Bovine leukemia virus infection in a juvenile alpaca with multicentric lymphoma

Abstract

Résumé

Case description

A 13-month-old intact male Huacaya alpaca (Vicugna pacos) was presented for evaluation of 2 mandibular masses and weight loss of 2 weeks duration. Prior to presentation, the alpaca had been treated for a suspected tooth root abscess with enrofloxacin [3 mg/kg body weight (BW), subcutaneously] and ketoprofen (1.1 mg/kg BW, intramuscularly). The animal originated from a farm in Virginia, USA with 250 alpacas and was kept in a field with 2 other alpacas.

The alpaca was underweight (body condition score 2/5, body weight 49.5 kg, average age-matched alpaca weight 63 kg). It had a marginally subnormal rectal temperature of 37.2°C, normal heart rate of 64 beats/min, and an elevated respiratory rate of 36 breaths/min with no increased respiratory effort. The mandibular masses consisted of a firm, ovoid, subcutaneous mass (2 cm × 1 cm) on the caudo-lateral aspect of the right mandible and a raised, ulcerated, gingival mass (2 cm × 1.5 cm) closely associated with, and causing displacement of the left incisors. The right mandibular lymph node was also enlarged (5 cm × 5 cm). The alpaca urinated a full stream of red urine followed by small blood clots. Abdominal palpation revealed enlarged kidneys.

Diagnostic tests included a complete blood (cell) count (CBC), serum biochemistry panel, ultrasound and radiographic examination of the mandible, and biopsies of the mandibular masses for histopathological examination.

The CBC and serum chemistry demonstrated marked azotemia [creatinine 158.4 μmol/L, reference interval (RI): 114.9–256.4 μmol/L], urea nitrogen [72.5 μmol/L (RI: 1.8–10.7 μmol/L)], mild hypoproteinemia [total protein 50 g/L (RI: 51–78 g/L)], and hypoalbuminemia [25 g/L (RI: 35–44 g/L)], with mild leukocytosis and anemia. Urinalysis confirmed hematuria, with intact red blood cells present on sediment evaluation, a specific gravity of 1.017, marked proteinuria, trace glucosuria and 3 to 10 white cells per high power field.

Ultrasound examination of the mandibular masses revealed well-defined areas with homogeneous hyper-echogenicity. Radiographically, the clinically apparent displacement of the left incisors was associated with normal bony structures. Abdominal ultrasonography and radiography confirmed enlarged kidneys, with multiple hyper-echoic nodular lesions present diffusely throughout the parenchyma, and enlarged mesenteric lymph nodes. The results of thoracic ultrasonography and radiology were unremarkable.

Pending the results of cytology and histopathology, the alpaca was treated with ceftiofur sodium (Naxcel; Pharmacia and Upjohn Company LLC, New York, New York, USA), 2.2 mg/kg BW, IV, q12h, omeprazole (Omeprazole for IV administration, Premier Pharmacy, Weeki Wachee, Florida), 1 mg/kg BW, IV, q24h and balanced isotonic intravenous fluid therapy (Lactated Ringer’s solution; Baxter Healthcare USP, Deerfield, Illinois, USA), 4 mL/kg BW per hour.

On day 2, the alpaca was inappetant and lethargic. Repeated laboratory evaluation demonstrated severe azotemia and persistent hematuria. By this time, cytology and histopathology of the mandibular masses had demonstrated lesions consistent with small cell lymphoma. The alpaca was humanely euthanized because of the poor prognosis associated with lymphoma in NWC; the median survival time following recognition of the disease has been reported as 1 month (range 1 wk to 3 mo) (1), with a shorter clinical course in young versus old animals (5).

Necropsy revealed variably sized, mostly homogeneously pale, masses at several sites: the gingiva of the left rostral mandible (2 cm × 1 cm); the right caudo-lateral aspect of the mandible (2 cm × 1.5 cm); on the surface of the liver; within enlarged mesenteric lymph nodes; multiple nodules scattered throughout both kidneys; surrounding the left kidney and adherent to the spleen; within the wall of the caudal vena cava; and attached to the omentum. Both kidneys were enlarged; the left was 14 × 25 × 19 cm with a weight of 2 kg, and the right was 18 × 11 × 12 cm with a weight of 1 kg.

Histopathology of all masses showed sheets of monomorphic round cells with distinct cell borders, scant basophilic cytoplasm, and large round stippled nuclei. The neoplastic cells had moderate anisokaryosis and 2 to 5 mitotic figures per 400× field. There was scattered single cell necrosis throughout the masses. Neoplastic cells infiltrated the pancreas, kidney, liver, omentum, and caudal vena cava. The diagnosis was disseminated small cell lymphoma.

Immunohistochemistry of the mandibular and liver masses was performed using antibodies against B (CD79α)- and T (CD3)-cell markers. Both CD79α and CD3 antibodies stained 5% of the round cells in the mandibular samples with focal areas of staining amounting to about 10% of the fields. The lymphoid cells from the liver had positive staining for CD79α in 10% to 15% of the cells; about 1% of the cells were CD3 positive. The conclusion was that the masses represented lymphoma of undetermined primary cell type, as most cells were not labelled with the markers used.

Serum collected antemortem and submitted to the Virginia Diagnostic Laboratory (VDL) was tested for antibody to BLV using an enzyme-linked immunosorbent assay (ELISA), (Veterinary Medical Research and Development, Pullman, Washington, USA) and agar gel immunodiffusion test (AGIDT), (Veterinary Diagnostic Technology Inc, Colorado, USA). Both assays showed positive results.

