Helicobacter pylori infection in the cat: evaluation of gastric colonization, inflammation and function.

BACKGROUND: Further elucidation of the consequences of Helicobacter pylori infection on gastric mucosal inflammation and gastric secretory function would be facilitated by an animal model that is susceptible to infection with H. pylori, is broadly similar in gastric physiology and pathology to people, and is amenable to repeated non-invasive evaluation. The goal of this study was to examine the interrelationship of bacterial colonization, mucosal inflammation and gastric secretory function in cats with naturally acquired H. pylori infection. MATERIALS AND METHODS: Twenty clinically healthy cats with naturally acquired H. pylori infection (cagA-, picB) and 19 Helicobacter-free cats were evaluated. Gastric colonization was determined by tissue urease activity, light microscopy, culture and PCR. The mucosal inflammatory response was evaluated by light microscopy, and by RT-PCR of the pro-inflammatory cytokines IL-1alpha, IL-1beta, IL-8 and TNF-alpha in gastric mucosa. Gastric secretory function was assessed by measuring pentagastrin-stimulated acid secretion, fasting plasma gastrin, and antral mucosal gastrin and somatostatin immunoreactivity. RESULTS: H. pylori colonized the pylorus, fundus and cardia in similar density. Bacteria were observed free in the lumen of gastric glands and were also tightly adherent to epithelial cells where they were associated with microvillus effacement. Mononuclear inflammation, lymphoid follicle hyperplasia, atrophy and fibrosis were observed primarily in H. pylori-infected cats, with the pylorus most severely affected. Neutrophilic and eosinophilic infiltrates, epithelial dysplasia, and up-regulation of mucosal IL-1beta and IL-8 were observed solely in infected cats. Fasting plasma gastrin concentrations and pentagastrin-stimulated acid output were similar in both infected and uninfected cats. There was no relationship of bacterial colonization density or gastric inflammation to plasma gastrin concentrations or gastric acid output. CONCLUSIONS: The pattern of colonization and the mucosal inflammatory response in cats with naturally acquired H. pylori are broadly similar to those in infected people, particularly children, and non-human primates. The upregulation of IL-8 in infected cats was independent of cagA and picB. Our findings argue against a direct acid-suppressing effect of H. pylori on the gastric secretory-axis in chronically infected cats.

Large unilamellar vesicles as trehalose-stabilised vehicles for vaccines: storage time and in vivo studies.

Liposomes, as a pharmaceutical formulation must display a long shelf life. The recombinant heat-shock protein from Mycobacterium leprae (18-kDa hsp) or its N-acylated derivative, when entrapped within or externally associated with large unilamellar vesicles, acts as a T-epitope source. Freeze-fracture electron microscopy shows unequivocally that trehalose avoids aggregation and fusion of these vesicles. Formulations containing trehalose retained up to 98% of the entrapped protein. The highest antibody level is obtained with formulations containing trehalose. The adjuvant effect depends on the liposomal membrane integrity.

Helicobacter pylori gastritis in cats with long-term natural infection as a model of human disease.

A natural infection with Helicobacter pylori (H. pylori) in domestic cats (Felis cattus) less than 2 years of age has been well described in a closed colony of animals. Six cats from this colony that were serially evaluated by culture, polymerase chain reaction, and light and electron microscopy for a period of 3 years demonstrated persistent gastric colonization with a single cag(-) vac(+) strain of H. pylori. In these cats, as well as five other 5- to 6-year-old cats that were examined, a long-term infection resulted in chronic diffuse lymphofollicular atrophic gastritis with areas of mucosal dysplasia in the antrum and predominantly midsuperficial gastritis in the body and cardia. Topographically, the distribution of lesions was similar in both young and older cats and closely resembled that found in humans, with the most severe changes occurring in the gastric antrum. Few granulocytes and no significant elevation in mast cells were seen in older H. pylori-infected cats compared with uninfected controls; however, marked increases in interepithelial globule leukocytes and numerous active mucosal lymphoid follicles were present in infected animals. Indices of gastritis were significantly greater in older infected cats when compared with uninfected controls and younger cats (P < 0.05). The antral cell proliferation index of infected older cats was significantly (P = 0.021) greater than that of uninfected controls. Apoptotic indices of the gastric antrum and body of infected cats were significantly (P = 0.01) increased versus controls. Chronic infection with H. pylori in cats shares many features of long-term H. pylori infection in humans, including the development of preneoplastic processes. This similarity provides useful, comparative insights into host-pathogen interactions.

