The safety of dirlotapide in dogs.

The safety of dirlotapide in dogs was evaluated in two studies with parallel designs. In an acute tolerance study, 24 beagles (six dogs per treatment) were treated orally once daily for 14 days with placebo or dirlotapide at 2.5, 5.0, or 10.0 mg/kg/day. In a margin-of-safety study, 38 overweight, neutered beagles were treated orally once daily for 3 months with dirlotapide at doses up to 0.5 mg/kg/day (six dogs), 1.5 mg/kg/day (12 dogs) and 2.5 mg/kg/day (six dogs). Control dogs received placebo at 0.3 mL/kg/day (10 dogs) and 0.5 mL/kg/day (four dogs). Results were similar for both studies, and no serious adverse events were observed. Dirlotapide was clinically well-tolerated in dogs at dosages up to 10 mg/kg/day for 14 days and 2.5 mg/kg/day for 3 months. Dirlotapide produced the expected decrease in food intake and body weight (up to 20-40%) without ill effects. Clinical, pathologic, and histopathologic findings were reversible and consistent with suppression of food intake and rapid weight loss produced by elevated dirlotapide dosages. In both studies, sporadic emesis and loose stools were observed in both placebo and dirlotapide-treated dogs. Incidence of emesis generally increased with dose and decreased with treatment time. Elevations in hepatic transaminase activity were seen in dogs treated with more than 1.5 mg/kg dirlotapide daily, but were not associated with clinical signs or microscopic evidence of hepatic degeneration or necrosis.

Histologic lesions in cynomolgus monkeys (Macaca fascicularis) naturally infected with simian retrovirus type D: comparison of seropositive, virus-positive, and uninfected animals.

Simian retrovirus (SRV) type D is a common cause of simian acquired immunodeficiency syndrome (SAIDS), a usually fatal immunosuppressive disease of macaques. Associated gross and histologic lesions have been well described for the rhesus macaque (Macaca mulatta) in experimental and natural infections. However, morphologic changes induced by this virus at the gross and light-microscopic level have not been documented in the cynomolgus macaque (Macaca fascicularis). In 1996, sporadic cases of anemia, weight loss, and diarrhea were noted in a colony of cynomolgus macaques in our research facility. Out of 28 animals, 24 tested positive for SRV by serology or virus isolation. Animals could mainly be classified into 1 of 2 categories: 1) positive for virus isolation but negative for SRV antibody and 2) negative for virus isolation but antibody positive. During the process of eliminating the virus from the colony, a complete postmortem examination was performed on the 24 infected animals that had to be culled. Twelve SRV-negative animals were available as controls. Minimal to mild follicular lymphoid infiltrates were seen in various organ systems in 75% of the negative animals, compared with moderate to marked infiltrates in 83% of infected animals. Lymphoid infiltrates were more common in the brain, bone marrow, and salivary gland of viremic animals and were rare to nonexistent in seropositive or negative animals. Lymphoid hyperplasia was present in 38% of the infected animals, whereas lymphoid depletion was seen in 47% of the infected animals. Overall, lesions were of greater severity in viremic animals than in virus-negative or seropositive animals. Overall, infected animals had lower, statistically significant hematocrit and lymphocyte values. Viremic animals had significantly lower hematocrit, white blood cell, lymphocyte, and neutrophil values than did controls. Only 1 out of 24 infected animals had clinical signs that were consistent with the definition of SAIDS, and none had evidence of opportunistic infections. Lesions were similar to those already reported in other species of macaques, but the absence of severe illness that was consistent with SAIDS in most viremic animals suggests that there may be a different manifestation of disease in the cynomolgus.

Phenotypic characterisation of a Neospora caninum temperature-sensitive strain in normal and immunodeficient mice.

