A retrospective analysis of renal carcinoma in the horse.

BACKGROUND: Renal carcinoma is a rare tumor of horses. HYPOTHESIS: Presenting complaints and clinical signs of this disease are vague and early diagnosis increases survival time. ANIMALS: Data were collected from the medical records of 4 horses presented to Washington State University as well as the 23 previously published case reports of horses with renal carcinoma. METHODS: Retrospective study. RESULTS: Renal carcinoma affects horses of all ages with most cases observed in geldings and Thoroughbreds. The most common presenting complaints are nonspecific and usually do not occur until late in the course of the disease. Routine laboratory results generally are unremarkable with no evidence of renal dysfunction. Urine and peritoneal fluid analyses are consistently abnormal, but the changes usually are nonspecific. Rectal palpation often allows detection of an abnormal kidney or a mass in the area of the kidney. Renal ultrasound examination is the most rewarding imaging procedure, and when combined with renal biopsy, antemortem diagnosis can be achieved. Renal carcinoma is both locally invasive and metastatic, necessitating careful staging for metastasis using thoracic radiography and abdominal ultrasound examination. If the tumor is localized to 1 kidney, nephrectomy is the treatment of choice. No chemotherapy or radiation treatment for renal carcinoma has been reported in the horse. Median survival for this series of cases was 11 days (0 days-1 year). CONCLUSIONS AND CLINICAL IMPORTANCE: Prognosis is poor to grave.

Ulcerative dermatitis, thrombocytopenia, and neutropenia in neonatal foals.

This report describes transient ulcerative dermatitis, severe thrombocytopenia, and mild neutropenia in 6 foals from 4 mares from geographically diverse regions of the United States. The foals presented at <4 days of age with oral and lingual ulcers, and crusting and erythema around the eyes, muzzle, and perineal, inguinal, axillary, trunk, and neck regions. There was a severe thrombocytopenia (0-30,000 platelets/microL), leukopenia (1900-3200 white blood cells/microL), and mild neutropenia (500-1800 neutrophils/microL). Four of the 6 foals had petechiae and ecchymotic hemorrhages and 3 had bleeding tendencies. Results of examination of a bone marrow biopsy from 1 foal were normal and results of a platelet surface immunoglobulin test in another were negative. Histopathology of the skin in all foals showed subepidermal clefting with subjacent vascular dilation, dermal hemorrhage, and superficial papillary necrosis. The foals were treated supportively with broad-spectrum antibiotics (5/6), corticosteroids (3/6), gastric ulcer prophylaxis (6/6), whole-blood transfusion (4/6), and platelet-rich plasma (1/6). The skin lesions and thrombocytopenia (>50,000 platelets/microL) improved in 2 weeks (4/6). Two foals had a decline in their platelet counts when the steroids were decreased and needed protracted treatment. All foals survived and were healthy as yearlings. Two mares that had 2 affected foals each, upon subsequent pregnancies to different stallions, had healthy foals when an alternate source of colostrum was given. The findings in the cases in this report suggest a possible relationship between colostral antibodies or some other factor in the colostrum and the thrombocytopenia and skin lesions, although further investigation is warranted to confirm or refute this hypothesis.

Effects of sodium citrate, low molecular weight heparin, and prostaglandin E1 on aggregation, fibrinogen binding, and enumeration of equine platelets.

OBJECTIVE: To investigate the effects of sodium citrate, low molecular weight heparin (LMWH), and prostaglandin E1 (PGE1) on aggregation, fibrinogen binding, and enumeration of equine platelets. SAMPLE POPULATION: Blood samples obtained from 4 Thoroughbreds. PROCEDURE: Blood was collected into syringes in the ratio of 9 parts blood:1 part anticoagulant. Anticoagulants used were sodium citrate, LMWH, sodium citrate and LMWH, or 300 nM PGE1/ml of anticoagulant. Platelet aggregation in response to ADP, collagen, and PGE1 was assessed, using optical aggregometry. Platelet activation was evaluated, using flow cytometry, to detect binding of fluorescein-conjugated anti-human fibrinogen antibody. Plasma concentration of ionized calcium was measured, using an ion-selective electrode. RESULTS: Number of platelets (mean +/- SEM) in samples containing LMWH (109.5+/-11.3 x 10(3) cells/microl) was significantly less than the number in samples containing sodium citrate (187.3+/-30.3 x 10(3) cells/microl). Increasing concentrations of sodium citrate resulted in reductions in platelet aggregation and plasma concentration of ionized calcium. Addition of PGE1 prior to addition of an agonist inhibited platelet aggregation in a concentration-dependent manner, whereas addition of PGE1 4 minutes after addition of ADP resulted in partial reversal of aggregation and fibrinogen binding. CONCLUSIONS AND CLINICAL RELEVANCE: A high concentration of sodium citrate in blood samples decreases plasma concentration of ionized calcium, resulting in reduced platelet aggregation and fibrinogen binding. Platelets tend to clump in samples collected into LMWH, precluding its use as an anticoagulant. Platelet aggregation and fibrinogen binding can be reversed by PGE1, which may result in underestimation of platelet activation.

