Gordon H. Theilen

Monoclonal antibodies to three epitopic regions of feline leukemia virus p27 and their use in enzyme-linked immunosorbent assay of p27

Three different monoclonal antibodies were developed against the major core protein (p27) of feline leukemia virus (FeLV). Each antibody was directed against a different epitope of the species-specific portion of FeLV-p27. The 3 antibodies reacted with 5 different isolates of FeLV but not with 7 other retroviruses (MuLV (Rauscher), MuLV (AKR), MPMV, MMTV, SMRV, BAEV, RD 114). These monoclonal antibodies could readily be adapted to an enzyme-linked immunosorbent assay (ELISA) for the specific measurement of FeLV-p27. When compared in an ELISA with conventional reagents, the battery of monoclonal antibodies proved to be as sensitive as conventional polyclonal antibodies.

Modes of transmission of caprine arthritis-encephalitis virus infection

Abstract A single feeding of milk containing 2×07 TCID50 of infectious caprine arthritis-encephalitis virus (CAEV) infected 50% of newborn kids. Lesser amounts were not infectious when given as a single ingestion, indicating that 2 × 107 TCID50 was near the minimal infective oral dose. Seroconversion following intravenous innoculation of infectious CAEV was observed to occur more rapidly (4 weeks post-innoculation), at a lower level of exposure (2 × 106 TCID50), and more efficiently (100%) when compared to other routes of exposure. The occurrence of maternal-fetal transmission was supported by experiments in which kids were removed from their mothers immediately at birth and reared in isolation on virus-free milk or milk replacers. Five percent of a group of 40 kids born to naturally CAEV-infected does and handled in this manner seroconverted within 8 weeks. This indicated that at least two kids in the group were infected prior to birth or during the birth process. When seronegative kids were housed in intimate contact with CAEV-infected animals, an additional 10% became seropositive within 20 weeks, suggesting that contact transmission of CAEV also occurs later in life between infected and susceptible goats. Intramammary infusion of 2 × 107 TCID50 infectiious CAEV resulted in seroconversion in three of three lactating seronegative does within 4–8 weeks post-infusion. Thus, goats may be infected by a single oral exposure to infectious CAEV, by contact with infected individuals as occurs in normal husbandry practices, by intramammary route as might occur during routing machine milking of lactating does, and from doe to fetus either prior to or during the birth process. Control of CAEV on commercial goat dairies will be enhanced by incorporation of the findings of these studies in a preventive program.

Possible immunoenhancement of persistent viremia by feline leukemia virus envelope glycoprotein vaccines in challenge-exposure situations where whole inactivated virus vaccines were protective

Abstract Kittens immunized with purified native FeLV-gp70 or -gp85 envelope proteins developed ELISA, but not virus neutralizing, antibodies in their serum to both whole FeLV and FeLV-gp70. Kittens vaccinated with envelope proteins and infected with feline sarcoma virus (FeSV) developed smaller tumors than nonvaccinates, but a greater incidence of persistent retroviremia. Similarly, FeLV-gp70 and -gp85 vaccinated kittens were more apt to become persistently retroviremic following virulent FeLV challenge exposure than nonvaccinates. Kittens vaccinated with inactivated whole FeLV developed smaller tumors after FeSV inoculation and had a lower incidence of persistent retroviremia than nonvaccinates. The protective effect of inactivated whole FeLV vaccine against persistent retroviremia was also seen with FeLV challenge-exposed cats. Protection afforded by inactivated whole FeLV vaccine was not associated with virus neutralizing antibodies, although ELISA antibodies to both whole FeLV and FeLV-gp70 were induced by vaccination.

