Brief Communication
Concurrent Inf ect ion with Toxoplasma gondii and Feline Leukemia Virus Antibody Response and Oocyst Production
Sharon Patton, MS, PhD, Alfred M. Legendre, DVM, MS, M. D. McGavin, DVM, PhD, and Diana Pelletier, BS
Four adult cats (two testing positive and two negative for feline leukemia virus FeLV) were fed Toxoplustnagondii tissue cysts collected from the brains of mice. Two control cats (1 FeLV+, 1 FeLV-) were not fed cysts. The cats infected with T. gondii shed thousands of oocysts but remained clinically and physically normal, with hemograms and clinical chemistry values essentially unchanged irrespective of their FeLV status. Infection with FeLV did not increase the duration of oocyst shedding. At necropsy no significant lesions were found. T. gondii antibodies were detected by three serologic tests in the cats fed tissue cysts. The time necessary for an antibody response to T. gondii was not altered by the FeLV infection. Indirect hemagglutination (IHA) was the least reliable of the serologic tests studied; it detected antibodies later in the infection, and titers were less than in the other tests. Latex agglutination (LA) detected antibodies a few days before IHA, but titers were less than in modified direct agglutination (MAT). MAT detected antibodies earliest in the infection and also measured antibodies in aqueous humor and cerebrospinal fluid. (Journal of Veterinary Internal Medicine 1991; 5:199-201)
IT IS WIDZLY believed that cats infected with feline leukemia virus (FeLV) may be predisposed to disease caused by other organisms like Tuxuplasma gundii because of virus-induced immunosuppression. The antibody response to certain antigens may be depressed in FeLV-infected cats.4 Reactivation of T. gundii infection with clinical signs was reported in a FeLV-positive cat.2 However, in a recent study,’ only six of 97 cats surveyed were concurrently infected with T. gundii and FeLV, and a study of 15 cases of clinical toxoplasmosis in cats did not detect FeLV antigen in the serum of any cat.6 The influence of FeLV infection on excretion of T. gunFrom the Departments of Environmental Practice (Patton, Pelletier). Urban Practice (Legendre), and Pathobiology (McGavin), University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee. This work was supported by The Institute of Agriculture, College of Veterinary Medicine, Center of Excellence for Livestock Disease and Human Health, Knoxville, Tennessee. The authors thank Rebecca Auk, Karen Puckett, and Shawna Doan for their excellent technical assistance. Reprint requests: Sharon Patton. MS, PhD, Department of Environmental Practice, College of Veterinary Medicine, PO Box 107 1, Knoxville, TN 37901-1071.
dii oocysts, the seventy of toxoplasmosis, or the production of antibodies to T. gundii in cats has not been studied. Mammals usually acquire infection with T. gundii after ingestion of oocysts from the environment or tissue stages in poorly cooked meat. Congenital toxoplasmosis and toxoplasmosis in immunosuppressed patients (those infected with human immunodeficiency virus and those receiving cytotoxic or immunosuppressive drugs or radiation) have potentially serious consequences for the human p ~ p u l a t i o nThe . ~ purpose of this study was to compare the development of anti-Tuxuplusma IgG, the excretion of T. gundii oocysts, and the clinical and pathologic consequences of toxoplasmosis in cats testing positive for FeLV antigen on enzyme-linked immunosorbent assay (ELISA)* to those in cats testing negative for FeLV antigen. Six adult cats (four females, two males), testing negative for T. gondii antibodies by indirect hemagglutination (IHA), latex agglutination (LA), and the modified
* Leukassay F, Pittman-Moore
199
Inc.. Washington Crossing, NJ.
200
PATTON ET AL.
