VACCINE FOR IMMUNIZING CATS
AGAINST TOXOPLASMA OOCYST SHEDDING
Background of the Invention
1. Field of the Invention
The present invention is broadly concerned with a method of immunizing cats against Toxoplas- mosis wherein use is made of a live, reproductively deficient mutant of Toxoplasma gondii. More par¬ ticularly, it is concerned with such a method, and the associated vaccine, wherein the mutant is desig¬ nated T-263 and has ATCC Accession No. 40615.
2. Description of the Prior Art Toxoplas osis is a parasitic disease, and research has indicated that the parasite has a complicated life cycle with the infection spreading to many animals. Oocysts (egg spores) are shed in the feces of domestic cats and certain types of wild cats. Oocysts are then spread by contact with the feces. Flies and cockroaches, which eat feces, can serve as transport agents, contaminating animals which do not directly encounter the cat feces. Mice and birds can be infected either from transport agents , or through direct contact and can then spread the infection to animals which prey on them. Humans can be infected by eating raw or rare meats, or by direct contact with infected cat feces , or contaminated soil.
Toxoplasma infections are quite prevalent, with one-quater to one-half of the adults in the United States and elsewhere asymptomatically infect¬ ed. While the presence of Toxoplasma infections has long been known, little was discovered about the transmission of Toxoplasma until the late 1930's and
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1940's when Toxoplasmosis was found in newborn babies in the U.S. However, the life cycle of Toxoplasma, and the central role played therein by cats, has now been conclusively established.
The spectrum of human diesease due to Toxoplasmosis was characterized by a combination of serologic, immunologic and epidemiological studies, and by isolation of the causative agent, Toxoplasma
-g£2—ondii*. In the acute infection where cells are destroyed by rapidly proliferating organisms, there may oceur fever, pneumonia, and inflammation of the heart muscle, liver and skin (rash). Toward the end of the period or following a subclinical acute infection, localized or generalized swelling of lymph nodes is observed, especially in women. In newborns infected in utero, a subacute disease picture is typical. In addition to the symptoms of acute Toxoplasmosis mentioned above, meningoence- phalitiβ ("brain fever"), often with hydrocephalus ("water on the brain") , and retinochoroiditis (in¬ traocular inflammation) are important. Most of the mothers who have given birth to infected babies had infections without symptons.
Thus, Toxoplasmosis deserves special attention because of the serious danger it raises for the unborn human baby. A pregant woman may have the infection and unknowingly infect the fetus. If not diagnosed and treated in time, her child may be born with permanent brain and eye damage. For this reason, efforts to prevent infection during preg¬ nancy are important.
Inasmuch as domestic cats are important spreaders of Toxoplasma oocysts which are shed in their feces, attempts have been made in the past to
immunize domestic cats against oocyst shedding. Generally speaking, prior successful immunizations have required primary infection of cats with Toxo¬ plasmosis, followed by the usual oocyst shedding and a buildup of immunity. However, this manner of immunization generates the very phenomenon sought to be avoided, i.e., oocyst shedding, and as such is deemed deficient. This is especially the case when it is considered that infectious oocysts tend to remain active for a period of months up to a year and a half. Meanwhile, attempts to immunize cats using bradyzoites of oocystless Toxoplasma strains have proven unsuccessful. A successful approach to immunization of cats is described in U.S. Patent No. 4,473,548, which involves chemoprophylactic treatment of cats after primary Toxoplasma infection, so as to sup¬ press oocyst shedding while giving immunity. In this procedure, cats are initially infected and thereafter monensin or salinomycin is orally admin¬ istered for essentially preventing oocyst shedding while permitting immunization to develop in the cats. Although monensin was well accepted by kit- tens without apparent toxicity, hesitancy existed in using monensin because of occasional toxicity pro¬ blems which have been described in other animals. Furthermore, human tolerance for monensin has not been investigated, and this constitutes another reason for the lack of acceptance of monensin pro¬ phylaxis.
Accordingly, there is a decided need in the art for a method of immunizing cats against
Toxoplasmosis which eliminates the problem of fecal
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1 oocyst shedding while avoiding use of prophylactic drug treatment.
Summary of the Invention 5 The present invention overcomes the pro¬ blems described above and provides a method (and a corresponding vaccine) for the immunization of cats against T. ondii challenge which eliminates the phenomonen of oocyst shedding in the vaccinated
10 cats.
Broadly speaking, the method of the inven¬ tion involves administering to cats (preferably orally) an effective amount of a vaccine comprising
1 ,. a specific mutant of T.gondii which has been found to immunize 84% of cats without the need of chemo¬ prophylaxis. This level of immunization is very similar to that found using the method described in U.S. Patent No. 4,473,548 (85%).