The in vivo persistence, immunogenicity and pathogenicity of a recently described temperature-sensitive (ts) strain from Neospora caninum, NCts-8, was investigated in normal and immunodeficient mice. Groups of BALB/c and SCID/Bg mice were infected s.c. with 5 x 10(6) wild-type NC-1, control NCts-8 (pass 0) or NCts-8 tachyzoites prepared at four in vitro passage levels (pass 7, 13, 21 and 28). For persistence and immunogenicity studies, BALB/c mice were bled and sacrificed at 4, 6 or 8 weeks p.i. Sera were analysed by IFAT and brain tissues examined for lesions by histology and tested for parasite presence by PCR. For pathogenicity studies, SCID/Bg mice were monitored by clinical signs and survival time. Results from parasite persistence experiments demonstrated microscopic lesions and PCR positive brain tissues in NC-1 infected mice. In contrast, brain tissues from NCts8-infected groups were consistently negative by histology and PCR. Based on IFAT titres, all parasite strains were immunogenic, although parasite-specific IgG levels were lower in the NCts-8 infected groups. Results from pathogenicity studies in SCID/Bg mice demonstrated a significantly (P < 0.0001) longer mean survival time in NCts-8 vs NC-1 infected groups. In addition, there was no significant difference in mean survival time between control NCts-8 and experimental passage NCts-8 infected mice. Collectively, these studies demonstrate that the NCts-8 strain maintains a stable phenotype following multiple passages in vitro, and possesses an attenuated, shorter persistence phenotype in vivo compared with the parental wild-type NC-1.

A poorly differentiated germ cell tumor (seminoma) in a Long Evans rat.

A large neoplasm that replaced 1 testis of a Long Evans Rat was noted at the final necropsy of a dietary 2-yr study. By light microscopy, the morphological features were consistent with a poorly differentiated seminoma. Ultrastructurally, the cells were polygonal, had a round nucleus, had straight cellular boundaries, and bore no resemblance to Sertoli cells. Although there was little evidence of spermatocytic differentiation, the presence of proacrosomal granules and vesicles, prominent Golgi apparatus, tight intercellular junctions, and a few centriolar pairs without axoneme development, in conjunction with the absence of lipid droplets or abundant smooth endoplasmic reticulum, supported the diagnosis of seminoma rather than Leydig cell tumor. The cells were S-100- and vimentin-positive, although cytokeratin- and alpha-fetoprotein-negative. Seminomas are extremely rare neoplasms in rats; this is the first report in this strain and the first extensive analysis of a rat seminoma without spermatocytic differentiation.

Sonographic renal findings in 20 dogs with leptospirosis.

Abdominal ultrasound examinations of 20 dogs with confirmed leptospirosis were reviewed retrospectively for renal abnormalities. Three dogs had a normal ultrasound examination. The remaining 17 dogs had sonographic abnormalities of the kidneys. These abnormalities, seen either alone or in combination, included renalmegaly (n=10), pyelectasia (n=9), increased cortical echogenicity (n=15), perinephric effusion (n=5), and a medullary band of increased echogenicity (n=6). At our institution, the medullary band of increased echogenicity has only been seen in dogs with leptospirosis and may therefore be a specific sonographic sign for this disease.

Digestion of host immunoglobulin and activity of midgut proteases in the buffalo fly Haematobia irritans exigua.

The digestion of blood by the buffalo fly (Haematobia irritans exigua) was monitored for 6h at 33 degrees C after a single meal. Following the meal, the concentration of soluble protein within the midgut increased to a peak at 2 hours then decreased steadily over the next 4h. The magnitude of the increase in soluble protein at 2h indicated a release of protein from another source; most likely from lysed red blood cells. The immunoglobulin (IgG) fraction of the blood meal was digested rapidly (50% within one hour of feeding) and fully digested within 4h. This is indicative of its accessibility to digestive enzymes within the midgut. In contrast, when flies had continuous access to blood, the concentration of IgG in the midgut remained at a more constant level. The loss of antigen-binding activity of a specific antibody was more rapid than complete degradation of the IgG, with 70% of binding activity lost within one hour of feeding. The level of trypsin activity in the midgut increased from pre-feeding levels to reach a peak at 2h before returning to basal levels after 6h. The pattern of trypsin activity follows closely that of the concentration of soluble protein in the midgut (r=0.88). The activity of leucine aminopeptidase in the midgut also increased immediately after feeding and remained elevated for 4 h before declining to a basal level after 6h. The rapid digestion of IgG and subsequent loss of antibody activity suggests that for a specific anti-buffalo fly antibody to be effective it would need to be able to either evade the digestive system or induce a rapid response.