agglutination test using whole formalin-fixed tachyzoites (MAT), were used in the study. The cats had participated in a FeLV exposure study for six months, and three of them tested positive for FeLV antigen on ELISA.* Four of the cats (two FeLV+, two FeLV-) were fed a suspension of tissue cysts (n = 12) prepared from the brains of mice that were previously inoculated with a goat isolate of T. gondii9 Two control cats (one FeLV+, 1 FeLV-) were not fed cysts. Cats were housed individually in fiber glass cages and fed dry cat food and water ad libitum. Feces were removed from the cages twice daily and examined for T. gondii oocysts by centrifugal flotation with sucrose s ~ l u t i o nAll . ~ six animals were killed at postinfection (PI) day 90. Immediately before death, cerebrospinal fluid (CSF) and aqueous humor were collected and titered without delay for antibody against T. gondii using IHA, LA, and MAT. Tests were not done on CSF from cat 4 because insufficient sample was collected. Body organs and tissues were examined for gross lesions at necropsy. Sections from spleen, lung, liver, kidney, heart, tongue, diaphragm, muscles, spinal cord, and brain were examined microscopically. Clinical observations were made daily for body temperature, gastrointestinal disturbance, fecal consistency, appetite, and general physical condition. Body weight was also recorded each week. Blood was collected weekly for complete blood counts and serum chemistry panels. Serum was stored at -20°C until titered for T. gondii antibodies using IHA, LA, and MAT. Both IHA and LA were performed using commercially available kits.? The formalin-fixed tachyzoites used as antigen in the MAT were supplied by bioMerieux Laboratory Reagents, Lyon, France. MAT was performed as previously described with mercaptoethanol added to the serum samples to remove IgM.7 Trypan blue dye was added to the diluent solution before it was mixed with the antigen so that agglutination could be easily seen. For all tests, twofold dilutions of serum were made ranging from 1:4 to 1:89 12. Sera with titers greater than 89 12 were titered to endpoint. Control sera$ (positive and negative) were included with each batch of serum tested. Titers less than 32 were considered nonspecific reactions. The four cats fed tissue cysts shed oocysts between PI day 7 and 16 (Table 1). Each cat shed several thousand oocysts each day. Except for a moderate loss of appetite for a few days PI, the cats remained clinically normal. There were no differences on physical examination between the cats fed T. gondii cysts and the control cats, irrespective of their FeLV status. The hemograms and clinical chemistry values collected throughout the study
t TPM-TEST, Wampole Laboratories, Cranbury, NJ, and ToxotestMT "Eiken," Tanabe USA, Inc.. San Diego, CA. $ The control sera used in this study were collected from laboratory personnel. They have reproducible titers on the serologic tests used in this study.
Journal of Veterinary Internal Medicine
remained within reference values before and after infection with T. gondii in all cats. At necropsy, no lesions associated with FeLV, diseases secondary to FeLV, or toxoplasmosis were found in any of the cats. The cats infected with T. gondii shed thousands of oocysts but remained clinically normal with no detectable lesions, grossly or microscopically, even when concurrently infected with FeLV. T. gondii antibodies were detected by all three serologic tests in the four cats fed tissue cysts (Table 1). After antibodies appeared in these cats, the sera remained positive for antibodies throughout the study. Antibodies were first detected by MAT at 2 1 days PI in two of the cats (cats 2 and 4) infected with T. gondii. This was five and six days after oocysts were no longer detectable in the feces. These same two cats did not develop positive IHA titers until 54 days PI, which was 39 and 40 days after oocysts were no longer detectable in the feces. With MAT, antibody titers increased each week. With IHA, the antibody titers increased slowly and remained relatively low, never exceeding 256 in any of the infected cats throughout the experiment. The earliest detectable IHA titer was at 35 days PI. The LA titers that appeared a few days before the IHA titers were less than the MAT titers, but higher than the IHA. One of the cats that was not fed tissue cysts occasionally had a titer of 4 on IHA and LA and 16 on MAT. Nonspecific titers