T (NWC) in North America.' Consequently, veterinarians

Lymphosarcoma in 10 New World Camelids Christopher K. Cebra, Franklyn 6. Garry, Barbara E. Powers, and LaRue W. Johnson We evaluated the medical information from 10 New World Camelids with lymphosarcoma. Additionally, tumor tissue from 5 animals was examined by electron microscopy for evidence of retroviral particles. Lymphosarcoma was the most common neoplasm of New World Camelids in our hospital. Both llamas and alpacas, from 4 months to 15 years of age, were affected. Emaciation (7 of 8) and palpable masses (9 of 10) were the most common physical examination abnormalities, and a left-shifted leukogram (7 of 7) and hypoalbuminemia (6 of 71 were the most characteristic clinihere are approximately 70,000 New World Camelids T (NWC) in North America.' Consequently, veterinarians are frequently asked to provide health care for these species. Many common diseases of NWC have been well described, but neoplasms rarely have been reported.'.' Lymphosarcoma (LSA) has been described in camelids and ruminants, including the dromedary: sheep,i goat: and Ilama.9~'0 Slaughterhouse surveys suggest that multicentric LSA is the most common malignant neoplasm in ruminants.' I,'' In cattle, there are sporadic non-bovine leukemia virus (BLV)-associated forms in younger animal^,^ as well as the BLV-associated, multicentric form in adults." There may be a genetic basis for the sporadic form in cattle.7.'4 Sheep and goats may develop LSA after experimental inoculation with BLV, or with blood or tissue products from BLV-infected COWS.'^^'^ Retrovirus-like particles have been identified ultrastructurally in ovine lymphocyte cultures obtained from flocks with multiple occurrences of LSA," and in tumor tissue from both sheep and goats from flocks with enzootic intranasal adenocarcinorna.'"'' However, with the exception of experimental infection and some sporadic herd outbreaks,17 LSA is not considered to have a viral origin in small ruminants.'.*" There are 2 reports of LSA in NWC, each describing a single mature llama (ages 4 and 10 years, respectively) with the multicentric form of the disease.','" However, from these 2 case reports no inferences can be made on the spectrum of the disease in NWC. The purpose of this study is to characterize the historical, clinical, and pathological features of LSA in 10 New World Camelids representing 2 species, the alpaca, Lamapacos, and the llama, Lama glama. Features of the disease that may aid in its timely diagnosis are also described. In addition, some tumor samples were examined ultrastructurally for the presence of retroviral particles. Materials and Methods The medical records of NWC with a histopathologic diagnosis of LSA admitted to the Colorado State University Veterinary Teaching Hospital (CSU-VTH) between January Ist, 1986 and June 30th, 1993 were reviewed (animals 1 to 5). Historical, physical examination, automated CBC (Coulter S+ IV: Coulter Corp, Hialeah, FL), and serum chemistry (Hitachi 704 and 91 1 Automatic Analyzers: Boehringer Mannheim, Indianapolis, IL) data were evaluated in these animals. Similar data were evaluated in 5 additional NWC with LSA identified from tissue submission to a veterinary diagnostic laboratory (animals 6 to 10). Hematoxylin and eosin-stained sections of formalin-fixed, paraf- copathologic findings. Lymphosarcoma was diagnosed by lymph node aspirate (n = 2). biopsy (n = 21, peritoneal fluid analysis (n = 2), or necropsy (n = 4). The clinical course after recognition of the disease was usually short, with a median survival of 1 month (1 week to 3 months). Organs with neoplastic infiltrates commonly included lymph nodes (n = 8). liver (n = 8). kidneys (n = 6). and lungs (n = 5). No retroviral particles were detected ultrastructurally. J Vet Intern Med 1995;9:381-385. Copyright 0 1995 by the American College of Veterinary Internal Medicine. fin-embedded tumor tissues from 8 NWC (animals 1 to 5 and 7 to 9) were examined by light microscopy by one of the authors (BEP). Multiple fields of each tissue section were examined, and the histological appearance of the tissue was described. In 2 cases (animals 6 and lo), the original tissue was not available for reevaluation. Electron microscopic examination was performed on paraffin blocks of tumor tissue retrieved from animals 1 through 5. Tissues were deparaffinized with xylene followed by decreasing concentrations of alcohol, then refixed in 3% glutaraldehyde followed by 1% osmium tetroxide. The tissues were then routinely processed and embedded in plastic media (Polybed 8 12; Polysciences Incorp, Warrington, PA). Ultrathin sections were stained with lead