Effect of feeding Mucuna pruriens on helminth parasite infestation in lambs.

ETHNOPHARMACOLOGICAL RELEVANCE: Mucuna pruriens is a tropical legume anecdotally reputed to have anthelmintic properties. This study was conducted to examine the validity of such claims. AIM OF THE STUDY: The aim of this study was to determine if ingestion of Mucuna seeds reduces helminth parasite infestation in lambs. MATERIALS AND METHODS: Thirty-six Dorper x Katahdin ram lambs were assigned to three treatments, a cottonseed meal based control diet, a diet in which Mucuna replaced cottonseed meal and the control diet with levamisole (7.5mg/kg body weight) administration. All diets were isonitrogenous and isocaloric. The 12 lambs in each treatment were assigned randomly to 4 pens, each containing 3 lambs. Lambs were trickle infected three times per week by gavage with infectious Haemonchus contortus larvae (2000 larvae/lamb) for 3 weeks. RESULTS: Levamisole treatment decreased fecal egg counts by 87% and abomasal worm counts by 83%. Mucuna intake did not statistically affect fecal egg counts or abomasal worm counts, though numerical (P>0.10) reductions of 7.4% and 18.1%, respectively were evident. Anemia indicators, feed intake, and lamb growth were unaffected by treatment. CONCLUSIONS: Levamisole reduced the Haemonchus parasite burden in lambs significantly but feeding Mucuna reduced the burden by levels unlikely to eliminate the clinical effects of parasitism.

Experimental transmission of a herpesvirus in Greek tortoises (Testudo graeca).

An experimental transmission study aimed at fulfilling Koch's postulates for a herpesvirus-associated stomatitis-rhinitis in Mediterranean tortoises is presented. Clinical, pathologic, serologic, and molecular studies were performed linking tortoise herpesvirus with the pathogenesis of stomatitis-rhinitis. Four adult Greek tortoises received either intranasally or intramuscularly two tortoise herpesvirus isolates by primary experimental infection and secondary challenge 11 months later. After the primary experimental infection and the secondary challenge, clinical signs of illness developed, which included conjunctivitis, diphtheritic oral plaques, and oral discharge. At 4 weeks after the secondary challenge, all tortoises were humanely euthanatized and evaluated. Although neutralizing antibodies developed after the primary experimental infection, they apparently did not prevent the later development of recurrent clinical signs. Polymerase chain reaction (PCR) and reverse transcription-PCR analyses allowed sensitive characterization of the systemic distribution of the herpesvirus DNA sequences and their presence in the cranial nerves and brains of the infected tortoises. Despite the failure to recover the herpesviruses used in the transmission study, the findings support the premise that tortoise herpes-virus is a primary pathogen of Greek tortoises.

Mycobacterium avium infection in an ostrich (Struthio camelus).

Acid-fast organisms were identified by histopathology of granulomatous lesions in an ostrich (Struthio camelus). The organisms were grown in Herrold's egg media with and without mycobactin and identified as Mycobacterium avium. An agar gel immunodiffusion (AGID) test for Mycobacterium avium paratuberculosis was performed for detection of antibody for M. avium in this infected ostrich and seven other ostriches that were in contact. The results of the AGID were consistent with the pathologic diagnosis of mycobacteriosis and the isolation of M. avium in the affected ostrich.

Persistent infectivity of a disease-associated herpesvirus in green turtles after exposure to seawater.

Herpesviruses are associated with several diseases of marine turtles including lung-eye-trachea disease (LETD) and gray patch disease (GPD) of green turtles (Chelonia mydas) and fibropapillomatosis (FP) of green, loggerhead (Caretta caretta), and olive ridley turtles (Lepidochelys olivacea). The stability of chelonian herpesviruses in the marine environment, which may influence transmission, has not been previously studied. In these experiments, LETD-associated herpesvirus (LETV) was used as a model chelonian herpesvirus to test viral infectivity after exposure to seawater. The LETV virus preparations grown in terrapene heart (TH-1) cells were dialyzed for 24 to 120 hr against aerated artificial or natural seawater or Hank's balanced salt solution (HBBS). Fresh TH-1 cells were inoculated with dialyzed LETV, and on day 10 post-infection cells were scored for cytopathic effect. Virus samples dialyzed up to 120 hr were positive for the herpesvirus DNA polymerase gene by polymerase chain reaction. Electron microscopy revealed intact LETV nucleocapsids after exposure of LETV to artificial seawater or HBSS for 24 hr at 23 C. LETV preparations remained infectious as long as 120 hr in natural and artificial seawater at 23 C. Similar results were obtained with a second culturable chelonian herpesvirus, HV2245. LETV infectivity could not be detected after 48 hr exposure to artificial seawater at 30 C. Since LETV and HV2245 remain infectious for extended periods of time in the marine environment, it is possible that FP-associated and GPD-associated herpesviruses also may be stable. These findings are significant both for researchers studying the epidemiological association of herpesviruses with diseases of marine turtles and for individuals who handle turtles in marine turtle conservation efforts.