Effects of intravenous administration of formaldehyde on platelet and coagulation variables in healthy horses.

OBJECTIVES: To assess safety and determine effects of IV administration of formaldehyde on hemostatic variables in healthy horses. ANIMALS: 7 healthy adult horses. PROCEDURE: Clinical signs and results of CBC, serum biochemical analyses, and coagulation testing including template bleeding time (TBT) and activated clotting time (ACT) were compared in horses given a dose of 0.37% formaldehyde or lactated Ringer's solution (LRS), IV, in a 2-way crossover design. In a subsequent experiment, horses received an infusion of 0.74% formaldehyde or LRS. In another experiment, horses were treated with aspirin to impair platelet responses prior to infusion of formaldehyde or LRS. RESULTS: Significant differences were not detected in any variable measured between horses when given formaldehyde or any other treatment. Infusion of higher doses of formaldehyde resulted in adverse effects including muscle fasciculations, tachycardia, tachypnea, serous ocular and nasal discharge, agitation, and restlessness. CONCLUSIONS AND CLINICAL RELEVANCE: Intravenous infusion of formaldehyde at doses that do not induce adverse reactions did not have a detectable effect on measured hemostatic variables in healthy horses.

Hepatic abscesses in three horses.

Hepatic abscesses were diagnosed in 3 adult horses. Two were < 4 years old and had evidence of concurrent immune-mediated conditions, including aseptic arthritis, immune-mediated thrombocytopenia, and immune-mediated anemia. Predisposing factors for hepatic abscess formation in these horses included prior abdominal surgery, proximal duodenitis/jejunitis, inflammatory bowel disease, and a penetrating foreign body in the large colon. Serum hepatic enzyme activities were within or slightly greater then reference limits in all 3 horses. The most pronounced and consistent abnormalities on CBC and serum biochemical analyses were hyperproteinemia, hyperglobulinemia, and a decreased albumin-to-globulin concentration ratio. Hepatic ultrasonography identified hepatic abscesses in all 3 horses. A variety of bacteria were isolated from these abscesses, including Staphylococus aureus and Bacteroides fragilis. One horse developed septic tibiotarsal arthritis, presumably as a result of intermittent bacteremia. Despite aggressive medical treatment, all horses were euthanatized because of a worsening condition and poor prognosis.

Increased interleukin-6 activity in the serum of ponies acutely infected with equine infectious anaemia virus.

Seven ponies were infected with the virulent wild-type Wyoming strain of equine infectious anaemia virus (EIAV). Infection status was monitored by serum reverse transcriptase activity, rectal temperature, and complete blood count. Preinfection serum and serum obtained during the initial febrile episode following infection were assayed for interleukin 6 (IL-6) activity. Postinfection IL-6 activity was significantly increased as compared to preinfection values. The magnitude of increase in IL-6 was positively correlated with reverse transcriptase activity (an indirect measure of viraemia) but was not correlated with rectal temperature. IL-6 production in response to EIAV infection may play a role in pathogenesis of disease, especially the hyperglobulinaemia and apparent polyclonal B cell activation in these horses.

The effect of supramaximal exercise on equine platelet function.