of 8 were also occasionally seen in the experimental cats in the weeks before infection with T. gondii. Only the MAT detected antibodies in the CSF and aqueous humor collected at death (Table 1). Dubey and Thulliez" recently evaluated the sensitivity of five tests for the detection of antibodies to T. gondii in cats fed tissues containing T. gondii cysts. Although T. gondii antibodies were detected by all five serologic tests in all the infected cats, the MAT was more sensitive for the serodiagnosis of toxoplasmosis. Antibody titers in LA, IHA, modified agglutination using acetone preserved antigen (MATA), and dye test (DT) were lower than those obtained with MAT and usually did not appear as rapidly. All of the experimentally infected cats shed oocysts, but none of them developed clinical toxoplasmosis. The initial appearance of antibodies in our study was similar to that in Dubey's study. Evidently, the time necessary for an antibody response to T. gondii was not altered by the FeLV infection. In our study, concurrent infection with isolates of T. gondii and FeLV produced no detectable disease in two healthy FeLV-infected cats. Perhaps this is not surprising because there appears to be considerable variation in the immunosuppressive ability of isolates of FeLV,3and the virus used in this study was not characterized. Also, clinical signs of disease might be produced if cats were inoculated with more bradyzoites or oocysts, or with a different isolate of T. gondii, because the virulence of T. gondii isolates also ~ a r i e s . ~
Vol. 5
. No. 3, 1991
20 1
TOXOPLASMA AND FeLV IN CATS
TABLEI . Effect of Coinfection With Feline Leukemia Virus and Toxopkasrnu gondii on the Antibody Response to T. gondii and the Excretion of Oocysts in Cats* Day Postinfection
Titers
Seroconversion Occurred
Cat
MinimumJMaximum Titers (Serum)
Cerebrospinal Fluid
Aqueous Humor
FeLV Status
Oocysts Shed
IHA
LA
MAT
IHA
LA
MAT
IHA
LA
MAT
IHA
LA
MAT
+ +
9-16 11-15 7-14 7-16 0 0
50 54 35 54 -
41 41 35 50 -
28 21 28 21 -
321256 641128 321256 32/64 010
3211024 128/1024 3214096 1281256 010 010
1024/40920 32140920 4096/81840 1024/40920
4 0 0 0 0 0
0 0 0 0 0 0
64 64 512 64 0 0
0 0 0 NS 0 0
0 0 0 NS 0 0
128 32 512 NS 0 0
I 2 3 4 5 6
~
-
+ ~
-
010
010 010
NS: no sample.
* Antibody response measured by indirect hemagglutination test (IHA), latex agglutination test (LA), and modified direct agglutination test (MAT).
This study confirmed that infected animals displaying no overt clinical signs may shed thousands of oocysts into the environment. These oocysts, which infect all other species of mammals and birds, remain viable for months.’ As a public health precaution, an examination of the feces for oocysts and other parasitic products should be performed routinely on all cats presented for examination. Serologic tests for IgG antibodies indicate only whether or not the cat has tissue stages of T. gondii, and, as these results confirm, infected cats that have shed oocysts can have no titer almost two months PI by some serologic tests. A detectable IgM titer specific for T. gondii, or a rising IgG titer, is necessary to prove a recent infection. IHA was the least reliable of the serologic tests studied, both because it detected antibodies later in the infection, and because titers were less than with the other two tests. MAT detected titers earlier in the infection and also detected antibodies in aqueous humor and CSF.
References I , Cotter S. Feline Viral Neoplasia. In: Greene, CE, ed. Clinical Microbiology and Infectious Diseases of the Dog and Cat. Philadelphia: WB Saunders, 1984; 490-5 13.
2. Schalm OW. Findings in Feline Bone Marrow: I . Effect of cyclophosphamide 2. Feline Infectious Anemia and Lymphosarcoma 3. Toxoplasmosis and Erythroleukemia. Feline Prac 1978; 8(4):18-22. 3 . Overbaugh J, Donahue PR, Quackenbush SL, et al. Molecular cloning ofa feline leukemia virus that induces fatal immunodeficiency disease in cats. Science 1988; 239:906-910. 4. Trainin Z , Wernicke D, Essex M. Suppression ofthe humoral antibody responses in natural retrovirus infections. Science 1983; 220:858-859. 5. Lappin MR, Marks A, Greene CE. et al. Serologic prevalence of select infectious diseases in 97 cats with suspected Toxoplusma zondii induced uveitis (Abstr). Proc 7th ACVIM Forum 1989; 67: I04 I . 6. Lappin MR, Greene CE, Winston S, et al. Clinical Feline Toxoplasmosis: Serologic diagnosis and therapeutic management of I 5 cases. J Vet Intern Med 1989; 3: 139-1 43. 