Feline Leukemia Virus Infection: Age-Related Variation in Response of Cats to Experimental Infection 1, 2 Edward A. Hoover," Richard G. Olsen," William D. Hardy, Jr.,4Joseph P. Schaller,3 and Lawrence E. Mathes3

1976.

Newborn kittens are highly susceptible to experimental FeLV infection and leukemogenesis by FeLV (1-5). The degree of susceptibility of older cats to FeLV is less certain, since definitive information concerning the relationship of age to susceptibility to experimental FeLV infection is limited. Horizontal transmission of FeLV and FeLV-related disease has been demonstrated conclusively in nature (6, 7), and seroepidemiologic studies indicate that many cats exposed to FeLV resist leukemogenesis (8-10). Age at time of exposure could be an important factor influencing the outcome of FeLV infection in cats. Therefore, the major objective of this study was to determine the response of cats of various ages to experimental infection with two strains of FeLV. Criteria used to evaluate susceptibility to FeLV were: I) detection of FeLV gsa in circulating leukocytes, 2) production of FOCMA and VN antibody, and 3) induction of Fe LV-related disease.

feline lymphoblast line FL-74 (13), which continuously produced FeLV-KT. Medium from the suspension cultures was concentrated by dialysis against Aquacide (Calbiochem, Los Angeles, Calif.) and virus separated by centrifugation at 40,000Xg onto a cushion of 45% sucrose. The final inoculum contained 107 FFU/ml and 1011 virus particles/ml, as determined by negative staining with phosphotungstic acid and enumeration byelectron microscopy. FeLV gsa in leukocytes.-The presence of FeLV gsa in circulating leukocytes was determined by the indirect immunofluorescence test described by Hardy et a!. (14). The presence of Fe LV gsa in leukocytes was indicative ofvirernia (15). The inoculation status of the cats was not known when the tests were performed. VN antibody assay.-VN antibody was determined by inhibition of focus induction by FeLV subgroup A (11) in 81C S+ L- cell cultures (12) as described by Schaller and Olsen (16). Reference neutralizing antiserum was goat antiserum against FeLV-KT, with a VN titer of 1:1,000 against FeLV subgroup A (16). The FeLV subgroup A reference virus was obtained from Dr. P. S. Sarma, National Cancer Institute, Bethesda, Md. FOCMA antibody assays.- The IMI test for antibody against FOCMA was that of Essex et a!. (17). The conditions for culture of the FOCMA-containing cell line FL74 were described in (13). The MCT for FOCMA antibody was' described in (18). For the test, 25 ILl of a suspension containing 8x 106 viable FL-74 target cells/rnl was incubated in microtiter plates, with 25 ILl rabbit serum as a source of complement and 25 ILl feline test serum in twofold serial dilutions. After incubation at 37° C for I hour, the plates were centrifuged at 500Xg for 5 minutes. Lysis of the FL-74 cell pellet was determined visually in a manner similar to that used for scoring a complement-fixation test. The highest positive serum dilution was considered that in which lysis of the cell pellet was 50% or greater. Inoculation of cats, sample collection.-The age, number,

MATERIALS AND METHODS

Animals.-We used 67 specific-pathogen-free cats from a breeding colony of hysterectomy-derived cats in the Department of Veterinary Pathobiology, The Ohio State University. Viruses.-FeLV strains used were FeLV-R (4), subgroup A (11), and FeLV-KT (3), subgroups A, B, and C (11). The FeLV-R inoculum was a 20% (wt/vol) homogenate of a thymic LSA, and represented the fourth in vivo passage of the original FeLV-R isolate. The inoculum contained 105 FFU Fel.V/ml as assayed in the 81C feline cell culture originated by Fischinger et a!. (12).

