Bovine Lymphoma

John A. Angelos

Bovine lymphoma (lymphosarcoma) is characterized in terms of the frequency of occurrence (sporadic or endemic/enzootic), age at onset, organ involvement, and associated etiologic agent.

Sporadic Lymphoma

Calf or Juvenile Form

The prevalence rate of juvenile lymphoma is unknown, but this form of lymphoma is rare. The cause of juvenile lymphoma is unknown and does not appear to be associated with BLV infection. The age at onset ranges from 3 to 6 months but can be seen in calves as young as 1 month or in cattle as old as 3 years. There have been reports of fetal involvement with the calf form.³ Calves generally present with a history of slight to moderate depression, weight loss, weakness (mainly in older calves and despite good appetite), or lymphadenopathy (mainly in younger calves). The onset of signs can be sudden (within a week).

Physical examination may reveal generalized bilateral enlargement of lymph nodes (deep cervical, parotid, subiliac [prefemoral], and mandibular) and occasionally hemal nodes. Rarely, node enlargement is restricted to a regional anatomic site and not generalized.

Hematologic features include a microcytic hypochromic anemia, a low hemoglobin (This heifer was serologically positive for BLV antibody, but the possibility of maternally derived antibody could not be ruled out. In another case report, an 18-month-old Friesian heifer with cutaneous lymphoid tumors also had extensive lymphoid nodules covering the external and luminal surfaces of the trachea.⁸ An 8- to 9-year-old beef cow with weight loss was reported with pleural and pericardial invasion with a lymphoid tumor of T-cell lineage; the animal tested negative for BLV by PCR.⁹ Four of 10 cattle 3 years of age or younger diagnosed with sporadic lymphoma had BLV provirus in tumor DNA as detected by PCR, and a possible role for BLV in some cases of sporadic lymphoma of cattle has been suggested.¹⁰ A 13-month-old alpaca affected with mandibular masses and weight loss of 2 weeks duration was diagnosed with disseminated small cell lymphoma involving the pancreas, kidney, liver, omentum, and the caudal vena cava; BLV proviral DNA was identified in normal liver and mandibular mass tissue adjacent to neoplastic lymphoid tissue, but it was not found in pancreatic or renal tissue.¹¹

Cutaneous Form

Cutaneous lymphoma is not as age specific as the other forms of sporadic lymphoma and may affect cattle between 1 and 3 years of age. The history may reveal an initial period of 1 to 3 months, during which cutaneous swellings are observed around the anus, vulva, escutcheon, shoulders, or flank. These signs may regress and subsequently recur.

At necropsy, a variety of organs may be involved, includ­ing heart, brain, skin, spinal cord, liver, lung, kidney, and abomasum. The massive lymphoid infiltration of the skin resembles the clinical manifestations of mycosis fungoides of humans. In a report of a 12-month-old red Holstein heifer with cutaneous T-cell lymphoma, serum activity of lactate dehydrogenase (LDH) was moderately increased despite normal to minimally increased activities of liver- and muscle-specific enzymes.¹²

Hemonode Enlargement (Hemal Lymph Node)

A lymphoproliferative condition associated with hemonode enlargement and some generalized lymphadenopathy has been described for cattle infected with a lentivirus¹³ that is now called bovine immunodeficiency virus (BIV).¹⁴ BIV belongs in the genus Lentivirus, subfamily Orthoretrovirinae, and family Retroviridae. BIV has molecular similarity to human immuno­deficiency virus and is associated with persistent lymphocytosis, lymphadenopathy, central nervous system (CNS) lesions, progressive weakness, and emaciation. Infected calves may develop enlarged superficial nodes (hemonodes), mainly in the cervical region anterior and dorsal to the prescapular lymph node, over the spine of the scapula, in the paralumbar fossa, and dorsal to the subiliac (prefemoral) lymph node. Lymphocytosis is possibly related to an increase in B lymphocytes. In utero transmission of BIV has also been documented.¹⁵

Adult Lymphoma (Bovine Leukemia Virus)

The adult, or enzootic, form of lymphoma is the most common neoplastic disease of cattle and is most commonly caused by a BLV infection.