When blood is collected into sodium citrate in the proportion of 9 parts blood:1 part sodium citrate, the concentration of plasma sodium citrate in the sample will depend on the packed cell volume (PCV) of the blood sample. This difference in plasma sodium citrate concentration secondary to alterations in PCV significantly affects human platelet aggregation responses. Since horses attain a high PCV in response to high-intensity exercise we investigated the effect of differences in sample plasma sodium citrate concentration on equine platelet aggregability. In addition, low molecular weight heparin (LMWH) was evaluated as an alternative anticoagulant for assessment of platelet aggregability during strenuous exercise in horses. Blood samples were collected pre-exercise and at fatigue after supramaximal treadmill exercise into either 3.8% sodium citrate (9 parts blood:1 part sodium citrate) or 20 u LMWH/ml of blood. Platelet aggregation responses to 1.25 mumol/l adenosine diphosphate (ADP) were measured via optical aggregometry. For samples collected into sodium citrate, aggregability was significantly less than pre-exercise values in samples collected at fatigue and in pre-exercise samples in which sodium citrate concentrations were adjusted to equal those in fatigue samples. However, samples collected into LMWH showed significantly increased platelet aggregability in samples collected at fatigue when compared to pre-exercise samples. In conclusion, higher plasma sodium citrate concentration had a marked inhibitory effect on equine platelet aggregation responses. Low molecular weight heparin was a good alternative anticoagulant for assessment of equine platelet function and results indicate that equine platelet aggregability was enhanced in response to supramaximal exercise.

Flow cytometric method for detecting thiazole orange-positive (reticulated) platelets in thrombocytopenic horses.

OBJECTIVE: To evaluate a method for detecting thiazole orange-positive (TO+, reticulated) platelets in equine blood, using flow cytometry. ANIMALS: 16 healthy, equine infectious anemia virus (EIAV)-negative horses and ponies; 9 thrombocytopenic, EIAV-positive horses and ponies; and 2 thrombocytopenic, EIAV-negative horses. PROCEDURE: Blood from healthy and thrombocytopenic horses was collected by jugular venipuncture. Appropriate sample requirement and incubation time for the assay were evaluated, using blood anticoagulated with EDTA or sodium citrate, or platelet-rich plasma in sodium citrate. The sample of blood or platelet-rich plasma was incubated with thiazole orange, and flow cytometric analysis was performed. Percentage of circulating TO+ platelets was determined from fluorescence (FL-1) logarithmic histograms. RESULTS: Healthy ponies (n = 9) had 1.28 to 2.83% (mean +/- SD, 2.03 +/- 0.50%) and horses (n = 7) had 0.9 to 3.44% (2.12 +/- 1.14%) TO+ platelets in circulation. Thrombocytopenic ponies (n = 7) had 11.14 to 48.41% (26.51 +/- 11.99%) and thrombocytopenic horses (n = 4) had 2.33 to 8.52% (6.19 +/- 2.68%) TO+ platelets in circulation. Mean platelet counts for the thrombocytopenic ponies and horses were 24,400 +/- 20,500 and 39,300 +/- 13,500 platelets/microliters, respectively (reference range, 94,000 to 232,000 platelets/ microliters). CONCLUSION: Thiazole orange-positive platelets can be detected in equine blood and percentages of TO+ platelets are increased in thrombocytopenic horses. CLINICAL RELEVANCE: Enumeration of TO+ platelets may prove to be a helpful noninvasive clinical measurement of bone marrow platelet production and aid in the assessment of platelet kinetics in thrombocytopenic horses.

Phorbol ester stimulation of equine macrophage cultures alters expression of equine infectious anemia virus.

Equine infectious anemia virus (EIAV) is a lentivirus that replicates predominantly in mature tissue macrophages. Viral expression is strongly influenced by the state of differentiation of the host cell. While blood monocytes can be infected, viral transcription is limited until the cell differentiates into a mature macrophage. Activation of mature macrophages infected with EIAV might also alter viral expression, presumably through binding of cellular transcription factors to viral nucleic acid sequences within the long terminal repeat (LTR). Using DNA amplification techniques, we compared LTR sequences of U.S. field strains of EIAV to sequences of a laboratory adapted strain of the virus. All field strain sequences were more closely related to Wyoming strain than to the Malmquist laboratory adapted strain or a previously sequenced infectious molecular clone of EIAV. Primary equine monocyte-derived macrophage cultures were infected with virulent and avirulent strains of EIAV and the effects of macrophage stimulation on EIAV expression were determined. Stimulation of macrophages with phorbol ester activated the cells to secrete tumor necrosis factor alpha (TNF alpha). This activation signal also resulted in a significant downregulation of viral expression as determined by supernatant reverse transcriptase activity. This effect occurred independent of the virulence of the virus strain used or the nucleic acid sequence of the viral LTR. This may represent an adaptive response of EIAV to evade the host immune response and establish a persistent infection.

