiltrate, and this occurred in the oldest fetus examined (gestational age 8 months), supporting the suggestion that maturity of the immune system is an important contributory factor (Ogino et al., 1992). Unfortunately, the present study gave no information on the spinal cord, in which neospora cysts and lesions have been described (Dubey et al., 1989; 1990a; Dubey and Lindsay, 1996). Unlike Dubey and Lindsay (1993), we found no haemorrhages in the cerebral parenchyma. In agreement with other reports (Barr et al., 1990; Wouda et al., 1997), lymphocytic hepatitis with a periportal tendency occurred, and was occasionally associated with focal necrosis. In 19 cases, lymphocytic myositis was observed, indicating the diagnostic value of diaphragmatic muscle samples. The average gestational age of the
62
E. Morales et al. Table 2 Immunohistochemical examination of brain, myocardium and liver from 53 of the 73 fetuses showing lesions consistent with neosporosis Immunolabelling
Positive Negative Inconclusive
Number (and %) of the 53 fetuses showing the stated immunolabelling result for tachyzoites in brain∗
myocardium
liver
19 (36) 17 (32) 15 (28)
24 (45) 24 (45) 5 (9)
25 (47) 16 (30) 12 (23)
∗ The brains of two (4%) of the 53 fetuses showed immunolabelled cysts.
aborted fetuses (5·5 months) was similar to that reported in California (Anderson et al., 1991b). Immunohistochemically, N. caninum organisms were identified most frequently and in greatest numbers in the liver, indicating the value of this tissue for demonstrating the parasite. The brain was helpful as a tissue in which tachyzoites and occasionally cysts could be demonstrated, although the parasites were scarce. The results differed, however, from those obtained in the Netherlands by Wouda et al. (1997), whose immunohistochemical study demonstrated neospora antigens in 85% of brain samples from neospora cases, as compared with 26% of liver samples and 14% of myocardial samples. The lesions described in the present study could not be considered pathognomonic for neosporosis, and it was not possible to demonstrate neospora antigens immunohistochemically in all tissues examined. However, the microgliosis and CNS necrosis were considered useful indicators of neosporosis, and strongly suggestive when taken together with myocarditis and hepatitis (Barr et al., 1990; Anderson et al., 1991b; Nietfeld et al., 1992). The findings in the present study and those of Wouda et al. (1997) suggest that for the diagnosis of neosporosis immunohistochemical examination of brain, liver and myocardium represents the method of choice. Finally, this study demonstrated the presence of bovine neosporosis in some of the main dairy farming regions of Mexico. Acknowledgments We are indebted to Dr J. P. Dubey (USDA Parasite Biology and Epidemiology Laboratory, Beltsville, MD, USA) and Dr A. M. Lo´pez (University of Prince Edward Island, Canada) for providing information and advice. We also thank Dr D. Haines (University of Saskatchewan, Canada) for providing
Neospora caninum-positive tissues, and J. G. Jime´nez (Facultad de Medicina Veterinaria y Zootecnia) for histological and immunohistochemical assistance. References Abbitt, B., Craig, T. M., Jones, L. P., Huey, R. L. and Eugster, A.K. (1993). Protozoal abortion in a herd of cattle concurrently infected with Hammondia pardalis. Journal of the American Veterinary Medical Association, 20, 444–448. Anderson, M. L., Barr, B. C., Blanchard, C. P., Dubey, J. P. and Conrad, P. A. (1990). Immunohistochemical diagnosis of protozoal abortion in cattle. Proceedings of the Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians. Denver, Colorado, October 7–9, 1990, Abstract 33. Anderson, M. L., Barr, B. C., Conrad, P. A., Thurmond, M. C., Picanso, J. and Dubey, J. P. (1991a). Bovine protozoal abortions in California. Bovine Practitioner, 2, 102–104. Anderson, M. L., Blanchard, P. C., Barr, B. C., Dubey, J. P., Hoffman, R. L. and Conrad, P. A. (1991b). Neospora-like protozoan infection as a major cause of abortion in California dairy cattle. Journal of the American Veterinary Medical Association, 198, 241–244. Anderson, M. L., Reynolds, J. P., Rowe, J. D., Sverlow, K. W., Packham, A. E., Barr, B. C. and Conrad, P. A. (1997). Evidence of vertical transmission of Neospora sp. infection in dairy cattle. Journal of the American Veterinary Medical Association, 210, 1169–1172. Barr, B. C., Anderson, M. L. and Blanchard, P.C. (1990). Bovine fetal encephalitis and myocarditis associated with protozoal infection. Veterinary Pathology, 27, 354– 361. Barr, B. C., Anderson, M. L., Dubey, J. P. and Conrad, P. A. (1991a). Neospora-like protozoal infections associated with bovine abortions. Veterinary Pathology, 28, 110–116. Barr, B. C., Conrad, P. A., Breitmeyer, R., Sverlow, K. W., Anderson, M. L., Reynolds, J., Chauvert, A. E. A., Dubey, J. P. and Ardans, A. A. (1993). Congenital Neospora infection in calves born from cows that had previously aborted Neospora-infected fetuses: four cases (1990–1992). Journal of the American Veterinary Medical Association, 202, 113–117.