Malignant fibrous histiocytoma and malignant histiocytosis in the dog--convergent or divergent phenotypic differentiation?

Malignant fibrous histiocytoma (MFH) and malignant histiocytosis (MH) are neoplasms with different histologic appearances and consequently a different putative cell of origin. Recently, the biopsy and necropsy services at the University of Pennsylvania have seen many canine soft tissue sarcomas that have the gross and histologic appearances of both MH and MFH within the same animal. A retrospective histologic evaluation of 263 cases diagnosed originally as either MH or MFH reclassified these neoplasms into 77 cases that were exclusively MH, 110 cases exclusively MFH, and 76 cases with features of both MH and MFH. Age, sex, breed predispositions, and distribution of lesions in organs were remarkably similar between the two categories. The hybrid neoplasms containing MH-like and MFH-like regions may be the result of divergent or convergent phenotypic differentiation.

Aggressive, undifferentiated sarcoma with widespread metastasis in a six-month-old Neopolitan mastiff.

A six-month-old Neopolitan mastiff presented for a rapidly growing cervical mass. Undifferentiated sarcoma was diagnosed at post mortem based on histopathology and immunohistochemistry. Metastases to mediastinum, pleura, lungs, liver, kidneys, omentum, mesentery, and multiple lymph nodes were present. Soft-tissue sarcomas are reported infrequently in children and young dogs. The cell of origin often is difficult to determine due to poor differentiation and rapid growth of these neoplasms.

Erythema multiforme major and disseminated intravascular coagulation in a dog following application of a d-limonene-based insecticidal dip.

Erythema multiforme major and disseminated intravascular coagulation developed in a dog 24 hours after exposure to a d-limonene-based insecticidal dip. Clinical signs included severe lethargy and weakness, ulceration of the oral mucosa, and erythematous serpiginous, annular, and arciform lesions on the head, trunk, and limbs. Clinicopathologic abnormalities included leukocytosis with neutrophilia, normocytic normochromic anemia, thrombocytopenia, prolongation of prothrombin and partial thromboplastin times, increased fibrin degradation products, hypoproteinemia, hyponatremia, hypochloremia, azotemia, high serum alanine aminotransferase and alkaline phosphatase activities, and high serum bilirubin concentration. Despite intensive supportive care, the dog developed severe intrathoracic and abdominal hemorrhage and died. Necropsy revealed severe diffuse epidermal necrosis and widespread hemorrhage within organs. Insecticidal dips containing d-limonene have the potential to induce various toxic effects, including, possibly, erythema multiforme major, and should be used cautiously.

Strongyloides stercoralis: histopathology of uncomplicated and hyperinfective strongyloidiasis in the Mongolian gerbil, a rodent model for human strongyloidiasis [corrected].

Tissues from corticosteroid-treated gerbils hyperinfected with Strongyloides stercoralis were compared grossly and microscopically to similar tissues from animals with uncomplicated strongyloidiasis. Gerbils with hyperinfection developed severe pulmonary alveolar haemorrhage with a variable degree of subacute eosinophilic interstitial pneumonia associated with numerous alveolar, vascular and interstitial larvae. Hyperinfection induced by corticosteroids, given either before inoculation of S. stercoralis larvae or after a chronic Strongyloides infection was established, produced similar lesions. In contrast, lungs from gerbils with uncomplicated Strongyloides infection had severe eosinophilic perivasculitis and vasculitis with very little haemorrhage, no pneumonia and no larvae. Sections of adult worms were present in the proximal part of the intestinal tract, lodged in spaces between mucosal epithelial cells. Adult worms were not associated with inflammation and were more common in the corticosteroid-treated gerbils. In corticosteroid-treated gerbils only, there were numerous larvae in the distal intestinal tract, throughout the intestinal wall and adjacent mesentery, within interstitial tissues and in lymphatic vessels. Significant inflammation with associated larvae was only present in the caecum and mesenteric lymph nodes, suggesting that the caecum was the main site for initiation of parenteral migration with subsequent invasion of the lymphatic system and lungs. The lesions in these gerbils were similar to those found in humans. Infection of gerbils with S. stercoralis is the best rodent model of human strongyloidiasis.