Large unilamellar vesicles as trehalose-stabilised vehicles forvaccines storage time and in vivo studies

Liposomes, as a pharmaceutical formulation must display a long shelf life. The recombinant heat-shock protein fromMycobacterium leprae (18-kDa hsp) or its N-acylated derivative, when entrapped within or externally associated with largeunilamellar vesicles, acts as a T-epitope source. Freeze-fracture electron microscopy shows unequivocally that trehaloseavoids aggregation and fusion of these vesicles. Formulations containing trehalose retained up to 98% of the entrappedprotein. The highest antibody level is obtained with formulations containing trehalose. The adjuvant effect depends on theliposomal membrane integrity.

Spontaneous leprosy in a wild-caught cynomolgus macaque.

Naturally occurring Mycobacterium leprae has been previously documented in only two species of nonhuman primates from West Africa--the chimpanzee and the sooty mangabey. We report here the first known case of spontaneous leprosy in an Asian macaque. A wild-caught cynomolgus macaque imported from The Philippines developed a reaction to a tuberculin skin test after 3 years at the California Regional Primate Research Center (CRPRC), University of California-Davis, Davis, California, U.S.A. Biopsies of concurrent skin lesions suggested a cutaneous mycobacterial infection. Diagnosis of the infection was obtained by a polymerase chain reaction (PCR) assay specific for M. leprae. Clinical presentation, histopathological findings, and ELISA serology for M. leprae-specific PGL-I and to the LAM mycobacterial antigens were consistent with those of human borderline (BB) leprosy. Longitudinal serologic data suggest that the cynomolgus macaque had subclinical leprosy at the time of arrival in the CRPRC quarantine. Intradermal tuberculin testing is the traditional method for screening nonhuman primate populations for mycobacterial infections. Exposure to nontuberculous mycobacteria, such as M. leprae, amy sensitize some individual primates to nonspecific mycobacterial antigens, resulting in false-positive tuberculin reactions. Susceptibility of the cynomolgus macaque and other nonhuman primates to M. leprae should be re-evaluated. Cynomolgus macaques and, possibly, other nonhuman primates may serve as valuable experimental models of leprosy in humans.

Distribution of technetium 99m-labeled red blood cells during isoflurane anesthesia in ferrets.

OBJECTIVE: To address the physiologic mechanism of isoflurane-associated reduction in hematologic variables in ferrets. ANIMALS: 6 young adult female ferrets. PROCEDURE: Distribution of 99mTc-labeled autologous erythrocytes was measured by serial in vivo imaging. Data were recorded in 4 ferrets, using a gamma camera, immediately prior to anesthesia, 15 minutes after 2% isoflurane anesthesia in O2 via endotracheal tube, 1 minute prior to and throughout a 10-minute phenylephrine infusion, 20 and 40 minutes after termination of the phenylephrine infusion, and 45 minutes after termination of anesthesia. Blood indices were also measured at times that paralleled those for imaging. One ferret served as a conscious control (no anesthetic administration), and another as an isoflurane control (no phenylephrine administration). RESULTS: In ferrets under anesthesia, splenic radioactivity increased from baseline of 10.2 +/- 2.0% to 38.4 +/- 3.2% (mean +/- SEM; P < 0.05) of the injected dose. Splenic radioactivity decreased to 13.4 +/- 3.8% of the injected dose during phenylephrine infusion and to near baseline for the recovery image. Splenic radioactivity in the conscious control remained constant throughout the study, whereas that of the anesthetized control was persistently increased throughout administration of isoflurane. Percentage reduction of the 15-minute sample values, compared with baseline values for all hematologic indices, was: RBC count, 33% (P < 0.05); hemoglobin concentration, 34% (P < 0.05); hematocrit, 35% (P < 0.05); and plasma protein concentration, 20% (P < 0.05). All RBC variables returned to within 7 to 14% of baseline by 45 minutes after termination of anesthesia. CONCLUSION: Isoflurane anesthesia causes splenic sequestration of RBC in ferrets that is partially reversed by phenylephrine infusion or termination of anesthesia. Thus, investigators and clinicians should be cautious when interpreting hematologic findings in isoflurane-anesthetized ferrets, and accordingly, fluid treatment and transfusion should be planned.

Effect of isoflurane on hematologic variables in ferrets.