7. Dubey JP and Beattie CP. Toxoplasmosis of Animals and Man. Boca Raton, FL: CRC Press Inc, 1988. 8. Legendre AM, Mitchener KL, Potgieter LND. Efficacy of a feline leukemia virus vaccine in a natural exposure challenge. J Vet Intern Med 1990; 4:92-98. 9. Patton S, Johnson SS, Puckett K. Prevalence of Toxoplusrnu gondii antibodies in nine populations of dairy goats: Compared titers using modified direct agglutination and indirect hemagglutination. J Parasitol 1990; 76:74-77. 10. Dubey JP, Thulliez, PH. Serologic diagnosis of toxoplasmosis in cats fed Toxoplusrna gondii tissue cysts. J Am Vet Med Assoc 1989; 19411297-1299,
Concurrent Inf ect ion with Toxoplasma gondii and Feline Leukemia Virus Antibody Response and Oocyst Production
Sharon Patton, MS, PhD, Alfred M. Legendre, DVM, MS, M. D. McGavin, DVM, PhD, and Diana Pelletier, BS
Four adult cats (two testing positive and two negative for feline leukemia virus FeLV) were fed Toxoplustnagondii tissue cysts collected from the brains of mice. Two control cats (1 FeLV+, 1 FeLV-) were not fed cysts. The cats infected with T. gondii shed thousands of oocysts but remained clinically and physically normal, with hemograms and clinical chemistry values essentially unchanged irrespective of their FeLV status. Infection with FeLV did not increase the duration of oocyst shedding. At necropsy no significant lesions were found. T. gondii antibodies were detected by three serologic tests in the cats fed tissue cysts. The time necessary for an antibody response to T. gondii was not altered by the FeLV infection. Indirect hemagglutination (IHA) was the least reliable of the serologic tests studied; it detected antibodies later in the infection, and titers were less than in the other tests. Latex agglutination (LA) detected antibodies a few days before IHA, but titers were less than in modified direct agglutination (MAT). MAT detected antibodies earliest in the infection and also measured antibodies in aqueous humor and cerebrospinal fluid. (Journal of Veterinary Internal Medicine 1991; 5:199-201)
IT IS WIDZLY believed that cats infected with feline leukemia virus (FeLV) may be predisposed to disease caused by other organisms like Tuxuplasma gundii because of virus-induced immunosuppression. The antibody response to certain antigens may be depressed in FeLV-infected cats.4 Reactivation of T. gundii infection with clinical signs was reported in a FeLV-positive cat.2 However, in a recent study,’ only six of 97 cats surveyed were concurrently infected with T. gundii and FeLV, and a study of 15 cases of clinical toxoplasmosis in cats did not detect FeLV antigen in the serum of any cat.6 The influence of FeLV infection on excretion of T. gunFrom the Departments of Environmental Practice (Patton, Pelletier). Urban Practice (Legendre), and Pathobiology (McGavin), University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee. This work was supported by The Institute of Agriculture, College of Veterinary Medicine, Center of Excellence for Livestock Disease and Human Health, Knoxville, Tennessee. The authors thank Rebecca Auk, Karen Puckett, and Shawna Doan for their excellent technical assistance. Reprint requests: Sharon Patton. MS, PhD, Department of Environmental Practice, College of Veterinary Medicine, PO Box 107 1, Knoxville, TN 37901-1071.
dii oocysts, the seventy of toxoplasmosis, or the production of antibodies to T. gundii in cats has not been studied. Mammals usually acquire infection with T. gundii after ingestion of oocysts from the environment or tissue stages in poorly cooked meat. Congenital toxoplasmosis and toxoplasmosis in immunosuppressed patients (those infected with human immunodeficiency virus and those receiving cytotoxic or immunosuppressive drugs or radiation) have potentially serious consequences for the human p ~ p u l a t i o nThe . ~ purpose of this study was to compare the development of anti-Tuxuplusma IgG, the excretion of T. gundii oocysts, and the clinical and pathologic consequences of toxoplasmosis in cats testing positive for FeLV antigen on enzyme-linked immunosorbent assay (ELISA)* to those in cats testing negative for FeLV antigen. Six adult cats (four females, two males), testing negative for T. gondii antibodies by indirect hemagglutination (IHA), latex agglutination (LA), and the modified
* Leukassay F, Pittman-Moore
199
Inc.. Washington Crossing, NJ.
200
PATTON ET AL.