Isolation and experimental transmission of a reovirus pathogenic in ratsnakes (Elaphe species).

A reovirus was isolated from juvenile Moellendorff's ratsnakes (Elaphe moellendorffi) and beauty snakes (Elaphe taenuris) that died soon after importation into the USA. Viper heart (VH2) cells inoculated with tissue homogenates showed cytopathic effects consisting of large syncytia formation followed by cell detachment from the monolayer. Tissue culture supernatants failed to hemagglutinate guinea pig and chicken erythrocytes at room temperature. Electron microscopy of purified virions revealed spherical to icosahedral particles measuring 70-85 nm in diameter with a double capsid layer. Preparations of the viral genome contained ten segments of dsRNA when analyzed by polyacrylamide gel electrophoresis. A juvenile black ratsnake (Elaphe obsoleta obsoleta) was experimentally inoculated with the isolate and was found dead 26 days post inoculation. Necropsy revealed diffuse subacute interstitial pneumonia with respiratory epithelial cell hyperplasia and syncytia. Reovirus isolated from this snake was used to inoculate another juvenile black ratsnake which was euthanized 40 days post inoculation. Pneumonia and multifocal subacute proliferative tracheitis were found on necropsy. Reovirus was isolated from the lung of this snake and was demonstrated by transmission electron microscopy. This is the first documentation of a pathogenic reptile reovirus and the first report of experimental transmission of a reovirus in snakes.

Genetic diversity in twenty variants of the avian polyomavirus.

To determine if different pathotypes of the avian polyomavirus (APV) exist and to compare the genomes of APVs originating from different geographic areas, dates, and species of birds, the partial sequences of 18 APVs were determined. New viral sequences were compared with three published APV sequences. Two of the new viruses had identical sequences. Forty point mutations were found at 31 loci. A 27-bp deletion was found in the VP2 and VP3 open reading frames of one virus. A duplication of the putative origin of replication and adjacent enhancer region was previously reported in one APV. Smaller duplications involving the origin in one APV and a second enhancer region in another were discovered. All duplications were in tissue culture-adapted viruses, suggesting they occurred during the isolation process. Excluding duplications and the deletion, maximum variation between viruses was small (11 bp). A maximum parsimony tree was constructed that contained three major branches. The three earliest isolates were on separate branches. The European viruses were confined to branch I, but APVs from the United States were on all three branches. Lovebird, budgerigar, and macaw APVs were also on each of the three branches, suggesting that species-specific pathotypes have not developed. Most nonsynonymous mutations occurred in a small portion of the VP2 and VP3 open reading frames, demonstrating a selection for these mutations. That a glycine at VP2 221 will inhibit virus replication in chicken embryo fibroblasts (CEFs) has been previously reported. In contrast, six of seven of the new APVs isolated in CEFs had a glycine at VP2 221.

Western immunoblot analysis of the antigens of Haemobartonella felis with sera from experimentally infected cats.

Cats were experimentally infected with a Florida isolate of Haemobartonella felis in order to collect organisms and evaluate the immune response to H. felis. Cryopreserved organisms were thawed and injected intravenously into nonsplenectomized and splenectomized cats. Splenectomized animals were given 10 mg of methylprednisolone per ml at the time of inoculation. Blood films were evaluated daily for 1 week prior to infection and for up to 60 days postinfection (p. i.). Blood for H. felis purification was repeatedly collected from splenectomized animals at periods of peak parasitemias. Organisms were purified from infected blood by differential centrifugation, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes for immunoblot analysis. Serum was collected from nonsplenectomized animals prior to and for up to 60 days p.i. and was used on immunoblots to identify antigens. The combination of splenectomy and corticosteroid treatment resulted in marked, cyclic parasitemias without concurrent severe anemia, providing an opportunity to harvest organisms in a manner that was not lethal to the animals. Several antigens (150, 52, 47, 45, and 14 kDa) were identified. An antigen with a molecular mass of approximately 14 kDa appeared to be one of the most immunodominant and was consistently recognized by immune sera collected at various times during the course of infection. These data suggest that one or more of these antigens might be useful for the serologic diagnosis of H. felis infections in cats.

Immunoprophylaxis of Rhodococcus equi pneumonia in foals.