Effects of isoflurane on the CBC in ferrets were studied. There was rapid decrease in all hematologic variables after induction of anesthesia. Percentage reductions in indices of the erythron (hematocrit, RBC count, hemoglobin concentration) exceeded those of plasma protein concentration and WBC count at the first postinduction time point. There was little additional decrease in these variables for the duration of anesthesia. The values had partially recovered to preanesthetic baseline at 45 minutes after anesthesia. Although these alterations appear to be well tolerated in healthy ferrets, care should be exercised when subjecting anemic, geriatric, or debilitated ferrets to isoflurane-induced anesthesia.

A technique for catheterization of the urinary bladder in the ferret.

The technique of catheterization of the urinary bladder, an important clinical skill for the diagnosis of urinary tract disorders, has not been described for the ferret. The bladder was catheterized in 23 ferrets (10 intact females; 11 spayed females; and 2 intact males) using a 3 1/2 French, red rubber urethral catheter fitted with a steel wire stylet. Ferrets were anaesthetized with isoflurane or ketamine (30 mg/kg IM) and xylazine (3 mg/kg IM). Females were positioned in ventral recumbency with the rear quarters elevated by a rolled surgical towel. The urethra was catheterized by direct visualization of the external urethral orifice using a vaginal speculum. The orifice was approximately 1 cm cranial to the clitoral fossa on the ventral floor of the vestibule. Blind passage was used in several spayed females. In males, the distal end of the penis was exteriorized from the prepuce and the external urethral orifice cannulated without stylet. No difficulty was encountered in advancing the catheter past the os penis. This catheterization technique allows urinary tract access for urine collection, pneumocystography, contrast cystography, double contrast cystography, and urine output determination in pharmacologic studies or in critical care of debilitated animals.

Estimation of glomerular filtration rate and evaluation of renal function in ferrets (Mustela putorius furo).

Three methods of determining glomerular filtration rate (GFR) were performed in adult ferrets, 9 months to 7 years old. Endogenous creatinine clearance was determined, using serum and urine creatinine values obtained during 24- and 48-hour collection periods from 27 ferrets housed in metabolic cages. Creatinine and radiolabeled inulin were administered to 12 female ferrets by constant IV infusion during isoflurane-induced anesthesia. Serial 20-minute urine collections, together with serum samples obtained at the midpoint of urine collection, provided measures for clearance calculations of these substances. Mean +/- SD endogenous creatinine clearance in ferrets for metabolic cage collections was 2.50 +/- 0.93 ml/min/kg of body weight. There were no significant differences between the 24- and 48-hour clearance rates. Mean inulin clearance was 3.02 +/- 1.78, and mean exogenous creatinine clearance was 3.32 +/- 2.16 ml/min/kg. Analysis of variance, using least-squared means adjustment, did not yield any significant differences between inulin and exogenous creatinine clearance rates. Exogenous creatinine clearance-to-inulin clearance ratio was 0.99 +/- 0.46, and there was significant correlation between the 2 methods (r = 0.82, P = 0.0001). Significant body temperature effects on inulin or exogenous creatinine clearance were not found. Infused inulin clearance, the generally preferred method for GFR calculation in mammalian species, was significantly (P = 0.0069) higher in younger (3.65 ml/min/kg) vs older ferrets (2.29 ml/min/kg). Results of this study indicate that inulin clearance is an adequate measure of GFR in ferrets as it is in other species. Compared with inulin clearance, exogenous creatinine clearance also provides a reliable estimate of GFR in ferrets.

Isolation and characterization of a thrombin-like enzyme from the venom of Crotalus durissus terrificus.

A thrombin-like enzyme was isolated in 6% yield from the venom of Crotalus durissus terrificus by ammonium sulfate precipitation followed by gel filtration on Sephadex G-75 and finally affinity chromatography on Sepharose-1,4-butanediol-diglycyl-p-aminobenzamide eluted with 0.15 M benzamidine. The enzyme behaved like a single component on SDS-PAGE corresponding to a molecular weight of 34 kDa. The specific activity of the enzyme toward bovine fibrinogen was 71 NIH U/mg protein. The pH optimum for the coagulation of human fibrinogen was 8.0. The enzyme hydrolyzes the alpha-chain of fibrinogen, has amidase activity on L-arginine-p-nitroanilide and L-arginine-7-amido-4-methyl-coumarin amino terminal blocked peptides and presents esterolytic activity on N-alpha-tosyl-L-arginine-methylester.