citrate and uranyl acetate, and examined for the presence of viral particles with a Zeiss EM 109 electron microscope (Carl Zeiss, Inc, Thomwood, NY). Results Study Population Over the period of the study, 1,I 56 NWC were admitted to the CSU-VTH. Of these, 55% were female, 34% were intact males, and 11% were castrated males; the median age was 3 years. Malignant neoplasms were histopathologically confirmed in 12 (1 %) of these NWC; the diagnoses included LSA (n = 5, animals 1 to 5), squamous cell carcinoma (n = 4) (1 with a concurrent mammary adenocarcinoma), intraocular melanoma (n = l), mastocytoma (n = l), and uterine leiomyosarcoma (n = 1). Neoplasia was, therefore, an infrequent diagnosis in this study population, but LSA represented the most common neoplasm seen. Five addi- From the Department of Clinical Sciences (Cebra, Garry, Johnson), and the Diagnostic Laboratory (Powers), College of Veterinary Medicine and Biomedical Sciences, Colorudo State University, Fort Collins, CO. Accepted Februav 25, 1995. Supported in part by the Colorado State University Llama Research Fund. The authors thank Drs Deme Erickson, Charles Stillion, Laura Freeman, and David Orton, and the llama owners who helped provide the injitrmation presented in this article. We also thank Jane Carmen,for the ultrastructural studies. Reprint requests: Christopher K. Cebra, VMD, Department of Clinical Sciences, College of Veterinary Medicine, Colorudo State University, Fort Collins, CO 80523. Copyright 0 1995 by the American College of Veterinary Internal Medicine 0891 -(i640/95/0906-0002$3.00/0 Journal of Veterinary Internal Medicine, Vol9, No 6 (November-December), 1995: pp 381-385 381

382 CEBRA ET AL tional NWC with LSA (animals 6 to 10) were identified as described previously. Signalment Lymphosarcoma was diagnosed histopathologically in 7 llamas and 3 alpacas, including 6 females, 1 castrated male, and 3 intact males; 3 females were pregnant. The median age was 3 years, with a range of 4 months to 15 years (Table I). The gender distribution of NWC with LSA was similar to that of the general hospital population, and all age groups were represented. The record-keeping system did not distinguish between total numbers of llamas and alpacas, so the population of NWC with LSA could not be compared with the overall study population on the basis of species. History The 10 NWC with LSA originated from 6 different states. Two alpacas were born on the same farm, but were unrelated. Prolonged anorexia and depression were the most common reasons for referral to the CSU-VTH (n = 4). A 20% decrease in body weight over a 3-week period was documented in llama 5. Blindness, oral ulcers, and enlarged mandibular lymph nodes had been noted in llama 2; this llama was the only NWC referred with a diagnosis of LSA, which had been based on a fine needle aspirate of a lymph node obtained by the referring veterinarian. The animal with normal attitude and appetite (alpaca 4) was admitted for inability to rise. The duration of clinical signs prior to admission had been approximately 1 week (n = 2) to 1 month (n = 3) (Table 1). The 5 animals that were not evaluated at the CSU-VTH were examined by herd veterinarians for the presence of submandibular or cervical masses (n = 3), or anorexia and lethargy (n = 1). Llama 7 was clinically normal, but had numerous hematologic and serum biochemical abnormalities on a regularly scheduled health screening. Field Therapy Prior to establishing a definitive diagnosis of LSA, most of these animals had been treated symptomatically. The majority were placed on a course of antibiotics. Dietary changes and supplements, vitamins, and rumenatoric agents had also been used to treat anorexia and weight loss. Other treatments included anthelmintics, nonsteroidal anti-inflammatory drugs, corticosteroids, plasma transfusion, and electrolytes. Submandibular and cervical masses were usually aspirated Table 1. Findings on 10 New World Camelids With Lymphosarcoma Animal No. Species Gender Age (y) Course of Disease Presenting Complaints Physical Examination Findings 1 Llama F(p) 5 1 wk Anorexia Emaciation Melena 2 Llama F 4 1 mo Anorexia Emaciation Blindness Gingivitis 3 Llama Mlg) 15 1 mo Anorexia Emaciation 4 Alpaca F(p) 2 2 wk Recumbency Emaciation Bilateral hind limb paresis and arreflexia internal mass 5 Llama M 1.15 1 mo Anorexia Emaciation Weight loss Heart murmur 6 Alpaca M 0.8 6 wk Anorexia Emaciation 7 Llama F(p) 10 3 mo Normal Obesity 8 Alpaca F 0.5 3 mo Submandibular mass 9 Llama M 0.33 2 wk Submandibular mass Emaciation 10 Llama F 7 1 mo Cervical mass Mass TachvDnea NOTE. F(p) denotes a pregnant female: M(g) denotes a gelded male.