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Barr, B. C., Conrad, P. A., Dubey, J. P. and Anderson, M. L. (1991b). Neospora-like encephalomyelitis in a calf: pathology, ultrastructure, and immunoreactivity. Journal of Veterinary Diagnostic Investigation, 3, 39–46. Conrad, P. A., Barr, B. C. and Sverlow, K. W. (1993). In vitro isolation and characterization of Neospora sp. from aborted bovine fetuses. Parasitology, 106, 239– 249. Delgado, G. R., Quintero, C. J. and Luna, A. A. de (1995). Estudio patolo´gico, microbiolo´gico y serolo´gico del aborto en bovinos Holstein de la Comarca Lagunera. Memorias del XIX Congreso Nacional de Buiatrı´a. Torreo´n Coahuila, Me´xico, 24–26 August, 1995, pp. 74–78. Dubey, J. P. (1992). A review of Neospora caninum and Neospora-like infections in animals. Journal of Protozoology Research, 2, 40–52. Dubey, J. P., Carpenter, J. L., Speer, C. A., Topper, M. J. and Uggla, A. (1988a). Newly recognized fatal protozoan disease of dogs. Journal of the American Veterinary Medical Association, 192, 1269–1285. Dubey, J. P., Hartley, W. J. and Lindsay, D. S. (1990a). Congenital Neospora caninum infection in a calf with spinal cord anomaly. Journal of the American Veterinary Medical Association, 197, 1043–1044. Dubey, J. P., Hattel, A. L., Lindsay, D. S. and Topper, M. J. (1988b). Neonatal Neospora caninum infection in dogs. Isolation of the causative agent and experimental transmission. Journal of the American Veterinary Medical Association, 193, 1259–1263. Dubey, J. P., Leathers, C. W. and Lindsay, D. S. (1989). Neospora caninum-like protozoon associated with fatal myelitis in newborn calves. Journal of Parasitology, 75, 146–148. Dubey, J. P. and Lindsay, D. S. (1993). Neosporosis. Parasitology Today, 9, 452–457. Dubey, J. P. and Lindsay, D. S. (1996). A review of Neospora caninum and neosporosis. Veterinary Parasitology, 67, 1–59. Dubey, J. P., Miller, S., Lindsay, D. S. and Topper, M. J. (1990b). Neospora caninum-associated myocarditis and encephalitis in an aborted calf. Journal of Veterinary Diagnostic Investigation, 2, 66–69. Helman, R. G., Stair, E. L., Lehenbauer, T. W., Rodgers, S. and Saliki, J. T. (1998). Neosporal abortion in Oklahoma cattle with emphasis on the distribution of brain lesions in aborted fetuses. Journal of Veterinary Diagnostic Investigation, 10, 292–295.