FeLV-KT was harvested from the medium of the VOL. 57, No.2, AUGUST 1976

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ABBREVIATIONS USED: FeLV=feline leukemia virus [strain Rickard (R), strain Kawakami-Theilen (KT)]; gsa=group-specific antigen; FOCMA=feline oncornavirus-associated cell membrane antigen; VN=vinis neutralizing; LSA=lymphosarcoma; FFU=focus-forming units; IMI=indirect membrane immunofluorescence; MCT=microcytotoxicity test; PI=post inoculation; FeSV=feline sarcoma virus. Received November 4, 1975; accepted February 13, 1976. Supported by Public Health Service grants CAI6599 and CA08748 (to W.D.H.) from the National Cancer Institute, contract NOI CP53571 from the Special Virus-Cancer Program of the National Cancer Institute, and by grant IM-27A from the American Cancer Society. 3 Department of Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210. 4 Laboratory of Veterinary Oncology, Memorial Sloan-Kettering Cancer Center, New York, N.Y. 10021. !

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ABSTRACT-Sixty-seven specific-pathogen-free cats of various ages (newborn, 2 wk, 1 mO,2 mo, 4 mo, and 1 yr) were inoculated ip with either the Rickard (R) or the Kawakami-Theilen (KT) strain of feline leukemia virus (FeLV). Susceptibility to FeLV was judged by induction of a) FeLV group-specific antigens (gsa) in leukocytes, b) FeLV-related disease, c) antibody to feline oncornavirus-associated cell membrane antigen (FOCMA), and d) virusneutralizing (VN) antibody. Susceptibility to FeLV decreased with age. Persistent viremia and FeLV-related disease developed in 100% of cats inoculated as newborns, in 85% of cats inoculated at 2 weeks to 2 months of age, and in 15% of cats inoculated at 4 months or 1 year of age. Cats susceptible to FeLV leukemogenesis became persistently FeLV gsa-positive (viremic) at 4 weeks post inoculation and thereafter and produced little or no FOCMA or VN antibody. Cats that resisted leukemogenesis by FeLV all developed persistent FOCMA and VN titers and never became FeLV gsa-positive. The disease in inoculated cats was influenced by virus strain; FeLV-R induced predominantly thymic lymphosarcoma, whereas FeLV-KT caused fatal non regenerative anemia without concurrent neoplasia.-J Natl Cancer Inst 57: 365-369,

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RESULTS Newborn Cats Inoculated With FeLV-R

FeLV gsa was detected in the leukocytes of all 24 inoculated cats that survived to at least 4 weeks of age (table I). Viremia appeared from 2 to 6 weeks PI (mean, 3.97 wk) and persisted until all cats died. Of the 24 cats, 20 (83%) developed thymic LSA; the remaining 4 (17%) died of bacterial infection or thymic atrophy and wasting disease (19). The period from inoculation until definitive clinical evidence of LSA was present was 13-34 weeks (mean, 23.5 wk). In 17 of 24 cats (71%) inoculated as newborns, no evidence of IMI FOCMA antibody was detected at the lowest serum dilution tested (l:4) at any point after inoculation (tables 1,2). In 7 cats (29%), IMI FOCMA TABLE

Specification

Fe LV gsa FOCMA antibody VN antibody FeLV-related disease

antibody titers (I0g2 of 2-4) were detected at some time during infection. Titers appeared between 5 and 20 weeks PI (mean, 13 wk) and persisted from 2 to 16 weeks (mean, 8 wk). In all instances, FOCMA antibody titers returned to less than 2 when LSA developed (table 2). The occurrence of FOCMA antibody with the MCT test paralleled data obtained with the IMI test (tables 2, 3). Titers developed in 5 of 24 cats (21%) between 2 and 20 weeks PI (mean, 12 wk) (table 2). Titers persisted for 2-10 weeks (mean, 7 wk) and were not detected in the terminal serum of any cat. VN antibody was not detected «2) at any time during FeLV infection in 24 cats (tables 1, 2). Newborn Cats Inoculated with FeLV-KT

All 4 cats inoculated with FeLV-KT died of nonregenerative anemia and erythroid hypoplasia (20) at 7-8 weeks of age (table 1). All cats became gsa-positive on week 4 or 6 PI (mean, 4.5 wk) and remained so. None of the 4 cats developed FOCMA or VN antibody. Two-Week-Old Cats Inoculated With FeLV-R