20 The specific mutant useful in the inven¬ tion has been designated T-263, and has been de¬ posited with the American Type Culture Collection; the mutant has been accorded ATCC Accession No. 40615. The mutant was one of a large number pro-
25 duced by exposing tachyzoites of Toxoplasma of the known llC strain to an alkylating agent, N-methyl- N1-nitro-N-nitrosoguanidine (Pfefferkorn, E.R. , and Pfefferkorn, L.C. Toxopla as gondii: Isolation and preliminary characterization of temperature-sensi¬
30 tive mutants. Experimental Parasitology 39, 365- 376, 1976.) In plaque assays, mutagenized tachy¬ zoites were selected for their resistance to adenine arabinoside, and one clone was selected for resist¬ ance to 5-fluoro-deoxyuridine. The adenine arabino-
35 side-resistant mutants were again mutagenized with
1 the same mutagen or with ethyl nitrosourea, and selected clones were tested for suitability as a vaccine. A total of 117 mutants were tested as vaccines in cats, and of this number only a single
^ mutant, T-263, met the dictates of the invention.
Description of the Preferred Embodiment
The following is a description of the mutagenesis, screening and testing of the T-263
10 Toxoplasma mutant useful in the invention. Litera¬ ture references cited herein are incorporated by reference. A. Materials and Methods
_. _. Mice utilized in the experiments were CF-1 mice obtained from SASCO, Inc. (Omaha, NE) . Young kittens were mostly obtained from random source pregnant cats.- These had been donated or were on loan to the Animal Care Unit at the University of
20 Kansas Medical Center in response to newspaper advertisements requesting pregnant cats or kittens for research. In addition, laboratory reared kit¬ tens from Theracon, Inc. Topeka, KS were used. All kittens were serologically tested for Toxoplasma
25 antibody, and examined for fecal parasites prior to use. All kittens used were seronegative; Toxocara eggs and Cystoisospora felis and C. rivolta oocysts were found occasionally, but no Toxoplasma oocysts were found. The kittens obtained from Theracon
30 showed C. rivolta oocysts only.
Toxoplasma was grown in a cell culture system composed of human skin fibroblasts. After exponential growth was reached and at a stage when 8 organisms were present per cell, a mutagen, ethyl-
35 nitrosourea was added. The ethyl-nitroso-urea
concentration was 300 micrograms per milliliter and the .duration of treatment was for four hours. Survival was measured at this point and the srvival rate- , as 3% compared to control cultures. At the end of the mutagen exposure the medium was removed and the organisms were grown for another two days in culture,, being subcultured every day. At this point dilutions of Toxoplasma were seeded into 96 well trays, and 117 clones were further analyzed.
Clones of Ara-A resistant Toxoplasma were grown in human fibroblast tissue cultures for short periods of time, but were normally maintained as chronic infections in mice. These were injected either subcutaneoulsy (sc) or intraperitoneally (ip) and to prevent illness and permit development of bradyzoites in tissue cysts, the mice were treated from days 3 to 14 with sulfamerazine-sodium (Sigma Chemical Co., St. Louis, MO) 15 mg/100 ml of water, given ad libitum to drink. After at least one month, a mouse infected with a particular strain was killed, bled to be checked for the development of antibody, and a brain smear examined by light micro¬ scopy for the presence of cysts of Toxoplasma. The carcass- of a mouse infected with a given candidate strain was then fed to one or several seronegative, weaned kittens and the feces were examined for the presence of oocysts over the next 30 days.
Feces were usually collected daily for examination from 5 days through 12 , and then usual¬ ly three times weekly for up to 30 days. Oocysts were concentrated by flotation in a sucrose solution of specific gravity of 1.15 and stored in 2% (v/v) sulfuric acid to permit sporulation. Intensity of shedding was graded on a scale of 1 to 4, with 1+
indicating only one oocyst found on a slide, 2+ being several to numerous oocysts on a slide, +3 being an average of one oocyst per high power field, and +4 being numerous oocysts per high power (=400x) field. In some experiments, oocysts were counted in a hemacytometer using standard techniques.
When a cat was found not to shed oocysts after primary infection with a candidate mutant, it was challenged (po) with an inoculum of T-265 brady¬ zoites, and again checked for oocyst production over a period of 30 days. When visual examination failed to identify oocysts after exposure, the apparent failure of shedding was verified by the inoculation of the combined sucrose float supernatants of the 5 to 12 day speciments into mice. The samples were neutralized with 3.3% (W/V) sodium hydroxide, using phenol red as an indicator, spun down, and the sediment was fed or injected (ip) into groups of several mice.
When mice died, impression smears were made of lung, liver, spleen and brain, stained with Giemsa, and examined for the presence of Toxoplasma. Survivors were bled after 21 days and tested sero- logically for the presence of antibody for Toxo¬ plasma.