Subsequently, neoplastic cells from the alpaca were tested for BLV using IS-PCR hydridization, a modification of an IS-PCR method previously used to successfully detect BLV in bovine tissues (modifications available from authors by request) (6). Specificity of the PCR assay for BLV was determined by previously showing lack of cross-reactivity with other retroviruses (Table 1). The IS-PCR was performed on deparaffinized formalin-fixed tissue sections of omentum, liver, kidney, the right mandibular mass, and pancreas. Primer sequences and PCR conditions were identical to those described previously (6). Positive and negative controls run simultaneously and under identical conditions as the experimental samples gave the expected positive and negative reactions, respectively. Positive controls included a smear of BLV-positive fetal lamb kidney (FLK) cells and a section of known BLV-positive mammary gland tissue from a cow with lymphoma. Negative controls included a smear of FLK and an adjacent serial section of each alpaca tissue reacted with the PCR mixture minus the primers. Specimens of liver and the right mandibular mass contained areas positive for BLV proviral DNA in normal tissue adjacent to the neoplastic lymphoid area. The omentum showed a weakly positive reaction within normal tissue. Bovine leukemia virus was not detected in the pancreas or kidney.

Table 1

Lack of cross-reactivity of primers from the bovine leukemia virus tax region with other retroviruses in standard solution PCR assays

Discussion

Lymphoma is reported to be one of the most common tumors in NWC, although, without confirmatory immunohistochemistry, 25% of cases may have been misdiagnosed as cases of primitive malignant round cell tumors (1,2,7). There is no age or gender predisposition; the age of affected animals in 1 retrospective study ranged from 0 (fetus) to 23 y (7). In the case reported here, the presenting clinical signs of weight loss and palpable masses are consistent with the most common presenting signs in another report on a series of cases (1). The additional observation of hematuria is a clinical feature that has not previously been reported in cases of lymphoma in NWC. Clinical pathology was consistent with other reports, but not necessarily a specific indicator of the tissue type affected (1,5,8). Ultrasonography and radiology were useful imaging modalities that indicated involvement of abdominal tissues, in addition to the mandibular masses that were detected on clinical examination. Previously, radiography has been used in NWC to demonstrate pleural effusion and lung lesions associated with lymphoma (9,10), but ultrasonography has indicated more accurately the location of potentially neoplastic tissues (5). Recent developments in diagnostic imaging techniques include computed tomography, which facilitates an even more precise assessment of tumor location (8). The definitive diagnosis was determined by histopathologic examination of neoplastic masses and the distribution of lesions was consistent with multicentric lymphoma, similar to most reports of lymphoma in NWC (1,4,7–14).

Immunohistochemical analysis has been performed on tissues from NWC with lymphoma to further determine the lymphocyte sub-type; T-cell, B-cell, and mixed tumors have been described mostly by using anti-CD3 for T-cells and anti-CD79α for B-cells (4,7,12–14). Unfortunately, in the alpaca described here, the results of immunohistochemistry did not provide a definitive identification by using these cell markers alone.

Because of the association between BLV and lymphoma in cattle (3), it was decided to test the alpaca for this virus. Although previous cases of lymphoma in NWC had been screened for retroviruses and none were detected (1,4), the ultra-structural methods used were potentially less sensitive than other tests routinely used in cattle. Methods of detecting BLV infection in cattle include PCR testing and serology (3). Serology was initially used to screen an antemortem blood sample taken from the alpaca, because it offers a rapid and inexpensive preliminary diagnosis, albeit tentative because the methods used are not validated in NWC. This is the first report of antibodies to BLV in NWC; previous surveys did not detect antibodies using the AGIDT in NWC from Argentina and Peru (15,16).

Polymerase chain reaction was selected to confirm the positive serological results. In particular, IS-PCR was selected because the reaction is performed directly on fixed tissue sections, the signal can be localized to a specific cell type and the risk of molecular contamination is eliminated. The primers were chosen to minimize cross-reactivity with other retroviruses yet maximize detection of any BLV variant. Bovine leukemia virus belongs to the retrovirus genus Deltaretrovirus along with human and simian T-cell leukemia viruses (17). Deltaretroviruses contain genomic regions that code for the major structural and enzymatic proteins common to all Retroviridae: gag, pol, and env (18). In addition, they contain a unique region tax, that codes for a viral regulatory protein and is also potentially oncogenic to the host cell. tax is not shared by any other genus of retro-viruses, including endogenous retroviruses (18,19). The use of tax primers in this study eliminated the possibility of cross-reactivity with other retroviruses outside of the Deltaretrovirus genus and this was confirmed by demonstrating non-reactivity in a panel of 8 representatives of other retroviral genera. In addition, the primers did not amplify proviral DNA of other Deltaretroviruses, confirming the specificity of the primers for BLV (Table 1). The presence of BLV proviral DNA in some of the alpaca tissues tested provides the first molecular evidence of infection with BLV in NWC. Although evidence of retroviruses was previously reported in an alpaca by transmission electron microscopy of tissues and reverse transcriptase assay, the virus was not fully characterized (20).

The source of viral infection for this alpaca is unclear. It did not have recent direct contact with other domestic ruminants; however, previous contact could not be ruled out. No other animals on the farm have been serologically tested, but this would be a useful step to determine the prevalence of BLV infection. A causal role for BLV in the pathogenesis of lymphoma could not be established as the virus was not detected within neoplastic cells. Further studies on BLV-positive lymphomatous NWC compared to normal controls would be necessary to establish a causal association.

Acknowledgment

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