LYMPHOSARCOMA IN NEW WORLD CAMELIDS 383 Table 2. Selected Laboratory Values for 7 New World Camelids With Lymphosarcoma Animal No. Reference 1 2 3 4 5 6 7 Range* PCV (%) 23 25 37 28 27 32 32 29-39 Protein (g/dl) 5.1 6.7 4 5 5.6 5.5 3.8 5.5-7 WBC (X IO~/~L) 47.5 31.6 6.8 11.1 16.4 21.1 14.9 7.5-21.5 Band (x 103/pL) 16.7 1.9 2.0 1.3 2.6 1.7 0.5 0-0.4 Seg (x IO~/~L) 22.3 1.3 1.1 7.4 9.3 8.9 9.0 4.6-16 Lymph (X IO3/pL) 3.8 20.9 0.2 1.7 1.8 7.2 2.3 1.0-7.5 Glucose (mg/dl) 160 268 260 247 165 146 141 90-1 40 BUN (mg/dl) 89 124 116 23 47 20 12 13-32 Creatinine (mg/dl) nd 12.3 3.9 2.2 3.3 0.9 1.4 1.5-2.9 Calcium (mg/dl) 9.2 11.3 5.8 8.0 8.2 9.0 8.3 7.7-9.4 Albumin (g/dl) 2.8 3.2 1.5 2.7 3.5 2.5 1.7 3.5-4.4 Globulin (g/dl) 1.8 2.6 1.8 1.6 2.0 2.8 2.1 1.7-3.5 Sodium (meq/l) 149 157 134 112 148 150 146 147-1 58 Potassium (meq/l) 3.0 4.5 3.9 4.1 4.2 4.3 5.3 4.3-5.6 AST (IU/L) 111 288 218 943 139 146 182 110-250 NOTE. nd denotes that the test was not performed. Abbreviations: WBC, white blood cell count; Seg, segmented neutrophil; lymph, lymphocytes; BUN, blood urea nitrogen; AST, aspartate aminotransferase. * Reference range for healthy adult NWC used by the CSU Clinical Pathology Laboratory. or lanced. Very little Auid was obtained, and the masses continued to grow or were complicated by secondary infection. In many cases, the animals appeared to respond transiently, but in all cases the disease ultimately progressed. Physical Examination Findings All 5 animals admitted to the CSU-VTH had increased respiratory effort or increased large airway sounds on auscultation. Two of 5 animals were febrile, 3 were tachycardic, and 3 were tachypneic (Table 1). There was no auscultable gastrointestinal motility in 3 of 4 anorectic llamas. Seven animals were severely emaciated, with prominent ribs, spine, and pelvic bones. Eight had at least 1 enlarged external lymph node or mass. Affected lymph nodes included the mandibular, retropharyngeal, superficial cervical, iliac, popliteal, and supraniammary. External masses in these animals were prominent, ranging in size from 9 to 180 cm. The paretic alpaca had no external lymph node enlargement, but several abdominal masses palpable per rectum had a homogeneous appearance on ultrasonographic examination. Other physical examination findings are listed in Table 1. Clinicopathologic and Imaging Findings CBC and serum chemistry profiles were performed on 7 animals (Table 2). Anemia (n = 4) and hypoproteinemia (n = 4) were common; leukocytosis was present in 2, and leukopenia in 1 animal. Lymphocytosis with atypical cells was seen only in llama 2. All animals had a left shift. Hyperglycemia (n = 7) and azotemia (n = 4) were common findings, as were bypodbuminemia (n = @ and bypokaa/emia (n = 4). Peritoneal fluid analysis was performed on animals 1, 5, and 6. Abdominocentesis was repeated in llama 5 because the original sample was interpreted as contaminated by hepa- tocytes; the liver of this animal was grossly enlarged at necropsy. The original fluid samples in animals 1 and 5 revealed high numbers of atypical lymphocytes, but the repeated sample revealed a more normal cell distribution. The fluid drawn from alpaca 6 had no abnormalities. A fine needle aspirate of a superficial cervical lymph node from llama 3 revealed pleiomorphic lymphocytes. Diagnostic excisional or needle biopsy samples were obtained from animals 8 and 10. Lumbosacral cerebrospinal fluid obtained from the paretic alpaca (animal 4) was interpreted as normal. Treatment and Clinical Course All 4 llamas admitted to the CSU-VTH were depressed and