An immunoprophylaxis program for R. equi infection of foals has been established on a number of thoroughbred breeding farms in Argentina over the past 4 years. Nearly 800 mares annually were immunized subcutaneously during the last 2 months of pregnancy with 2-3 doses of a vaccine containing soluble antigens of R. equi, including the virulence associated protein (VapA) and 'equi factors' exoenzymes. The mortality from R. equi pneumonia in the foals from vaccinated dams dropped from an average of 3% in the 5 years before the vaccination program was initiated to an average of 1.2% in the 4 years during which the program was applied (P < 0.02). On 3 farms, an additional 380 foals of vaccinated dams annually over 3 years also received at 25 days of age 600-1200 ml of hyperimmune plasma from donors immunized with this vaccine, and as well at 4 days of age in foals with poor transfer of R. equi antibodies from their dams. The average foal mortality because of R. equi in the 380 foals annually to which hyperimmune plasma was administered dropped from 5.8% on these 3 farms to 0.2% (P < 0.05). Active vaccination of foals of unvaccinated mares on an enzootic farm at 20, 30, and 40 days of age did not protect them from mortality due to R. equi pneumonia. Serology was done by complement fixation and an agar gel immunodiffusion (AGID) tests using antigens prepared in the same manner as the vaccine antigens. The immune responses among hyperimmune plasma donors varied considerably as did the responses of vaccinated mares. Of 1117 serum samples with normal post suckling gammaglobulin levels (> 600 mg%) collected at 2 days of age from foals of vaccinated mares, 36% showed a negative or weak positive AGID reaction, while the remainder had positive to strongly positive reactions.

Immunoperoxidase detection of ophidian paramyxovirus in snake lung using a polyclonal antibody.

In a retrospective study of proliferative interstitial pneumonia in viperid and nonviperid snakes, formalin-fixed, paraffin-embedded lungs from 52 snakes were screened for immunohistochemical reactivity to ophidian paramyxovirus. All snakes were from zoological collections that experienced mortalities attributed to paramyxovirus infection. Of the 52 snakes, 47 had pulmonary lesions compatible with ophidian paramyxovirus infection. Histologic changes in affected lungs included hyperplasia and hypertrophy of septal and faveolar epithelial cells, loss of ciliated cells, mixed leukocytic interstitial infiltrates, fibrinonecrotic exudate in the lumen of proximal and distal faveolar compartments, and occasional epithelial syncytial cell formation or intraepithelial eosinophilic intracytoplasmic inclusions. Lungs were immunohistochemically stained for paramyxovirus antigens by utilizing rabbit polyclonal antibodies against a paramyxovirus isolate from a black mamba (Dendroaspis polyepis polyepis). Virus infection in 6 snakes was confirmed by virus isolation from frozen lung tissue. Of the 6 lungs from which paramyxovirus was isolated, 5 lungs stained positively for viral antigens utilizing antisera to the black mamba isolate. Altogether, 36 lungs stained positively for paramyxovirus antigens. There was multifocal to diffuse linear staining of the lumenal surface of faveolar epithelium, and there were multiple foci of granular cytoplasmic staining. Immunohistochemical staining of formalin-fixed lungs from snakes with proliferative interstitial pneumonia was helpful as a routine diagnostic test for substantiating a diagnosis of ophidian paramyxovirus infection.

Antigens of Vibrio vulnificus.

Rabbit antisera raised against a formalinized culture of Vibrio vulnificus were examined for the development of IgM and IgG antibody by ELISA analysis of serum samples harvested weekly for 5 weeks following the initial injection. V. vulnificus antigens recognized by the sera in three fractions of the culture were identified by immunoblot analysis. The major species recognized in the culture supernatant solution by both IgM and IgG had a molar mass of 58 kD. In fractions derived from the bacterial pellet, IgM recognized a species of 56 kD, while IgG reacted strongly with 29.5, 56, 59, 68, 87, and 186 kD species.

Chronic upper respiratory tract disease of free-ranging desert tortoises (Xerobates agassizii).

Seventeen desert tortoises, Xerobates agassizii, with upper respiratory tract disease were examined; thirteen were euthanatized for necropsy. Four normal control desert tortoises from a clinically healthy population were similarly evaluated. Hemoglobin and phosphorus values were significantly (P less than or equal to 0.05) lower and serum sodium, urea, SGOT, and cholesterol values were significantly higher in ill tortoises compared to controls. No significant differences in concentrations of serum or liver vitamins A and E were found between the two groups. While no significant differences were found for concentrations of lead, copper, cadmium, and selenium, the livers of ill tortoises had higher concentrations of mercury and iron. Lesions were found consistently in the upper respiratory tract (URT) of ill tortoises. In all ill tortoises dense infiltrates of lymphocytes and histiocytes obscured the mucosal epithelium and underlying glands. The mucosal epithelium was variably dysplastic, hyperplastic, and occasionally ulcerated. Electron microscopic studies revealed small (350 to 900 nm), pleomorphic organisms resembling Mycoplasma sp., in close association with the surface epithelium of the URT of ill tortoises. Pasteurella testudinis was cultured from the nasal cavity of all ill tortoises and one of four control tortoises. A Mycoplasma sp. was cultured from the nasal passageways of four ill tortoises and was ultrastructurally similar to the pleomorphic organism present on the mucosa in tissue section.