Lindsay, D. S. and Dubey, J. P. (1989). Immunohistochemical diagnosis of Neospora caninum in tissue sections. American Journal of Veterinary Research, 50, 1981–1983. Lindsay, D. S., Dubey, J. P. and Cole, R. A. (1993). Neospora-induced protozoal abortions in cattle. Compendium on Continuing Education for the Practicing Veterinarian, 15, 882–889. McAllister, M. M., Dubey, J. P., Lindsay, D. S., Jolley, W. R., Willis, R. A. and McGuire, A. M. (1998). Dogs are definitive hosts of Neospora caninum. International Journal of Parasitology, 28, 1473–1478. McIntosh, W. D. and Haines, M. D. (1994). Neospora infection in an aborted fetus in British Columbia. Canadian Veterinary Journal, 35, 114–115. Morales, S. E., Ramı´rez, L. J., Trigo, T. F., Ibarra, V. F., Puente, C. E. and Santacruz, M. (1997). Descripcio´n de un caso de aborto bovino asociado a infeccio´n por Neospora sp. en Me´xico. Veterinaria Me´xico, 28, 353–357. Nietfeld, J. C., Dubey, J. P. and Anderson, M. L. (1992). Neospora-like protozoan infection as a cause of abortion in dairy cattle. Journal of Veterinary Diagnostic Investigation, 4, 223–226. Ogino, H., Watanabe, E., Watanabe, S., Agawa, H., Navita, M., Haritani, M. and Kawashima, K. (1992). Neosporosis in the aborted fetus and newborn calf. Journal of Comparative Pathology, 107, 231–237. Pare´, J., Thurmond, M. C. and Hietala, S. K. (1996). Congenital Neospora caninum infection in dairy cattle and associated calfhood mortality. Canadian Journal of Veterinary Research, 60, 133–139. Thornton, R. N., Thompson, E. J. and Dubey, J. P. (1991). Neospora abortion in New Zealand cattle. New Zealand Veterinary Journal, 39, 129–133. Venturini, L., Di Lorenzo, C., Venturini, C. and Romero, J. (1995). Anticuerpos anti Neospora sp. en vacas que abortan. Veterinaria Argentina, 12, 167–170. 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, 180–185.
Received, November 1st, 1999 Accepted, March 10th, 2001
Neosporosis in Mexican Dairy Herds: Lesions and Immunohistochemical Detection of Neospora caninum in Fetuses E. Morales∗, F. J. Trigo∗, F. Ibarra†, E. Puente‡ and M. Santacruz§ ∗Departamento de Patologı´a and †Departamento de Parasitologı´a, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Auto´noma de Me´xico, 04510, Me´xico, D.F., ‡Pfizer, S.A. de C.V. Insurgentes Sur 1685, 2 Piso, Col. Guadalupe Inn, 01020, Me´xico, D.F. and §Centro Agropecuario Industrial de Tizayuca, S.A. Parque Industrial Tizayuca, Hidalgo, Me´xico Summary Of 211 aborted bovine fetuses collected from Mexican dairy herds between January 1996 and March 1999, 73 showed microscopical lesions consistent with neosporosis. Of these 73 fetuses, 58 (79%) showed lymphocytic myocarditis, 39 (53%) showed microgliosis and multifocal necrosis in the brain, 39 (53%) showed lymphocytic hepatitis, and 19 (26%) showed lymphocytic myositis. Immunohistochemical examination of brain, myocardium and liver from 53 of the same 73 fetuses demonstrated Neospora caninum antigens in 41 (77%), of which 19 (46%) gave positive results in one of the three sites, 15 (37%) in two, and seven (17%) in three. The results indicated the presence of neosporosis in a number of the main dairy farming regions of Mexico. 2001 Harcourt Publishers Ltd
Introduction Neospora caninum, a protozoon first recognized in the USA, causes disease in dogs (Dubey et al., 1988a; Dubey, 1992), dairy cattle (Anderson et al., 1991a,b; Barr et al., 1991a; Nietfeld et al., 1992), beef cattle, sheep, horses, goats and deer (Dubey and Lindsay, 1996). In non-human primates and numerous other hosts, experimental N. caninum infection induces clinical disease (Dubey and Lindsay, 1996). N. caninum was first isolated from dogs (Dubey et al., 1988a,b), and later from bovine aborted fetuses (Conrad et al., 1993). Neosporosis in cattle is characterized by abortion, neonatal paralysis, or the birth of symptomless carrier calves (Anderson et al., 1991a,b; Pare´ et al., 1996). Cattle may be infected transplacentally or as the result of ingesting oocysts produced by dogs, which represent the definitive host (Barr et al., 1993; Pare´ et al., 1996; Anderson et al., 1997; McAllister et al., 1998). Correspondence to: E. Morales 0021–9975/01/050058+06 $35.00