inappetent. Llama I died shortly after admission and llama 2 was euthanized 12 hours after admission. Symptomatic, emergency therapy consisting of antibiotics, anti-inflammatories, and IV fluids was initiated in animals 4 and 5 prior to the diagnosis of LSA; these animals survived only 1 and 2 days, respectively. The rapid clinical course in these animals may have reflected the advanced state of the disease on admission to the CSU-VTH. Longer clinical courses were noted in animals 6 to 8 and 10 (Table 1). Specific antineoplastic therapy was attempted only in llama 3, using dexamethasone (1 mg/kg IV), followed by a combined chemotherapy regimen of cyclophosphamide (200 mg/mz IV), vincristine (0.5 mg/m2 IV), and prednisolone (0.5 mg/kg IV q 24 h). This llama s condition improved for 1 day, but then he deteriorated and was euthanized 4 days after presentation. Necroppsl. FZhdZh~X Necropsy results are summarized in Table 3. A complete postmortem examination was not performed on animals 6-10, but representative samples were sent to a veterinary diagnostic laboratory by the herd veterinarians. For animals 4,

384 CEBRA ET AL Case No. 1 2 3 4 5 6 7 8 9 10 Table 3. Necropsy Findings on 10 New World Camelids With Lymphosarcoma Sites of Lesions ~~~ ~~~ ~ Pericardium, great thoracic vessels, lymph nodes, spleen, omentum, mesentery, liver, third stomach compartment, lungs, pleura, renal cortices, bladder Epicardium, lymph nodes, spleen, intestines, liver, lungs, mediastinum, renal cortices Lymph nodes, liver, lung, renal cortices Epicardium, myocardium, lymph nodes, mesentery, peritoneum, liver, lumbar spinal extradural space Epicardium, endocardium, myocardium, lymph node, liver, lung, renal cortices Lymph nodes, liver Lung Lymph nodes, liver, kidney, mesovarium, pancreas Lymph nodes, liver, kidney, mesentery, lip Peritracheal mass 6, 7, and 9, the diagnosis of LSA was made at necropsy. Neoplastic tissue masses ranged in size from 9 to 2,100 cm2. Lymph nodes (n = 8), liver (n = 8), renal cortices (n = 6), lung (n = 5), and heart (n = 4) were the most common sites of neoplastic involvement. The infiltrates were diffuse to multifocal. Lung infiltrates tended to be subpleural, perivascular, and peribronchiolar. Liver infiltrates were randomly distributed and extensive. Lymph nodes were effaced by neoplastic cells, such that the cortex and medulla could not be distinguished. The infiltrates consisted of a monotonous to mildly pleiomorphic population of round cells with scant to moderate basophilic cytoplasm and medium to large round to slightly oval nuclei. Nucleoli were small to medium in size, and single or multiple. Mitoses were numerous, usually 5 to 10 per magnification field X 400. Tissues retrieved from paraffin blocks from animals 1 to 5 were examined by electron microscopy for the presence of viral particles. Ten fields were examined from each section, but no viral particles were seen. Discussion Lymphosarcoma has been described in many species of large and may be the most common malignant neoplasm in many of these species."." This report shows that, although neoplasms are uncommon in NWC, LSA was the most commonly identified malignant neoplasm in this clinic. To our knowledge, this report also documents the disease in alpacas for the first time. Although camelids 6 through 10 and the 2 previous reports','" show that LSA is found in NWC outside of our clinic population, the paucity of reports suggest that this is an infrequent diagnosis. A search of the nationwide Veterinary Medical Data Base revealed only I additional llama with LSA, medical details of which have been published previously.'" The lack of reports may represent that this disease is infrequent or underdiagnosed, and inferences about the frequency of LSA in the general NWC population cannot be made from this study. In conventional ruminant species, cutaneous, multicentric, thymic, and alimentary forms of LSA have been de- ~cribed.