Cats were bled from the ear before expos¬ ure to tissues of infected mice, at least 30 days thereafter, and following challenge infection. Mice were bled from the retroorbital sinus while under ether anesthesia. Antibody titers were determined by ther Sabin-Feldman dye test (Frenkel and Jacobs,
"Ocular Toxoplasmosis", A.M.A. Arch. Ophth. 1958), using tachyzoites of the RH strain of Toxoplasma,
1 maintained as acute infections in mice and passed thrice weekly (ip).
After a candidate mutant strain had failed to produce oocysts in 3 cats after primary exposure,
*■* and had immunized these 3 cats, an attempt was made to detect whether that strain was capable of forming gametes. Mouse tissues containing bradyzoites of the Ara-A resistant (non-oocyst forming) vaccine candidate strain were mixed with tissues containing
10 bradyzoites of a FUDR resistant oocyst-producing strain -f the mixture was fed to one or several kit¬ tens. Production of oocysts incorporating both Ara-A and FUDR resistance indicated recombination of
_. _. the two genomes, because of production of one gamete by the candidate strain (Pfefferkorn, L.C., and Pfefferkorn, E.R. Toxoplasma gondii: Genetic recom¬ bination between drug resistant mutants. Experi¬ mental Parasitology 50, 305-316, 1980.)
20 Presence of cysts of Toxoplasma in brain of mice was determined microscopically; one or several brains were fed to kittens; infection was evaluated by oocyst shedding, and in its absence, by seroconversion. Oocyst numbers were estimated
25 visually, and quantitated by titration in 6 mice and after exposure to excystation fluid in fibroblast cultures. The number of mice with Toxoplasma in smears and of the seropositive animals were combined to determine the IDrr*' In cell cultures the pres¬
30 ence of plaques in certain dilutions served as indicator of infection.
B. RESULTS
A total of 117 mutagenized araA^ mutants
35 were tested in cats as described above. 106 mutants
gave rise to oocyst shedding and were discarded. Of these, 85 mutants gave rise to oocysts shedding in the first cat, 16 in the second cat, 3 in the third. Two mutants were tested in 11 or 12 cats, with shedding observed in 6 of 11 and, 2 of 12.
The infection with 10 mutants was not accompanied by oocyst shedding. Of these, infec¬ tions with 6 mutants did not confer immunity, and 3 mutants were lost. One mutant, denominated T-263, was tested in a total 37 cats, none of which shed oocysts, determined visually and by mouse inocula¬ tion. When these cats were challenged, 31 or 83.7% were immune. This includes a group of 5 kittens challenged after 6 months, of which 3 were immune. Results of these tests are set forth in the follow¬ ing Table:
Results of
Vaccination of Cats Challenge with (Compl
Oocyst Shedding Reciprocal antibody titer Oocyst shedding Recipro visually mouse inoc. Range Geometric mean visually mouse innoc Range
T-263 0/37 0/37 <2-128 9.2 6/37 6/37 <2-1
Vaccine
r-265 __ 16/16 16/16 16-5
P - <0.001
In order to test for presence of either male or female gametes by T-263, bradyzoites of this
R ara-A mutant were fed to cats simultaneously with bradyzoites of T-237, an FUDR oocyst producer. The resultant oocysts were cloned and tested for ara-A resistance. In three different experiments, 3%, 1.5%, and 0.01% of oocysts were found to be ara-A resistant, with 0.1%, 0.1%, and 0.01% of oocysts doubly resistant.
To assess the sensitivity of visually determining the number of oocysts, a suspension was counted and titrated. In the presence of a visual count of 50,000 oocysts, oral titration in mice yielded 10,000, subcutaneous titration 19, and titration in human skin fibroblast cultures 4,400 infectious units.
As described, the vaccine comprising the T-263 mutant immunized essentially 84% of the cats challenged. This is a slightly lower, but statis¬ tically insignificant, immunization protection than that afforded by infection with normal bradyzoites which gives concomitant oocyst shedding (88-94%) . (Frenkel, J.K. , and Smith, D.D. Immunization of cats against shedding of Toxoplasma oocysts. Jour¬ nal of Parasitology 68(5) :744-748, 1982.) A par¬ ticular advantage of the new vaccine and method is that it eliminates chemophrophylaxis with monensin or the like; at the same time, immunization in ac c o rd ance with the invention does not lead to oocyst formation . The occurrence of the ara-A gene and of genetic recombination , although in low inci¬ d ence , sugges ts that the T -263 mutant forms one gamete , and according the mutant has been designated
"reproductively deficient" to characterize this attribute.
The T-263 mutant to be used for vaccina¬ tion purposes has been successfully propagated in mice in order to develop the uniquely infectious bradyzoites in cysts. Alternative methods would be the use of other small laboratory animals such as rats; or propagation in certain cell cultures, in which cyst formation occurs spontaneouly or in the presence of antibody (Hoff et al. "Toxoplasma gondii Cysts in Cell Culture", J. Parasitol. 63:1121-24, 1977). In addition, while the bradyzoite cysts have been fed directly, if desired the vaccine may in¬ clude a suitable carrier.