In California, New Zealand and the Netherlands, neosporosis is the most commonly diagnosed cause of bovine abortion (Anderson et al., 1991b; Thornton et al., 1991; Dubey and Lindsay, 1996). Within the American continent, apart from the USA, bovine neosporosis has been identified only in Canada (McIntosh and Haines, 1994), Mexico (Abbitt et al., 1993; Delgado et al., 1995; Morales et al., 1997) and Argentina (Venturini et al., 1995). The diagnostic techniques used consist of (1) histopathological and immunohistochemical examination of tissues from fetuses or newborn animals, and (2) ELISA or immunofluorescence to demonstrate specific antibody in fetal serum and fluids, or in maternal serum (Dubey and Lindsay, 1996). The most significant and distinctive microscopical lesions are lymphocytic encephalomyelitis with multifocal microgliosis and necrosis in the brain and spinal cord. In addition, lymphocytic myocarditis and myositis may be found, as well as lymphocytic periportal hepatitis, with or without foci of hepatocellular necrosis. Occasionally there 2001 Harcourt Publishers Ltd
59
Bovine Neosporosis
is inflammation and necrosis in other organs. Cysts are usually confined to the central nervous system (CNS), with clusters of tachyzoites in muscular tissue, liver and the CNS (Dubey et al., 1989, 1990a, b; Barr et al., 1990, 1991b, 1993). Histopathology and immunohistochemistry, which are reliable diagnostic methods, have been used to identify methods for diagnosis in individual cases, but epidemiological studies based on these techniques are few (Lindsay and Dubey, 1989; Anderson et al., 1990; Barr et al., 1990, 1991b; Wouda et al., 1997) and in Mexico there is little information about this disease. The first report was of six fetuses from a herd of 800 cows in the northeast of Mexico (Abbitt et al., 1993), and subsequently fetuses with distinctive neosporosis lesions were reported from diagnostic laboratories in Torreon Coahuila (Delgado et al., 1995) and Mexico City (Morales et al., 1997). In view of the meagre data on bovine neosporosis in Mexico, this report presents epidemiological information on the disease, based on histopathological and immunohistochemical examination of tissues from aborted fetuses. Materials and Methods Animals Aborted fetuses (n=211) from the main dairy production states of Hidalgo, Coahuila, Queretaro, Guanajuato, Durango, Aguascalientes, Jalisco, Estado de Mexico, Tamaulipas, Tlaxcala and Chihuahua were submitted to the Faculty of Veterinary Medicine and Zootechnics for diagnosis between January 1996 and March 1999. Histopathology Samples of brain, myocardium, diaphragmatic muscle, liver, lung, kidney and spleen were fixed in 10% neutral buffered formalin and processed by routine methods. Sections (5 m) were cut and stained with haematoxylin and eosin (HE), and selected sections of brain were also stained by the periodic acid-Schiff (PAS) method. Immunohistochemistry Of 73 fetuses with distinctive lesions of neosporosis, 53 (73%) were subjected to immunohistochemical examination of the brain, myocardium and liver (i.e., the organs most severely affected) to identify the parasite. Serial sections (3 m) were mounted on poly-L-lysine-coated slides, heated at 56°C, dewaxed in xylene, and rehydrated through graded
ethanols. Protease (Sigma Chemical Company, St Louis, Missouri, USA) was applied for 10 min at 37°C, followed by washing for 5 min with running deionized water. Endogenous peroxidase was quenched by immersion in 3% H2O2. Slides were blocked by the application of normal rabbit sera (Dako Corporation, Carpinteria, California, USA) for 10 min. The sections were then treated for 30 min at 37°C with goat anti-N. caninum primary antibody (Veterinary Medical Research and Development, Pullman, Washington, USA), diluted 1 in 3000. A secondary biotinylated antibody (Dako) diluted 1 in 600 was applied for 20 min at 37°C, followed by an avidin–biotin peroxidase complex (ABC) (Dako) for 20 min at 37°C. To develop the immunological reaction, the slides were treated for 5 min with a diaminobenzidine solution (DAB) (Zymed Laboratories, South San Francisco, California, USA). The sections were then counterstained with haematoxylin. They were considered positive when structures consistent with the morphology of the parasite were immunolabelled by this method. As a negative control, antiN. caninum antibody was replaced by normal goat serum (Dako) and positive controls consisted of sections of canine brain containing clusters of N. caninum tachyzoites (supplied by Dr Deborah Haines, University of