Sea star factor blocks development of T-dependent antibody secreting clones by preventing lymphokine secretion.

Sea star factor (SSF), a protein of 39 kDa purified from macrophage-like coelomocytes of the echinoderm Asterias forbesi, has potent immunosuppressive effects on T-dependent but not T-independent antibody responses in vivo. SSF at a concentration of 0.5 microgram/ml markedly inhibits T-dependent antibody production in vitro by fluorescein (Flu)-specific B cells responding in clonal microculture to antigenic stimulation with Flu-conalbumin via the conalbumin-specific T cells D10.G4.1 (D10). At this concentration of SSF, Ig secretion induced by a T cell-independent stimulus, lipopolysaccharide (LPS), is not affected. Inhibition of antibody production in T-dependent microcultures by SSF can be completely overcome in a dose-dependent fashion by addition of lymphokine-rich supernatants from stimulated cultures of D10 cells. The possibility that SSF suppresses production of requisite cytokine growth factors from T cells was substantiated by the finding that SSF diminishes concentrations of stimulatory cytokines detectable in supernatants from antigen-stimulated cultures. Nevertheless, levels of intracytoplasmic mRNA for IL-4 and IL-5 are not detectably altered by concentrations of SSF that suppress antibody production. Furthermore, when cultures of D10 cells stimulated in the presence of SSF are subjected to freezing and thawing to release intracytoplasmic lymphokines, total levels of stimulatory cytokines are not lower than those in cultures without SSF. These results suggest that SSF inhibits antibody responses by limiting the availability of lymphokines produced by helper T cells. The mechanism for this inhibition may involve either direct effects of SSF on T cells or a block in effective T cell-B cell interaction.

Histamine receptors on bovine peripheral blood lymphocytes.

Histamine receptors on bovine peripheral blood lymphocytes (PBL) were detected by three different methods: a rosetting technique, binding to histamine-bearing Sepharose beads and immunofluorescence staining. The rosetting technique used histamine-rabbit serum albumin (H-RSA) conjugated to bovine red blood cells to detect histamine receptors and this showed that 10.8% of bovine PBL were positive. A method using H-RSA conjugate coupled Sepharose beads also detected histamine receptor bearing PBL but was not quantitative. The indirect immunofluorescence method, by which the subpopulation of histamine receptor bearing lymphocytes can be easily double stained to concurrently identify the B cell marker, revealed that PBL, the B cell and T cell fraction of bovine PBL contained 18.4, 52.8 and 9.3% histamine receptor bearing cells, respectively. This method was found to be more stable and more easily quantifiable than the other two methods. Blocking tests using the histamine H1 receptor antagonist diphenhydramine and the histamine H2 receptor antagonist cimetidine suggested that bovine PBL have both H1 and H2 receptors on their surfaces. Addition of histamine into cultures of PBL at the concentration range 10(-6) to 10(-3) M suppressed the response of PBL to the mitogen phytohemagglutinin. The histamine induced suppression of mitogenesis could be reduced partially by the H2 receptor antagonist cimetidine, but not by the H1 antagonist diphenhydramine. It is possible that histamine induced suppression of PBL mitogenesis was mediated by H2 receptors on T cells.

Effects of cattle tick (Boophilus microplus) infestation on the bovine immune system.

The immunosuppressive effect of experimental Boophilus microplus infestation on bovine peripheral blood lymphocytes (PBL) and on host antibody production to a protein antigen (ovalbumin) was examined. Boophilus microplus infestation caused a marginal decrease in the percentage of T lymphocytes in PBL, which was observed in both lightly (5000 larvae) and heavily (40,000 larvae) infested cattle, and began at the second infestation and continued until the end of the fourth infestation. The percentage of B lymphocytes in heavily tick-infested cattle was less than that in non-infested control cattle after the fourth infestation. The response of PBL from tick-infested cattle to phytohemagglutinin (PHA) was always less than that of tick-free cattle after the second infestation. No noteworthy differences were detected between the three stages of tick infestation, that is, 1 week before the peak of adult engorgement, the middle of the peak and 1 week after all ticks had dropped. Boophilus microplus saliva (100 microliters ml-1) suppressed 47% of the response of bovine PBL to PHA in vitro. This suppressive effect of saliva may contribute to the lower responsiveness of PBL from tick-infested cattle. Antibody production by tick-infested cattle was examined during the third and fourth heavy tick infestation. Tick-infested cattle showed a diminished response against ovalbumin after the second immunization. The immunosuppressive effects of tick infestation may play an important role in tick survival or in the transmission of tick-borne diseases in the field.