agglutination test using whole formalin-fixed tachyzoites (MAT), were used in the study. The cats had participated in a FeLV exposure study for six months, and three of them tested positive for FeLV antigen on ELISA.* Four of the cats (two FeLV+, two FeLV-) were fed a suspension of tissue cysts (n = 12) prepared from the brains of mice that were previously inoculated with a goat isolate of T. gondii9 Two control cats (one FeLV+, 1 FeLV-) were not fed cysts. Cats were housed individually in fiber glass cages and fed dry cat food and water ad libitum. Feces were removed from the cages twice daily and examined for T. gondii oocysts by centrifugal flotation with sucrose s ~ l u t i o nAll . ~ six animals were killed at postinfection (PI) day 90. Immediately before death, cerebrospinal fluid (CSF) and aqueous humor were collected and titered without delay for antibody against T. gondii using IHA, LA, and MAT. Tests were not done on CSF from cat 4 because insufficient sample was collected. Body organs and tissues were examined for gross lesions at necropsy. Sections from spleen, lung, liver, kidney, heart, tongue, diaphragm, muscles, spinal cord, and brain were examined microscopically. Clinical observations were made daily for body temperature, gastrointestinal disturbance, fecal consistency, appetite, and general physical condition. Body weight was also recorded each week. Blood was collected weekly for complete blood counts and serum chemistry panels. Serum was stored at -20°C until titered for T. gondii antibodies using IHA, LA, and MAT. Both IHA and LA were performed using commercially available kits.? The formalin-fixed tachyzoites used as antigen in the MAT were supplied by bioMerieux Laboratory Reagents, Lyon, France. MAT was performed as previously described with mercaptoethanol added to the serum samples to remove IgM.7 Trypan blue dye was added to the diluent solution before it was mixed with the antigen so that agglutination could be easily seen. For all tests, twofold dilutions of serum were made ranging from 1:4 to 1:89 12. Sera with titers greater than 89 12 were titered to endpoint. Control sera$ (positive and negative) were included with each batch of serum tested. Titers less than 32 were considered nonspecific reactions. The four cats fed tissue cysts shed oocysts between PI day 7 and 16 (Table 1). Each cat shed several thousand oocysts each day. Except for a moderate loss of appetite for a few days PI, the cats remained clinically normal. There were no differences on physical examination between the cats fed T. gondii cysts and the control cats, irrespective of their FeLV status. The hemograms and clinical chemistry values collected throughout the study
t TPM-TEST, Wampole Laboratories, Cranbury, NJ, and ToxotestMT "Eiken," Tanabe USA, Inc.. San Diego, CA. $ The control sera used in this study were collected from laboratory personnel. They have reproducible titers on the serologic tests used in this study.
Journal of Veterinary Internal Medicine
remained within reference values before and after infection with T. gondii in all cats. At necropsy, no lesions associated with FeLV, diseases secondary to FeLV, or toxoplasmosis were found in any of the cats. The cats infected with T. gondii shed thousands of oocysts but remained clinically normal with no detectable lesions, grossly or microscopically, even when concurrently infected with FeLV. T. gondii antibodies were detected by all three serologic tests in the four cats fed tissue cysts (Table 1). After antibodies appeared in these cats, the sera remained positive for antibodies throughout the study. Antibodies were first detected by MAT at 2 1 days PI in two of the cats (cats 2 and 4) infected with T. gondii. This was five and six days after oocysts were no longer detectable in the feces. These same two cats did not develop positive IHA titers until 54 days PI, which was 39 and 40 days after oocysts were no longer detectable in the feces. With MAT, antibody titers increased each week. With IHA, the antibody titers increased slowly and remained relatively low, never exceeding 256 in any of the infected cats throughout the experiment. The earliest detectable IHA titer was at 35 days PI. The LA titers that appeared a few days before the IHA titers were less than the MAT titers, but higher than the IHA. One of the cats that was not fed tissue cysts occasionally had a titer of 4 on IHA and LA and 16 on MAT. Nonspecific titers of 8 were also occasionally seen in the experimental cats in the weeks before infection with T. gondii. Only the MAT detected antibodies in the CSF and aqueous humor collected at death (Table 1). Dubey and Thulliez" recently evaluated the sensitivity of five tests for the detection of antibodies to T. gondii in cats fed tissues