Saskatchewan, Saskatoon, Canada). Results Histopathology Of the 211 fetuses examined, only 104 (49%) had detectable lesions. Of these, 73 had lesions consistent with neosporosis, the remainder showing changes suggestive of bacterial or viral infection. The 73 suspected neospora cases came from Hidalgo (38 cases), Coahuila (29), Aguascalientes (2), Queretaro (1), Guanajuato (1), Estado de Mexico (1) and Tlaxcala (1). The neospora lesions were classified as mild, severe or moderate. In mild cases, foci composed of only a few inflammatory cells were present in the myocardium or liver, or a few foci of microgliosis, with or without necrosis, were seen in the brain. In severe cases, there was an abundance of inflammatory cells in the myocardium or liver, or foci of microgliosis, with or without necrosis. Moderate cases fell between the two extremes of mild and severe. The most significant and consistent lesions of neosporosis were those in the myocardium, brain, liver and diaphragmatic muscle. The type and
60
E. Morales et al. Table 1 Type and severity of lesions consistent with neosporosis in 73 bovine fetuses Severity of lesions
Mild Moderate Severe
Number (and %) of the 73 fetuses showing microgliosis and necrosis
myocarditis
hepatitis
myositis
28 (38) 9 (12) 2 (3)
26 (36) 21 (29) 11 (15)
26 (36) 11 (15) 2 (3)
11 (15) 6 (8) 2 (3)
severity of these lesions are summarized in Table 1. Fifty-eight (79%) of the 73 fetuses displayed lymphocytic myocarditis (multifocal or diffuse), of variable severity, the mild form being the most common, followed by moderate, and then severe. In none of these cases were tachyzoites visible in muscle fibres with HE staining. Focal necrotic lesions and foci of microgliosis were observed in cerebral tissue from 39 fetuses (53%), in some cases the necrosis being surrounded by microglial cells and lymphocytes (Fig. 1). Although these lesions were scattered throughout the CNS, they were found most frequently in the cerebral cortex; they were sometimes multifocal and were commonly near blood vessels. In only one fetus (8 months of gestation) was lymphocytic perivascular infiltration detected, and in only six fetuses (8%) were neospora cysts, of different sizes, found. Each was round or oval in shape and 21×21 m to 49×33 m in size, with a thick PAS-positive wall (Fig. 2). An inflammatory reaction surrounding a tissue cyst, with associated degenerative changes, was seen only once. With HE staining, free tachyzoites were not seen. The severity of CNS lesions was variable, with mild changes occurring most commonly, followed by moderate, and then severe. Thirty-nine fetuses (54%) displayed lymphocytic periportal hepatitis of variable severity, mild lesions being the most common. Randomly distributed focal necrosis was also seen in six of these 39 cases. Parasitic structures were not identified on HE-stained sections of liver. In 19 fetuses (26%), multifocal, generally mild, lymphocytic myositis was seen in diaphragmatic muscle, but no parasitic structures were found. Only in three cases (4%) was a mild pulmonary inflammatory reaction appreciable, consisting of alveolar macrophages with some lymphocytes. The kidney and spleen showed no significant changes. In only 47 of the 73 fetuses could the gestational age be ascertained; it ranged from 3 to 8 months (mean 5·5).
Fig. 1. Fetal brain with a central necrotic focus surrounded by microglial cells. HE. ×400.
Immunohistochemistry Brain, myocardium and liver from 53 of the 73 fetuses with microscopical lesions consistent with neosporosis were examined immunohistochemically. N. caninum antigen was identified in 41 (77%) of the 53 fetuses, immunolabelling being detected in one organ in 19 (46%) of the 41 positive cases, in two organs in 15 (37%), and in all three organs in seven (17%). In the brain, neospora cysts,
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Bovine Neosporosis
(36%) of the 53 fetuses, sometimes associated with microgliosis and necrosis. In the myocardium, tachyzoites were identified in 24 (45%) of the 53 fetuses, mainly inside myocardial fibres, where they were associated with infiltrates or areas of necrosis, or both. In the liver, tachyzoites, either isolated or in clusters, were identified in 25 (45%) of the 53 fetuses (Fig. 3, Table 2).
Discussion
Fig. 2. Neospora caninum cyst in the brain of an aborted bovine fetus. Note the absence of inflammation. PAS. ×1000.