Potent immunosuppression by secretory/excretory products of larvae from the sheep blowfly Lucilia cuprina.

Secretory/excretory products (sec/ex) of parasitic larvae of the sheep blowfly Lucilia cuprina potently inhibited proliferation of peripheral blood leucocytes stimulated by mitogens in vitro. Suppression of proliferation was not due to irreversible damage because cells cultured for 24 h in high concentrations of sec/ex appeared viable (assessed by Trypan blue exclusion) and did not show impaired proliferation after washing. Furthermore, suppression induced by sec/ex could be overcome by increasing concentrations of mitogen. The inhibitory activity could be demonstrated in cultures where sec/ex was added at different times during the culture period. Inhibitory activities in sec/ex were heat-labile and sensitive to treatment with trypsin. In addition to effects in vitro, sec/ex was strongly immunosuppressive in vivo. Sheep given combined injections of myoglobin and sec/ex had markedly lower anti-myoglobin antibody levels in sera than sheep that received injections of myoglobin alone. There was no significant antibody response to sec/ex itself. Immunosuppressive moieties in sec/ex produced by blowfly larvae may promote parasite survival by inhibiting the immune response of host sheep.

Reduced growth of Lucilia cuprina larvae fed serum from sheep treated with anthelmintics.

The effect of three commonly used anthelmintics, levamisole hydrochloride, ivermectin and closantel, on the development of the sheep blowfly, Lucilia cuprina, was determined. Sheep were treated with each anthelmintic using the manufacturers' recommended dose for helminth control. Both ivermectin and closantel significantly (p < 0.05) reduced the growth rate of larvae of L cuprina cultured in vitro on serum from these sheep. Levamisole hydrochloride had no effect. Ivermectin was effective for less than 6 days after treatment, whereas closantel significantly reduced larval growth 21 days after treatment. Dose-response experiments showed that lower concentrations of both ivermectin and closantel were not as effective in reducing larval growth.

The survival and fecundity of buffalo flies after treatment of cattle with three anthelmintics.

Two anthelmintics with known insecticidal action (ivermectin and closantel) and one with no recorded effect on insects (levamisole) were tested to evaluate their effects on buffalo fly (Haematobia irritans exigua). Blood from animals given closantel or levamisole had no significant effect on mortality of buffalo flies in an in-vitro assay. In contrast, blood from animals given ivermectin showed a dose-dependent effect on the mortality of buffalo flies. At 24 h after one injection of the recommended dose of ivermectin, 98% of the flies applied to cattle in an in-vivo assay are killed. Blood from cattle injected with ivermectin killed 95% of flies 8 d after injection and still killed 15% of flies at 18 days after injection. Surviving flies laid almost no eggs and this effect on flies was significant up to 33 d after injection. The results indicate that ivermectin may be useful to control buffalo fly populations in the field.

Acquired immune response of cattle exposed to buffalo fly (Haematobia irritans exigua).

Naturally acquired immunity to buffalo fly (Haematobia irritans exigua) infestation was examined in cattle. Animals exposed to flies had serum antibodies to buffalo fly antigens at levels that correlated with the intensity of exposure. Two weeks of intense exposure to buffalo fly induced an increase in peripheral blood eosinophil numbers and a concomitant rise in serum antibody levels in exposed animals. Antigens specific for antibody induced by natural exposure were identified using antisera from exposed cattle to probe Western blots of whole fly homogenate separated using SDS-PAGE. Similar immunoreactive bands were found with buffalo fly saliva. Immunoreactive proteins were partially purified from whole fly homogenates by anion-exchange chromatography. Fractions eluted from columns were screened using Western blots probed with serum from exposed animals. Exposed animals showed immediate hypersensitivity to partially purified antigens and to buffalo fly saliva. Flies which fed on exposed animals with high serum levels of antibody to fly antigens did not show greater mortality than flies fed on unexposed animals.