Epidemiology

Horizontal transmission of BLV has been demonstrated by the transfer of blood from infected cattle to susceptible cattle. Iatrogenic (reuse of common hypodermic needles between animals, reuse of syringes, dehorners, ear notchers or ear tattoo pliers, castration instruments, or even nose tongs) are considered to be risk factors in transmission; however, some studies were unable to demonstrate transmission after use of a common needle.^24-26 A role for Tabanids in BLV transmission has also been demonstrated.²⁷ A reduction in seroprevalence to BLV was documented following management changes that presumably reduced exposure to BLV-contaminated blood; these changes included switching from gouge to electrical burning dehorners, single use of needles and obstetrical sleeves, disinfection of tattoo equipment, replacement of whole milk feeding with high-quality milk replacer, and heat treatment of colostrum before feeding.^28,29 Infection after use of a Tb needle dipped in blood of a BLV-infected animal could be prevented by wiping the needle with cotton before injection,³⁰ and transmission is considered less likely if instruments are disinfected. Rectal palpation has been postulated as a means of transmitting BLV, and experimental studies found that under extreme conditions of blood contamination and mucosal irritation, BLV can be transmitted rectally.^31-35 Under conditions typical of routine palpation during pregnancy examination, however, BLV may not necessarily be transmitted to any measurable degree.^33,35,36 Risk of spread from respiratory secretions is generally considered low, yet transmission from inhalation of BLV shed in nasal secre­tions is considered possible.^37-40 Herd size might be positively correlated with a high rate of lymphoma, which may reflect higher rates of BLV infection in large herds or a preponder­ance of susceptible pedigrees in some of the herds studied.^41,42 Close contact and heavy stocking/comingling of infected and susceptible animals also increases the incidence of horizontal transmission.^43,44 The presence of persistent lymphocytosis (PL) cattle greatly increases the chances of horizontal transmission.¹⁹ PL is defined as an absolute lymphocyte count at least three standard deviations above the normal mean count that persists for at least 3 consecutive months.

Vertical transmission from infected dams to their fetus across the placenta can occur; however, studies have shown this occurs in only up to about 10% of pregnancies.^15,45 While transmission in colostrum and milk has been shown,^15,46 colostral antibodies also impart a protective effect against BLV infection in calves from BLV-infected dams.⁴⁷ Colostrum replacers can contain BLV antibodies that temporarily impart BLV seropositivity to calves; presence of BLV proviral DNA has also been detected in colostrum replacers, but it is unlikely that infectious BLV 4849

could survive the manufacturing process.,

Susceptibility to BLV infection is associated with BoLA type,^50,51 which may explain why higher rates of lymphoma are found in certain families of cattle. The progression of disease to persistent lymphocytosis in some BLV-infected cows has been linked to a shift away from cell mediated immunity toward a humoral response.^52,53 Polymorphisms at the MHC class II BoLA-DRB3.2 gene have been associated with differing circulating proviral loads in blood of Argentinian Holstein cattle and can be used to define BLV-resistant versus susceptible genotypes in these cattle.⁵⁴

Antigenic and molecular evidence for the presence of BLV in mammary glandular epithelium has been demonstrated.⁵⁵ The effect of BLV persistent mammary infection on the mammary gland is not completely understood. Although one study identified increased average milk production in BLV- infected versus uninfected cattle,⁵⁶ others report negative associations between milk production and BLV status.^23,57

Diagnosis

Cattle presented with the adult or enzootic form of lymphoma usually are older than 4 years of age but may be as young as

2 years. Cattle are often presented with a history of loss in condition, an abrupt drop in milk production (over a period of a few days), enlarged peripheral nodes, exophthalmos, or partial to complete anorexia, particularly with regard to grain or concentrates.