Fig. 3. Liver with a cluster of immunolabelled Neospora caninum tachyzoites ABC, with antiserum to N. caninum. ×1000.
without associated lesions, were identified in two (4%) of the 53 fetuses. Tachyzoites, either isolated or in clusters, were identified in the brain of 19
From the histopathological results, which were similar to those in other reports (Barr et al., 1990, 1991a; Anderson et al., 1991a; Dubey and Lindsay, 1993; Lindsay et al., 1993), it may be concluded that the tissues most frequently affected, and therefore most useful for diagnostic purposes, are CNS, myocardium, skeletal muscle and liver. Some authors point out that myocarditis tends to be severe but is easily concealed by autolytic changes (Barr et al., 1990; Dubey and Lindsay 1996; Wouda et al., 1997). In the present study, however, myocarditis was commonly mild; some cases displayed autolytic changes. In a study of neospora lesions in the brain of six aborted bovine fetuses, Helman et al. (1998) found most lesions in the cerebrum, followed by the cerebellum and then the medulla. In the present study, the distribution of lesions in the brain was not specifically examined, but most lesions were seen in the cerebral cortex. The neospora cysts, as in other reports (Dubey and Lindsay, 1996), were scarce and only one was associated with an inflammatory reaction; this reaction was possibly due to a break in the cyst wall, with consequent liberation of the contents, as suggested previously (Dubey et al., 1990a; Dubey, 1992). In only one case was there an appreciable perivascular lymphocytic infiltrate, and this occurred in the oldest fetus examined (gestational age 8 months), supporting the suggestion that maturity of the immune system is an important contributory factor (Ogino et al., 1992). Unfortunately, the present study gave no information on the spinal cord, in which neospora cysts and lesions have been described (Dubey et al., 1989; 1990a; Dubey and Lindsay, 1996). Unlike Dubey and Lindsay (1993), we found no haemorrhages in the cerebral parenchyma. In agreement with other reports (Barr et al., 1990; Wouda et al., 1997), lymphocytic hepatitis with a periportal tendency occurred, and was occasionally associated with focal necrosis. In 19 cases, lymphocytic myositis was observed, indicating the diagnostic value of diaphragmatic muscle samples. The average gestational age of the
62
E. Morales et al. Table 2 Immunohistochemical examination of brain, myocardium and liver from 53 of the 73 fetuses showing lesions consistent with neosporosis Immunolabelling
Positive Negative Inconclusive
Number (and %) of the 53 fetuses showing the stated immunolabelling result for tachyzoites in brain∗
myocardium
liver
19 (36) 17 (32) 15 (28)
24 (45) 24 (45) 5 (9)
25 (47) 16 (30) 12 (23)
∗ The brains of two (4%) of the 53 fetuses showed immunolabelled cysts.
aborted fetuses (5·5 months) was similar to that reported in California (Anderson et al., 1991b). Immunohistochemically, N. caninum organisms were identified most frequently and in greatest numbers in the liver, indicating the value of this tissue for demonstrating the parasite. The brain was helpful as a tissue in which tachyzoites and occasionally cysts could be demonstrated, although the parasites were scarce. The results differed, however, from those obtained in the Netherlands by Wouda et al. (1997), whose immunohistochemical study demonstrated neospora antigens in 85% of brain samples from neospora cases, as compared with 26% of liver samples and 14% of myocardial samples. The lesions described in the present study could not be considered pathognomonic for neosporosis, and it was not possible to demonstrate neospora antigens immunohistochemically in all tissues examined. However, the microgliosis and CNS necrosis were considered useful indicators of neosporosis, and strongly suggestive when taken together with myocarditis and hepatitis (Barr et al., 1990; Anderson et al., 1991b; Nietfeld et al., 1992). The findings in the present study and those of Wouda et al. (1997) suggest that for the diagnosis of neosporosis immunohistochemical examination of brain, liver and myocardium represents the method of choice. Finally, this study demonstrated the presence of bovine neosporosis in some of the main dairy farming regions of Mexico. Acknowledgments We are indebted to Dr J. P. Dubey (USDA Parasite Biology and Epidemiology Laboratory, Beltsville, MD, USA) and Dr A. M. Lo´pez (University of Prince Edward Island, Canada) for providing information and advice. We also thank Dr D. Haines (University of Saskatchewan, Canada) for providing
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Received, November 1st, 1999 Accepted, March 10th, 2001