A molecular genetic approach to the study of Venezuelan equine encephalitis virus pathogenesis.

Viral pathogenesis can be described as a series of steps, analogous to a biochemical pathway, whose endpoint is disease of the infected host. Distinct viral functions may be critical at each required step. Our genetic approach is to use Venezuelan equine encephalitis virus (VEE) mutants blocked at different steps to delineate the process of pathogenesis. A full-length cDNA clone of a virulent strain of VEE was used as a template for in vitro mutagenesis to produce attenuated single-site mutants. The spread of molecularly cloned parent or mutant viruses in the mouse was monitored by infectivity, immunocytochemistry, in situ hybridization and histopathology. Virulent VEE spread through the lymphatic system, produced viremia and replicated in several visceral organs. As virus was being cleared from these sites, it began to appear in the brain, frequently beginning in the olfactory tracts. A single-site mutant in the E2 glycoprotein appeared to block pathogenesis at a very early step, and required a reversion mutation to spread beyond the site of inoculation. The feasibility of combining attenuating mutations to produce a stable VEE vaccine strain has been demonstrated using three E2 mutations.

Production and characterization of a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes.

An IgG1 mouse monoclonal antibody, designated 1.646, is described which recognizes a cytoplasmic antigen of equine mononuclear phagocytes. Indirect fluorescent antibody staining of peripheral blood leukocytes reveals a granular cytoplasmic staining, predominantly in adherent blood mononuclear cells. Indirect fluorescent antibody staining is positive for alveolar and peritoneal macrophages. In some horses, a few neutrophils are also stained. In equine tissue samples stained by immunohistochemistry, the distribution of positive cells is consistent with the distribution of tissue macrophages. The most intense and reliable staining occurs with splenic and lymph node macrophages. Hepatic Kupffer cells also stain with antibody 1.646, although the intensity of that staining is somewhat variable between horses. A granular pattern of staining typical of lipofuscin deposition is also seen in liver sections. There is also pale staining of some biliary and renal tubular epithelium. Equine erythrocytes, platelets and lymphocytes are not recognized by this antibody, and neither are monocyte/macrophages of human, canine or feline origin. Antibody 1.646 recognizes two proteins (150 and 30 kDa) of equine monocyte-derived macrophages when assayed by Western immunoblot. Because of the distribution of staining (tissue mononuclear phagocytes, lipofuscin-containing storage granules, biliary and renal tubular epithelium, and some neutrophils) we hypothesize that antibody 1.646 recognizes a cytoplasmic antigen that is closely associated with lysosomal membranes.

Wild-type equine infectious anemia virus replicates in vivo predominantly in tissue macrophages, not in peripheral blood monocytes.

In situ hybridization of tissues from two horses infected with the wild-type Wyoming strain of equine infectious anemia virus (EIAV) identified the liver, spleen, lymph nodes, kidney, lung, and adrenal gland as the primary host tissue sites for viral transcription during acute infection. Combined immunohistochemistry, with a monoclonal antibody recognizing a cytoplasmic antigen of equine mononuclear phagocytes, and in situ hybridization for viral RNA identified most infected cells as mature tissue macrophages. In contrast, in situ hybridization of adherent peripheral blood mononuclear cells collected from horses on various days during the first 2 weeks postinfection with the Wyoming strain of EIAV failed to detect any viral RNA in these cells. For the two horses described here, serum reverse transcriptase activity correlated directly with the degree of replication detected in tissue macrophages on the day of sacrifice. These results suggest that unlike other lentivirus infections in which mature tissue macrophages accumulate cytoplasmic viral RNA to a high level but fail to produce infectious virions, mature tissue macrophages are the likely primary source of the high titer of viremia present during acute infection with EIAV. No significant posttranscriptional block of viral replication in tissue macrophages appears to occur with EIAV.