Common sites of lymphoid tumor predilection in adult lymphoma include heart (usually right atrium), abomasum, uterus, kidney, and spinal cord (epidural space); other common sites include the retrobulbar space and rumen/reticulum.⁵⁸ Physical examination often reveals an organ system failure resulting from tumor involvement. Thoracic auscultation may reveal cardiac dysrhythmia, tachycardia, tachypnea, and hyperpnea. Epicardial lymphosarcoma has been described in

3 dairy cows 3 to 19 months following diagnosis and treatment for idiopathic hemorrhagic pericardial effusion; at the time of initial presentation with pericardial effusion, none of 3 affected cows had cytologic evidence of neoplasia in pericardial fluid.⁵⁹ Dependent, pitting edema is common when cervical or supramammary lymph nodes are involved. Involvement of the intestinal lymphatics is often associated with generalized dependent edema anterior to the udder. Peripheral lymph nodes typically found to be enlarged are the prescapular, subiliac, and supramammary nodes. Feces may be scant, pasty, or watery and probably reflect the presence of lesions within the GI tract. Melena may be present in animals with ulceration of the abomasum and lymphoid cell infiltration in the abomasal wall. The main ultrasound finding in a case of abomasal lympho­sarcoma was enlargement of lymph nodes caudal to the reticulum, as well as severe enlargement of the abomasum with thickening of the abomasal wall.⁶⁰ Rectal palpation can be useful in cases lacking peripheral node enlargement or exophthalmos. Tumor masses palpated in the abdomen typically are multiple and range in size from only slightly enlarged lymph nodes to massive lesions half a meter in diameter. The internal iliac nodes are involved in most cattle with abdominal tumors. Tumors tend to be firm but not hard and may feel slightly lobulated. Differentiation by palpation of lymphoma tumors from other masses is highly subjective. Exploratory laparotomy may be indicated as an additional diagnostic tool in difficult cases, particularly in valuable cattle.

The hemogram of cattle with lymphoma often is generally unremarkable, except in the case of PL animals in which circulating lymphocytes remain elevated for a prolonged period. Approximately 30% of cattle with BLV infection develop persistent lifelong lymphocytosis, composed mostly of B cells.^61,62 Lymphocytotic cattle have higher BLV proviral loads in peripheral blood lymphocytes than nonlymphocytotic cattle, although some nonlymphocytotic cattle may have high BLV proviral loads and could represent obstacles to successful eradication efforts.⁶³ Because neutrophil concentrations can be similar between BLV-antibody positive versus BLV-antibody negative cattle while lymphocyte concentrations are increased in BLV-antibody positive cattle, recommendations have been made to avoid the use of calculated neutrophil-to-lymphocyte ratios in favor of direct comparison of patient absolute leukocyte concentrations to updated lymphocyte reference intervals from healthy BLV-negative cattle.⁶⁴ Anemia may be present in cattle with GI hemorrhage as a result of abomasal wall lymphoma and ulceration with bleeding; such cases would also be likely to exhibit melena. Fibrinogen levels have been inconsistent in lymphoma, and therefore its measurement may be helpful only in differentiating an abscess.

Although helpful in some cases, cytology of aspirates of tumors or tumorous nodes may not always be a reliable diagnostic tool. Discrimination cannot be made between a normal node responding to an infectious agent and a node involved with neoplastic lymphocytes; in both situations, cells resembling young, poorly differentiated lymphocytes may be present. Examination of nontumorous active lymph nodes may reveal an elevated proportion of large, young lymphocytes that could resemble those of lymphoma. In a retrospective study, the sensitivity of antemortem diagnostic testing found peripheral lymph node biopsy to be 100% sensitive while fine-needle aspirate was only 41% sensitive.⁵⁸ When compared with core­needle biopsy of peripheral lymph nodes, fine-needle aspiration was more specific and more predictive for a positive test.⁶⁵ Cytology and culture of aspirates can be helpful in differentiat­ing a tumor from an abscess. Failed attempts at collection of cerebrospinal (lumbosacral) fluid in cattle with recumbency or rear limb weakness may be associated with the presence of lymphoid tumors in the vertebral canal; cytologic examination of cells from the collection needle in such cases may reveal abnormal lymphoid cells.

At necropsy lymphomas are found enclosed in a capsular-like tissue that, when sectioned with a knife, results in an eversion of tumor stroma. Cut tumor tissue is cream colored and friable, with little binding structure. Centers of tumors may appear necrotic and mushy, whereas peripheral regions are firmer and pink to white. Histopathology provides the only definitive diagnosis. Tissue should be biopsied by surgical removal of as much of the node (or mass) as possible.

