Robert R. Marshak

PEDIGREE STUDIES IN BOVINE LYMPHOSARCOMA

As early as 1897, European veterinarians’ reported the occurrence of lymphosarcoma in cow families in which both a dam and a daughter were affected. Early in the 20th Century, German workers2 noticed a higher incidence of the condition in some breeds than in others and late? reported on a familial incidence of lymphosarcoma. However, as lymphosarcoma became more widely recognized, gradually less importance seemed to be placed on its hereditary aspects. In the United States, Karlson4 studied the familial aggregation of lymphosarcoma cases in cattle herds in Minnesota. He reported on a Milking Shorthorn herd of 25 animals in which five cases of lymphosarcoma had occurred within a six-month period. Two of the affected animals were half-sisters; the relationship of the other affected animals was not mentioned but there may have been relationships. He also reported on an Aberdeen-Angus herd of 100 head in which three lymphosarcoma cases had been diagnosed. Two of these cases were in full sisters and one was in a daughter of one of the sisters. Finally, he described a Jersey herd in which six cases of lymphosarcoma occurred in a single family during a ten-year period. In 1962 Larson5 reported on additional Minnesota cattle herds in which lyniphosarcoma occurred in related animals. Familial aggregations of lymphosarcoma cases were also reported by Starr and Young6 who studied a Holstein family in which a normal cow produced two affected daughters, an affected granddaughter, and an affected grandson; and a Guernsey family in which four successive female generations succumbed to lymphosarcoma. In Denmark, Bendixen’ observed that lymphosarconia (“leukosis enzootica bovis”) occurred with greater frequency in the daughters of affected cows than in the daughters of unaffected cows. Our pedigree studies were undertaken in an attempt to elucidate some of the factors involved in the multiple occurrence of lymphosarcoma in certain dairy cattle herds in Pennsylvania and surrounding states.

Studies on the development of persistent lymphocytosis and infection with the Bovine C-type Leukemia Virus (BLV) in cattle

Abstract Determination of BLV infection rates in several cattle populations demonstrated that BLV is readily detectable in cases of bovine leukosis; in multiple-case leukosis herds; in leukosis contact herds; and in limited numbers of animals in leukosis-free herds. Animals with and without persistent lymphocytosis can be infected with BLV. The results of a reciprocal foster nursing experiment with 114 dairy cattle distributed through 9 different combinations of colostrum-deprived calves and foster dams showed that persistent lymphocytosis is a function of the genetic composition of the calf and not influenced by the persistent lymphocytosis status of the foster dam. Examination of sera from this experimental population for the presence of BLV infection indicated that more than 87 % of the animals had become infected by the end of the experiment. No correlation was demonstrable between persistent lymphocytosis and BLV infection. Calves derived from herds free of persistent lymphocytosis and BLV infection developed BLV infection without the simultaneous development of persistent lymphocytosis when raised in the foster nursing environment.

OCCURRENCE OF LYMPHOCYTOSIS IN DAIRY CATTLE HERDS WITH HIGH INCIDENCE OF LYMPHOSARCOMA

European veterinarians were the first to suggest a relationship between bovine lymphosarcoma and the occurrence of lymphocytosis in the peripheral blood of clinically normal cattle in herds where cases of lymphosarcoma had appeared.’S2 p 9 v 4 They believe that lymphosarcoma is a slowly advancing, horizontally transmitted, infectious disease (“leucosis enzootica bovis”), and that the lymphocytosis represents subclinical infection. In Denmark, for purposes of herd evaluation, a “Leukosis Key” (TABLE l ) , based upon total lymphocyte counts, is used to classify clinically healthy cattle from “leucosis” herds into normal, suspect, and positive groups. This work was undertaken to study the occurrence and significance of lymphocytosis in multiple-case lymphosarcoma herdst in the eastern United States. Fur purposes of comparison, it was necessary to establish values for absolute lymphocyte counts in a statistically valid sampling of cattle of various ages from “normal”

ETIOLOGICAL STUDIES ON BOVINE LYMPHOSARCOMA

herds of the four major dairy breeds.

Evidence in support of a virus etiology for bovine leukemia

European workers believe that the incidence of lymphosarcoma, or “leukosis” t of cattle, has increased since World War I1 in the cattle populations of Denmark, Sweden, and Germany. They also believe that “leukosis” is a slowly advancing infectious disease with a long incubation period in which the initial sign is an increase in the absolute lymphocyte An infectious etiology for lymphosarcoma has also been suggested by Goetze et al. 1956’; by the reported isolation of a virus from “leukosis” cattle by Thorell, 1957,’O Montemagno et ul., 1957,11 and Papparella, 195912; by the cattle transmission experiments of Rosenberger, 196lS; and by the recent reports of McKercher, 1962,13-14 Jarrett, 1962,16 and Sorenson, 1962.I6-l7 However, proof that an infectious agent is involved in the etiology of bovine lymphosarcoma is still lacking. Our group has recently reported on attempts to characterize lymphosarcoma with regard to clinical manifestations, pathologic alterations and familial distribution of cases in high incidence herds.18 Analysis of pedigree data from cattle with lymphosarcoma in multiple-case herds indicates that the disease occurs in related animals. Such a familial aggregation of cases is suggestive of genetic susceptibility to lymphosarcoma and/or vertical transmission of an infectious agent. We have been attempting to exploit the clinical, hematological, and epidemiological data in developing criteria for the selection of cattle to be used in etiological studies of lymphosarcoma. Cattle are considered to be “normal” or “noninfected” 1 if they: 1. Show no clinical signs of lymphosarcoma. 2. Are derived from herds with no past history of lymphosarcoma. 3. Are derived from herds not showing generalized lymphocytosis on re-