Amputation and Doxorubicin for Treatment of Canine and Feline Osteogenic Sarcoma

Abstract Fourteen dogs and 3 cats with naturally occurring oesteogenic sarcoma of the appendicular skeleton were treated with adjuvant chemotherapy using doxorubican HCl following treatment of the primary tumor by amputation or disarticulation. Doxorubican HCl was given intravenously every 21 days at a dosage of 30 mg/m 2 body surface area commencing 3 weeks after amputation, and continuing for 6 months or until metastatic disease was recognized radiographically. The median duration of clinical signs prior to surgical treatment for 14 dogs was 51 days (mean, 54 days ; range, 7–127 days ). The median time interval between surgery and onset of radiographically evident metastatic disease for 9 dogs was 73 days (range 50–160 days ). Median post-operative survival for 14 dogs was 104 days (range 52–368 days ); 35% were alive 6 months post-operatively, 21% were still alive at 9 months and 14% survived more than one year. Times to onset of metastatic disease and survival times were calculated from day of surgical treatment. For 3 cats duration of preoperative clinical signs were 24, 70 and 95 days ; intervals prior to metastatic disease for 2 cats were 133 and 274 days ; and survival times were 143, 180 and 414 days .

Chemoimmunotherapy for canine lymphosarcoma

Thirty‐two dogs with naturally occurring multicentric lymphosarcoma were randomly assigned to one of two treatment groups. One half of the animals received combination chemotherapy plus vitamin injections (controls) while the other half received identical chemotherapy plus injections of chemically‐modified tumor cell extract in Freunds complete adjuvant (vaccinates). Clinical staging revealed no bias between groups but showed that prognosis could be closely correlated with the severity of disease at initial presentation. Twenty dogs (62%), including 11 vaccinates and 9 controls, responded favorably to chemotherapy and were evaluated for length of first remission and total survival time. Both parameters were significantly longer in vaccinated dogs than in controls. These data suggest that immunological stimulation may be a helpful adjunct to conventional therapy in selected types of cancer when immunological principles are observed.

Production of monoclonal antibodies: The effect of hybridoma concentration on the yield of antibody-producing clones

The relationship between the number of hybrid clones in each well of the primary culture, the yield of antibody-producing wells and the short-term stability of the polyclonal cultures was tested. The results of the study demonstrate that a higher yield of antibody-secreting clones was obtained by employing lower density of hybrids and that instability of antibody secretion by polyclonal cultures is not the result of overgrowth of non-secreting clones.

TUMOR VACCINES FOR IMMUNOTHERAPY OF CANINE LYMPHOSARCOMA

Canine hemolymphatic disease is a group of neoplasms that are frequently encountered in the veterinary practice. Priester and Mantel l reported that it was the fourth most common neoplasm in the dog. Lymphosarcomas (malignant lymphoma) comprise the majority of hemolymphatic tumors, whereas granulocytic leukemia is relatively rare in the dog. Dorn et aL8 reported an incidence of lymphosarcoma of 241 100,000 dogs in a defined population in two California counties. There is increased frequency of the disease with age. Canine malignant lymphoma is usually seen as a generalized lymphatic disease. In a series of 146 dogs with lymphosarcoma from our clinic, 84% had a generalized lymph node involvement. The liver and spleen were involved, respectively, in 54 and 62% of all the animals. Involvement of just the organs of the abdomen or of the thorax or the skin accounts for 14% of our 146 cases. Most of the lymphosarcoma animals presented to our clinic are in advanced stages of disease, usually in stages I11 and IV according to the criteria of Squire et aL3 Until recently, lymphosarcoma was classified as an untreatable fatal disease of dogs. There are little data in the literature that provide information that pertains to spontaneous remissions and to survival after diagnosis in untreated dogs. Unfortunately, most dogs with lymphosarcoma are euthanized, so data from the literature and from our own experience with untreated animals are scarce. Nevertheless, it is recognized that without treatment, dogs rarely survive beyond 30 days after clinical diagnosis (TABLE 1). A report from Scotland indicated a longer duration of life, but the survival time was calculated from the time the owners first noted signs of illness rather than from time of established diagnosis by a veterinarian. However, even there, the mean survival time was less than 2 months. Of 91 dogs presented to our clinic with lymphosarcoma and not treated, owners requested that 5 1 be immediately euthanized. Seventeen were hospitalized for clinical work-up. These dogs died with a mean time of 2.5 days. Seventeen other dogs in less advanced disease were sent home without treatment. The latter group survived from 5 to 133 days, with an approximate mean of 30 days (TABLE 1 ) .