Standard serologic tests to diagnose BLV infection include agar gel immunodiffusion (AGID) and enzyme-linked immu­nosorbent assay (ELISA). The ELISA is more sensitive than the AGID.⁶⁶ Serologic tests identify antibodies to a BLV 51-kilodalton (kD) envelope glycoprotein (gp51) or 24 kD core protein (p24). Low antibody response to p24 was observed in cattle with low BLV proviral loads.⁶³ ELISA test kits that detect antibodies in milk samples are available commercially. The use of a milk ELISA to measure anti-BLV antibodies has been used successfully to determine lactation-specific BLV prevalence in dairy herds.⁶⁷

Diagnosis is also possible by use of PCR to detect BLV nucleic acid,^68-71 as well as by a combined PCR-ELISA that is more sensitive than staining of PCR amplicons with ethidium bromide.⁷² In a majority of calves that were experimentally infected with BLV, calves became PCR positive sooner than they seroconverted (as determined by a commercial BLV antibody test).⁷³ However, the measurement of antibodies to p24 antigen and whole-BLV antibodies by ELISA has been suggested as a cost-effective tool in BLV control programs as compared with real-time PCR methods.⁷⁴ ELISA-positive cattle that were not detected by PCR testing have been reported; this is most likely related to a low number of infected circulating lymphocytes at the time of sample collection.⁷⁵ The PCR by itself is considered unreliable for routine detection of BLV in herds with a high prevalence of disease,⁷⁶ and the ability of PCR to detect a BLV-infected animal is improved by means of a nested PCR, which was reported to be effective in diagnos­ing infection when seroconversion had not yet occurred.⁷⁷ A loop-mediated isothermal amplification (LAMP) assay has also been developed to diagnose BLV infection in cattle; this test has sensitivity at least as good as the conventional single PCR and has the advantage of not requiring specialized thermal cycling equipment.⁷⁸ Novel quantitative real-time PCR methods that use Coordination of Common Motifs (CoCoMo) primers to measure proviral loads of known and novel BLV variants were found to be more sensitive than some of the more tra­ditional real-time PCR methods and antibody-based methods to detect BLV^79,80 A luminescence syncytium induction assay (LuSIA) has also been shown to accurately detect and quan­titatively measuring bovine leukemia virus infection.⁸¹

The presence of antibodies to gp51 is generally considered to be a prerequisite to a diagnosis of lymphoma, except for cows during the periparturient period when circulating antibod­ies may fall below the level detectable by a serologic test.⁸² The mere presence of BLV antibodies does not necessarily mean that an animal has lymphoma, which is found in only about 1.7% of BLV-infected cattle.⁸³ However, serology can be helpful in predicting the chance of lymphoma in cattle seropositive to the gp51 antigen. Sera from cattle with lym­phoma generally have high titers to the gp51 antigen (a score of 3 or 4 on AGID), compared with those of infected cattle without lymphoma (score of 1 or 2 on AGID). In addition, cattle that do not have lymphoma tend not to have antibodies to the p24 antigen of BLV, as detected by AGID. The percentage of cattle with histologically confirmed lymphoma but in which lymphoma was not diagnosed by a gp 51-positive and p24-negative test result (false-negative rate of the diagnostic test) has been found to be only 0.21%, using the AGID test.⁴² Unfortunately, most diagnostic laboratories do not have the capability to perform the test using the p24 antigen.