Observations on a heifer with cutaneous lymphosarcoma

Virus‐like particles resembling the C‐type particles of murine leukemia were detected in tissue biopsies and more frequently in thin sections of pellets following density gradient centrifugation of milk from leukemic cows and cows in a high incidence leukemic herd. These particles had a buoyant density in potassium citrate and sucrose gradients of 1.15 to 1.16 Gm/cc. Fractions in which the particles were found contained both DNA and RNA, with RNA in higher concentration. Virus interference assays on tissue cultures derived from leukemic and leukemia‐free cattle yielded equivocal results. Bone marrow cultures were uniformly resistant to challenge with vesicular stomatitis virus. Interferon was identified in the supernatant fluid of two resistant cultures. Immunofluorescence studies with anti‐Rauscher virus globulin and a globulin from a leukemic calf suggested possible antigenic similarity when the fluorescent globulins were used to stain tissue cultures derived from leukemic cattle.

Comparative aspects of bovine leukemia.

Cutaneous lesions are exceedingly rare in cattle with lymphosarcoma in the United States. Such cases are of particular interest because of a widely held belief, on epidemiologic grounds, that the etiology and pathogenesis of “skin leucosis” differs from the more typical adult form of the disease. Except for cutaneous lesions, a 20‐month‐old grade Holstein heifer originating on a Pennsylvania farm showed the clinical configuration, pathological alterations and laboratory findings commonly observed in cattle with generalized lymphosarcoma. It is, therefore, difficult to accept the view that the etiology and pathogenesis of lymphosarcoma in this animal with skin manifestations (one of many possible expressions of the disease) was fundamentally different from other forms of lymphosarcoma.

DISCUSSION OF PART VII

The discovery of particles resembling mouse leukemia virus in cows milk from a herd with a high incidence of leukemia and in lymph node cells of leukemic cattle and the finding of similar viruslike particles in human leukemic plasma and in pasteurized market milk are disquieting developments calling for intensive investigation to determine the biologic significance of the particles. The features of bovine leukemia are reviewed, and experiments are described which suggest the presence of a “resistance factor” in leukemic milk.

Virus-Like Particles in Cow's Milk From a Herd With a High Incidence of Lymphosarcoma

The Organizing Committee’s decision to devote a full section to the subject of bovine leukemia attests to the rapidly growing interest in this relatively untouched area of oncological research. There is as yet pitifully little in the way of hard scientific fact, but at least we are beginning to approach the fringes of the problem and from several promising directions. Although no one today can point with certainty to an etiologic relationship between bovine leukemia and an infectious agent, the virus theory seems to be uppermost in the minds of our participants. Most investigators will agree that there are strong teleological grounds for such a view. The field of viral oncology is now so complex and its techniques so sophisticated that the virologist who elects to work on problems as difficult and untried as bovine leukemia is well advised constantly to sharpen his awareness of the quicksands which lie at every step. Even if we grant the premature assumption that a leukemic virus is present, the forseeable difficulties in attempting to unmask such an agent are staggering. Consider the following points: (1) there is reason to believe that the disease has a long natural incubation period, perhaps as long as five years; (2) the host must be of a certain as yet undefined genetic constitution, and we are dealing with a species in which inbreeding (in the relatively pure sense of the inbred laboratory animal) does not exist; (3) if an agent is isolated from the tissues of a leukemic cow, how can one demonstrate its relationship to leukemia, since the cow is an animal with a broad, incompletely characterized virus spectrum; and (4) consider working with such an isolate in a laboratory animal or tissue culture system with its own perhaps incompletely defined virus population. The odds against rapid progress seem formidable indeed and emphasize the need for early and continuing attempts to clarify the relationship between a presumed oncogenic isolate and the host from which it supposedly originated. Hopefully, the authors of these papers and other investigators of bovine leukemia will make stronger efforts to compare their various agents serologically amongst themselves and with other appropriate laboratories and will continually attempt to relate their isolates to the presumed animal of origin. In this regard, a detailed, systematic classification and confirmation of all bovine viruses is urgently needed as a baseline for the evaluation of isolates. Tumor development in laboratory animals inoculated with materials from leukemic cattle must be interpreted with extreme caution as the procedure employed may only have incited the experimental animal’s own oncogenic agents. Of far greater importance are more and better organized attempts at experimental intraspecies transmission of bovine leukemia, particularly to new born calves and in-utero bovine fetuses. The preliminary work on the ultrastructure of normal and leukemic bovine lymphocytes by Sorenson in the United States and by Jarrett in Scotland indi-