~," These are named based on the predilection sites for tumor growth, with reticuloendothelial organs commonly involved in the multicentric form. The multicentric form was documented in all cases reported here, except for llama 10, on which a complete necropsy was not performed. The widespread metastases and short clinical course seen in many of these NWC suggest that the disease was in an advanced stage prior to diagnosis. In spite of the experiences reported here, antemortem diagnosis of LSA should be readily achieved if this differential diagnosis is considered during the clinical workup. Although the clinical signs in affected animals are usually nonspecific, a thorough physical examination frequently reveals lymphadenopathy and weight loss, which may not be apparent because of the long hair coat. is a particularly noteworthy finding in NWC because their peripheral lymph nodes are normally small and difficult to detect by palpation. Other causes of generalized lymphadenopathy in NWC seem to be infrequent, but include infectious lymphadenitis. Submandibular swelling may also be seen with mandibular osteomyelitis, actinobacillosis, tooth root abscesses, or right heart failure. Other causes of persistent weight loss include poor nutrition, poor dentition, parasitism, juvenile llama immunodeficiency syndrome, Johne's disease, and chronic inflammatory bowel disease (eosinophilic enteritis).22 Previous reports support the diagnostic value of body fluid cytology and tissue mass aspirates or biopsies from NWC with LSA.9,1" Further clinicopathologic evaluation revealed consistent abnormalities, although many were not specific to LSA. Left shift was seen much more commonly than lyrnphocytosis on evaluation of the leukogram. Damage to mucosal surfaces, immunosuppression, tumor necrosis, or cytokine production caused by the neoplasm may have led to the increased counts of band neutrophils. Hyperglycemia and hypokalemia were likewise nonspecific abnormalities commonly seen on serum biochemical analysis, while azotemia, hypoalbuminemia, and increased asparate aminotransferase activity reflected disruption of specific tissue function. Although all age groups were represented, and the gender distribution of NWC with LSA resembled those of the general clinic population, the small number of animals presented here may have been insufficient to identify trends in these regards. Pregnancy is also unlikely to be a risk factor, although this was impossible to test because of the medical record system. There are no reports on the role of viruses in LSA in NWC. No ultrastructural evidence of retroviral cause for the LSA was seen in these NWC. There is currently no evidence of natural or artificial transmission of retroviruses to NWC.23. In summary, NWC with multicentric LSA were typically evaluated for anorexia and depression; severe emaciation and peripheral lymphadenopathy were detected during physical examination. The distribution of lesions (ie, multicentric form) was very similar to that seen in cows with either enzootic leukosisx or in calves,' goats,' or sheep' with sporadic multicentric lymphoma. In camelids less than 1 year of age, there was often a single, large submandibular mass,