Control

Although there are not curative treatments for lymphoma in cattle, supportive therapy may be indicated to reduce discomfort and prolong life long enough to remove valuable ova/embryos or calves or to harvest semen. Presently no vaccines offer effective protection from BLV infection and control. As such, eradica­tion of BLV requires reduction of blood transmission through iatrogenic means and through physical contact among cattle.⁸⁴ Basic disinfecting practices of instruments and equipment used on cattle such as dehorners, ear notchers, tattoo pliers, nose tongs, and others should help reduce the transmission of BLV between animals. In one study, disinfection of instruments with chlorhexidine and sodium hypochlorite was implemented as part of a set of corrective actions that were successful at reducing new cases of BLV.⁴⁴ Avoiding the reuse of hypodermic needles and syringes between animals is also recommended to reduce transmission through blood contamination. Transmission between infected and susceptible cattle can also be reduced by segregating infected and noninfected cattle,^44,85,86 and segrega­tion of infected animals reduces seroconversion of uninfected neighbor cattle.⁸⁷ Separation of cattle by 10 feet is preferred, but a single fence may reduce sufficiently the degree of contact necessary for transmission. If potential for rectal transmission is a concern, palpation sleeves may be rinsed or replaced between cows. Although insect transmission of BLV is theoretically pos­sible⁸⁸ and has been demonstrated experimentally by interrupted feeding of tabanids from cattle with persistent lymphocytosis to uninfected cattle,²⁷ studies of natural infection did not link flies with new natural infections.^89,90 Vector control to curtail stable fly feeding was, however, reported to be effective at reducing BLV transmission.⁹¹ Although data have not shown the use of common needles to be an important means of BLV transmission, it would be prudent to use individual sterile needles for treatment, testing, or vaccination. As animals with PL are potential sources for increasing horizontal and vertical transmission of BLV, routine testing of the herd and culling of PI animals should reduce infection rates.

Reduction of vertical transmission via milk or colostrum should address testing and culling PL- and BLV-positive cows and reducing BLV exposure of calves via infected colostrum or milk. In a sheep model of BLV infection, freezing and thawing of leukocytes from colostrum of a BLV-infected dairy cow appeared to be effective at inactivating the infectivity of BLV-infected leukocytes.⁹² As calves born to BLV-positive cows are exposed to BLV during parturition, and because antibodies to BLV in colostrum from BLV-positive cattle appear to be protective against BLV infection, neonatal BLV transmission may be reduced by feeding a calf born to a BLV-positive cow the colostrum from its dam.⁴⁷ In Japan, feeding colostrum from the dam was also found to be protective against within- farm BLV transmission.⁹³ Because BLV proviral loads in blood rise following birth of calves to dams with high proviral loads, early identification and culling of infected calves could help prevent virus transmission.⁹⁴ Heat treating colostrum (65^o C for 30 minutes) appears to be an effective means of inactivating BLV and should be considered when developing a control program.^29,95 Colostrum replacers may contain antibodies to BLV that render calves receiving such replacers to be seroposi­tive to BLV; in one study these antibodies waned by 4 months of age.⁴⁸

Transmission of BLV has not been shown in cows insemi­nated artificially using commercially prepared frozen semen.^96-100 Although infection resulting from the use of an infected bull in natural breeding may be possible,^101,102 one study found that bulls that were BLV PCR positive in peripheral blood had no evidence of BLV in semen by PCR testing.¹⁰³ Embryo transfer recipients should be tested for BLV before transfer.¹⁰⁴ Embryo transfer using noninfected recipients offers a means of producing phenotypically preferred cattle from BLV-infected cows and controlling in utero infection.^{89,96,105,106} It is possible to produce transferrable in vitro-fertilized embryos that are free of BLV provirus from oocytes that are exposed to BLV during maturation, fertilization, or after fertilization through a washing procedure.¹⁰⁷

For most herds, control and eradication of the infection would require modification of facilities, alteration of manage­ment practices, and serologic surveillance at least annually. Planning a control program should include a cost/benefit analysis to evaluate potential return on the investment. Knowledge of costs incurred as a result of BLV including costs of lost milk production are important in decision making relative to implementation of BLV control programs.^108,109 When considering the potential for BLV infection to cause immune suppression,^110,111 the various ways that BLV infection could cause economic losses in cattle populations are especially diverse. Financial benefit of a BLV control program has been docu­mented in herds in which the prevalence of BLV infection is 12.5% or higher.¹¹² The elective culling of BLV-infected cattle based on positive lymphocyte BLV antigen expression in vitro was reported to be effective in preventing transmission of BLV infection.¹¹³