containing T. gondii cysts. Although T. gondii antibodies were detected by all five serologic tests in all the infected cats, the MAT was more sensitive for the serodiagnosis of toxoplasmosis. Antibody titers in LA, IHA, modified agglutination using acetone preserved antigen (MATA), and dye test (DT) were lower than those obtained with MAT and usually did not appear as rapidly. All of the experimentally infected cats shed oocysts, but none of them developed clinical toxoplasmosis. The initial appearance of antibodies in our study was similar to that in Dubey's study. Evidently, the time necessary for an antibody response to T. gondii was not altered by the FeLV infection. In our study, concurrent infection with isolates of T. gondii and FeLV produced no detectable disease in two healthy FeLV-infected cats. Perhaps this is not surprising because there appears to be considerable variation in the immunosuppressive ability of isolates of FeLV,3and the virus used in this study was not characterized. Also, clinical signs of disease might be produced if cats were inoculated with more bradyzoites or oocysts, or with a different isolate of T. gondii, because the virulence of T. gondii isolates also ~ a r i e s . ~
Vol. 5
. No. 3, 1991
20 1
TOXOPLASMA AND FeLV IN CATS
TABLEI . Effect of Coinfection With Feline Leukemia Virus and Toxopkasrnu gondii on the Antibody Response to T. gondii and the Excretion of Oocysts in Cats* Day Postinfection
Titers
Seroconversion Occurred
Cat
MinimumJMaximum Titers (Serum)
Cerebrospinal Fluid
Aqueous Humor
FeLV Status
Oocysts Shed
IHA
LA
MAT
IHA
LA
MAT
IHA
LA
MAT
IHA
LA
MAT
+ +
9-16 11-15 7-14 7-16 0 0
50 54 35 54 -
41 41 35 50 -
28 21 28 21 -
321256 641128 321256 32/64 010
3211024 128/1024 3214096 1281256 010 010
1024/40920 32140920 4096/81840 1024/40920
4 0 0 0 0 0
0 0 0 0 0 0
64 64 512 64 0 0
0 0 0 NS 0 0
0 0 0 NS 0 0
128 32 512 NS 0 0
I 2 3 4 5 6
~
-
+ ~
-
010
010 010
NS: no sample.
* Antibody response measured by indirect hemagglutination test (IHA), latex agglutination test (LA), and modified direct agglutination test (MAT).
This study confirmed that infected animals displaying no overt clinical signs may shed thousands of oocysts into the environment. These oocysts, which infect all other species of mammals and birds, remain viable for months.’ As a public health precaution, an examination of the feces for oocysts and other parasitic products should be performed routinely on all cats presented for examination. Serologic tests for IgG antibodies indicate only whether or not the cat has tissue stages of T. gondii, and, as these results confirm, infected cats that have shed oocysts can have no titer almost two months PI by some serologic tests. A detectable IgM titer specific for T. gondii, or a rising IgG titer, is necessary to prove a recent infection. IHA was the least reliable of the serologic tests studied, both because it detected antibodies later in the infection, and because titers were less than with the other two tests. MAT detected titers earlier in the infection and also detected antibodies in aqueous humor and CSF.
References I , Cotter S. Feline Viral Neoplasia. In: Greene, CE, ed. Clinical Microbiology and Infectious Diseases of the Dog and Cat. Philadelphia: WB Saunders, 1984; 490-5 13.
2. Schalm OW. Findings in Feline Bone Marrow: I . Effect of cyclophosphamide 2. Feline Infectious Anemia and Lymphosarcoma 3. Toxoplasmosis and Erythroleukemia. Feline Prac 1978; 8(4):18-22. 3 . Overbaugh J, Donahue PR, Quackenbush SL, et al. Molecular cloning ofa feline leukemia virus that induces fatal immunodeficiency disease in cats. Science 1988; 239:906-910. 4. Trainin Z , Wernicke D, Essex M. Suppression ofthe humoral antibody responses in natural retrovirus infections. Science 1983; 220:858-859. 5. Lappin MR, Marks A, Greene CE. et al. Serologic prevalence of select infectious diseases in 97 cats with suspected Toxoplusma zondii induced uveitis (Abstr). Proc 7th ACVIM Forum 1989; 67: I04 I . 6. Lappin MR, Greene CE, Winston S, et al. Clinical Feline Toxoplasmosis: Serologic diagnosis and therapeutic management of I 5 cases. J Vet Intern Med 1989; 3: 139-1 43. 7. Dubey JP and Beattie CP. Toxoplasmosis of Animals and Man. Boca Raton, FL: CRC Press Inc, 1988. 8. Legendre AM, Mitchener KL, Potgieter LND. Efficacy of a feline leukemia virus vaccine in a natural exposure challenge. J Vet Intern Med 1990; 4:92-98. 9. Patton S, Johnson SS, Puckett K. Prevalence of Toxoplusrnu gondii antibodies in nine populations of dairy goats: Compared titers using modified direct agglutination and indirect hemagglutination. J Parasitol 1990; 76:74-77. 10. Dubey JP, Thulliez, PH. Serologic diagnosis of toxoplasmosis in cats fed Toxoplusrna gondii tissue cysts. J Am Vet Med Assoc 1989; 19411297-1299,