Biological and biochemical characterization of a new isolate of feline sarcoma virus: Theilen-pedersen (TP1-FeSV)

A new feline sarcoma virus designated Theilen-Pedersen (TP1-FeSV) has been isolated from a spontaneous, slowly growing fibrosarcoma of a domestic short-haired 4-year-old castrated cat. The virus codes for a gag-onc fusion protein of 83,000 molecular weight phosphorylated in vivo at serine, threonine, and tyrosine residues. Cells transformed in vitro with TP1-FeSV exhibit five- to 10-fold elevated levels of phosphotyrosine over FeLV-infected cells. The gag-onc polyprotein has associated with it a tyrosyl protein kinase activity which in vitro results in autophosphorylation of the molecule at tyrosine residues. The fusion protein cannot be labeled metabolically with [3H]glucosamine and tunicamycin has no effect on the electrophoretic mobility of the in vivo [32P]orthophosphate-labeled fusion protein. The fusion protein, in common with the gag precursor Pr65gag, can be metabolically labeled with palmitic acid.

ELECTRON MICROSCOPIC OBSERVATIONS OF BOVINE LYMPHOSARCOMA.

The histological characteristics of normal lymphoid tissues as well as lymphosarcoma from cattle are well established.102 Likewise cytologic features of normal and neoplastic lymphoid cells have been investigated by many technics for light microscopy? The present study was undertaken to determine the ultrastructure of bovine lymphosarcoma in the anticipation that certain features of the neoplastic cells would be characteristic or a t least distinctive from normal lymphoid cells. It was also anticipated that electron microscopy might give information concerning the possible etiologic factors involved in bovine lymphosarcoma and specifically information as to whether or not viruses were associated with these neoplastic cells.

EPIZOOTIOLOGY OF LYMPHOSARCOMA IN CALIFORNIA CATTLE

The question remains unresolved of whether bovine lymphosarcoma is an infectious disease, with or without a genetic predisposition, or whether some unknown carcinogen is responsible. The disease has been reportzd to be prevalent in certain areas of the world,l*z and investigators have described the spread of lymphosarcoma from one part of a country to a n ~ t h e r . ~ Cattle with lymphosarcoma have been incriminated as the carriers of an infectious agent because multiple cases of lymphosarcoma follow the introduction of these so-called “carrier” animals into herds previously free of the d i~ease .~ Herds with frequent occurrence of the disease have been referred to as “leucosis herds” or “multiple incidence herds.” In such herds, an annual mortality from this disease of 5 to 10 per cent has not been uncommon. Hematologic studies conducted on every animal in multiple-incidence herds have shown elevations above normal in average total leukocyte counts, usually due to increased numbers of lymphocytes, in contrast to findings in control studies of lymphosarcoma-free herds.6.6-7 Reports from Sweden indicate that man may spread the disease via biological products prepared from leukemic animals for the immunization of cattle against piroplasmosis.’ Furthermore, Swedish investigators have suggested the likely possibility that a concomitant chronic reticuloendothelial disease like piroplasmosis may help precipitate leukosis. Reports from Germany indicate the strong possibility of experimental transmission of the disease via gross neoplastic and whole-blood tissues used as the source of inoculum, though such transmission may take years to manifest itself? Some investigators have reported diseased animals to have a strong familial relation~hip,~ whereas others have not emphasized this relation~hip.~ It is probable that the disease, if viral in nature, may be transmitted vertically, like avian lymphomatosis’” and murine leukemiall* or it may be transmitted by contact112 but this too must be resolved. Census, death certificates, and cancer registry data have made the epidemiological study of leukemias possible in mar1.1~ Similar studies of the animal leukemias are difficult until more complete descriptive data are established upon which associations of cause and effect might be made,’* and until the