Persistent viremia occurred in 8 of 9 cats at 4 weeks PI and thereafter. Of the gsa-positive cats, 7 died of LSA between 26 and 34 weeks PI (mean, 31 wk), and 1 cat died of bacterial septicemia at 4 weeks PI. FOCMA antibody developed only in the 1 cat that remained gsanegative. One-Month-Old Cats Inoculated With FeLV-R

Viremia was persistent in 4 of 6 cats at 4 weeks PI and thereafter. Persistent FOCMA antibody titers developed in the 2 cats that remained gsa-negative (table 1). Thymic LSA occurred in all viremic cats after 16-36 weeks PI (mean, 30 wk). Two-Month-Old Cats Inoculated With FeLV-R

All 4 cats became FeLV gsa-positive and developed thymic LSA between 16 and 35 weeks PI (mean, 27 wk). Three viremic cats developed transient FOCMA antibody titers 10-13 weeks PI (table 1). Four-Month-Old Cats Inoculated With FeLV-R

Of 10 cats given FeLV-R, 1 became persistently viremic at 4 weeks PI and died of thymic LSA at 40 weeks (table I). This cat had IMI FOCMA titers (I0g2) of 2-4

1. -Response of cats of various age groups to inoculation with FeL V Number of cats affected/ total No. of cats inoculated with FeLV-KTa

Number of cats affected/total No. of cats inoculated with FeLV-Ra Newborn

2 Weeks

1 Month

2 Months

4 Months

1 Year

Newborn

4 Months

24/24 7/24" 0/24 24/24

8/9 1/9 NT" 8/9

4/6 2/6 NT" 4/6

4/4 3/4" NT" 4/4

1/10 10/10 8/10 1/10

1/6 6/6 5/6 1/6

4/4 0/4 0/4 4/4

1/4 4/4 3/4 1/4

a Cats surviving to at least 4 weeks PI. Since FeLV gsa and FOCMA conversion generally occurs at 4 weeks PI, valid determination of the response of cats cannot be made before this time. " Titers were transient, persisting 2-16 weeks. c NT = not tested.

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and inoculation status of the cats is in table 1. In addition, II mother cats that suckled FeLV-inoculated litters were tested. All FeLV inoculations were ip, and the quantity of inoculum was the same for all age groups. For FeLV-R, 1.3 ml of a preparation containing 105 FFU /rnl was used. This dose was oncogenic in previous experiments. For FeLV-KT, 1.0 ml of a preparation containing 107 FFUI ml was used. Blood was collected weekly from all cats for the first 8 weeks, biweekly from 8 weeks to 5 months, then monthly. At each sampling period, FeLV gsa in leukocytes and FOCMA antibody were determined by the IMI and MCT tests. VN antibody was determined at 4week intervals for most animals. All pre-exposure blood smears or sera from this group and all other groups of cats were negative for gsa, FOCMA, and VN antibody. The cats were housed in individual cages as follows: newborn litters-entire litter in one cage for 8 weeks, then 2 cats per cage; 2- to 8-week-old cats-2 per cage; 4month- and I-year-old cats-I per cage. All cages were in the same room. No special order of progression was used in cleaning, feeding, or handling the cats, but feeding and litter utensils were never interchanged between cats. Necropsy and histopathologic examination was done on each animal that died or became moribund. Hemograms and thoracic radiographs were performed where indicated for diagnosis.

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SUSCEPTIBILITY OF CATS TO EXPERIMENTAL FELV INFECTION

TABLE 2. -Inoculation of newborn cats with FeL V-R: Response of highly susceptible cats that develop persistent viremia and thymic LSA o Cat #742-2" PI week No.

+ + + + + + + + + + + + +

FOCMA antibodyc

FOCMA antibodyd

Feline leukemia virus infection: age-related variation in response of cats to experimental infection.

Feline Leukemia Virus Infection: Age-Related Variation in Response of Cats to Experimental Infection 1, 2 Edward A. Hoover," Richard G. Olsen," Willia...
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