Erosive polyarthritis in two greyhounds.

Clinical signs of disease, treatment, laboratory findings, and gross and microscopic changes of erosive polyarthritis in 2 Greyhounds are described. A microscopic feature that may help distinguish this condition from other types of arthritis is extensive necrosis of deep articular cartilage zones, with relative sparing of the more superficial surface cartilage. We believe that the disease in the 2 dogs of this report was identical to that encountered previously in Britain and Australia. Bacteriologic culture and serologic investigation failed to reveal the causative agent.

Initiation and ultrastructure of a reptilian fibroblast cell line obtained from cutaneous fibropapillomas of the green turtle, Chelonia mydas.

Two fibroblastic cell lines were established from explants of fibropapillomas of each of two different green turtles (Chelonia mydas). These cells, designated GTFP (Green Turtle Fibropapilloma), were subcultured approximately 30 times at 30 degrees C in Eagle's minimal essential media supplemented with 2 to 10% fetal bovine serum. The ultrastructural morphology of the cultured fibroblasts is described. The cells contained abundant rough endoplasmic reticulum, polyribosomes, and mitochondria; collagen fibrils were visible in the extracellular space. No viruslike particles or evidence of other pathogenic agents could be demonstrated by electron microscopy in any of the cultured cells examined.

Postoperative examination of the biceps femoris muscle sling used in excision of the femoral head and neck in dogs.

Twenty-four dogs underwent unilateral excision of the femoral head and neck. An adjunctive biceps femoris muscle sling procedure was done in 16 dogs. In eight dogs (controls), the flap was dissected and returned to its original position. Four dogs with muscle slings were euthanatized on days 2, 5, 30, and 60, respectively. Four control dogs were euthanatized on day 2 and four dogs on day 5. Limb function did not differ consistently between dogs with muscle slings and control dogs. There was marked swelling and edema of the affected limb in half the dogs with muscle slings but not in the controls. Postoperative temperature elevations were significantly higher in dogs with muscle slings on days 1 and 2 (p less than 0.05). Infection was documented in four dogs with muscle slings. Flaps from control dogs had only minor gross and histologic abnormalities. On days 2 and 5, flaps from dogs with muscle slings appeared congested and swollen beneath and distal to the ostectomy site, with infarction involving 50 to 90% of the muscle mass. On days 30 and 60, the muscle slings were atrophic and fibrous, and by day 60 a synovial membrane covered the surfaces of all slings within the pseudarthrosis. Muscle fiber loss was attributed to infarction, necrosis, and disuse atrophy.

Effects of Newcastle disease virus administration to mice on the metabolism of cerebral biogenic amines, plasma corticosterone, and lymphocyte proliferation.

Newcastle disease virus (NDV) administration to mice increased concentrations of plasma corticosterone, with a maximal effect at 8 h. This elevation of plasma corticosterone concentrations was not observed in hypophysectomized animals in which the completeness of the hypophysectomy was verified by functional tests. NDV administration consistently increased concentrations of free tryptophan in all brain regions examined (prefrontal cortex, hypothalamus, and brain stem). It also caused an activation of cerebral catecholamine and indoleamine metabolism as determined by measurement of the amines and their catabolites. 3-Methoxy,4-hydroxyphenylethyleneglycol (MHPG), the major catabolite of norepinephrine (NE), homovanillic acid (HVA), a major catabolite of dopamine (DA), and 5-hydroxyindoleacetic acid (5-HIAA), the major catabolite of serotonin (5-HT), were all increased in both hypothalamus and brain stem. Ratios of catabolites to the parent amine, considered to be an index of utilization of the neurotransmitters, were increased for NE, DA, and 5-HT in the hypothalamus and for DA and 5-HT in the brain stem. This pattern of changes resembles that observed following stressors such as footshock or restraint. There were also significant increases of tryptophan, HVA, dihydroxyphenylacetic acid (DOPAC), and 5-HIAA in hypophysectomized relative to sham-operated mice. The NDV treatment also increased thymus weights and markedly decreased the proliferative responses of isolated spleen cells to phytohemagglutinin, concanavalin A, pokeweed mitogen, and Escherichia coli lipopolysaccharide. These changes were not caused by increased circulating corticosterone because they were present at equal magnitude in hypophysectomized mice. Thymosin alpha 1 concentrations in the plasma were not altered by NDV or hypophysectomy. These results indicate that administration of NDV to mice can initiate neurochemical and endocrine responses like those observed during stress and can also cause immunosuppression. They are thus consistent with the hypothesis that a virus can be a stressor.