Mousepox resulting from use of ectromelia virus-contaminated, imported mouse serum.

Mousepox was identified in a single mouse-holding room in early 1999 after a group of 20 CAF1/Hsd mice were inoculated SC with a killed murine spindle cell tumor line, S1509A. The cell line had been used without complications multiple times and was determined to be free of viral contamination on the basis of results of mouse antibody production testing. Of the 20 mice inoculated, 12 mice died by postinoculation day 8. Severe lymphoid and hepatic necrosis was observed in select mice subjected to histologic examination. Ballooning degeneration of epithelial cells with intracytoplasmic eosinophilic inclusion bodies was observed in the skin overlying the inoculation site of the single mouse from which this tissue site was evaluated. Presence of ectromelia virus was confirmed by use of immunohistochemical and polymerase chain reaction analyses, and the virus was isolated after serum, pooled from 5 of the index cases, was inoculated into an immune-naive mouse. Investigation into the source of virus contamination included inoculating mice with aliquots of various S1509A freeze dates; chemically defined media and supplements, including fetal bovine serum; and two lots of pooled commercial mouse sera, after heat inactivation at 56 degrees C for 30 minutes used as a medium supplement. One lot of pooled commercial mouse serum was identified as the source of ectromelia virus. This lot of serum was inadvertently used to feed S1509A cells that were subsequently inoculated into mice. We determined that the contaminated serum, which was purchased in late 1998, originated from China. The serum was imported into the United States as a batch of 43 L in early 1995. The serum was blended into a single lot and filtered (0.2 microm) before distribution to major suppliers throughout the country. The serum was sold or further processed to obtain a variety of serum-derived products. Because murine serum is generally sold in small aliquots (10 to 50 ml), we speculate that several thousand aliquots may have been derived from this batch of serum and, if inoculated into mice, would likely result in additional mousepox outbreaks.

Monoclonal antibody production in murine ascites. I. Clinical and pathologic features.

BACKGROUND AND PURPOSE: Murine ascites production has been associated with appreciable morbidity and mortality, thus raising animal-welfare concerns. To address these concerns, the clinicopathologic changes associated with in vivo production of monoclonal antibodies in mice were characterized, and results were compared among cell lines. METHODS: Five hybridoma cell lines were grown in groups of 20 mice. Fourteen days prior to inoculation with 10(6) hybridoma cells, mice were primed with 0.5 ml of pristane given intraperitoneally; 12 mice were sham treated (controls). Ascites fluid was collected a maximum of three times by abdominal paracentesis. Clinical observations and pre- and postabdominal tap body weights were recorded. Necropsies were performed on all mice. RESULTS: For all groups combined, overall survival to tap 1 was 98%, to tap 2 was 96%, and to tap 3 was 79%; survival among groups ranged from 90 to 100% for tap 1, 85 to 100% for tap 2, and 35 to 100% for tap 3. Disseminated intra-abdominal seeding with irregular soft tissue and/or solid tumor masses was observed at necropsy. CONCLUSIONS: Significant clinicopathologic changes were associated with monoclonal antibody production in mice, and differences between various hybridoma cell lines were apparent.

Monoclonal antibody production in murine ascites. II. Production characteristics.

OBJECTIVE: To characterize monoclonal antibody production parameters of five hybridoma cell lines in murine ascites for correlation with clinicopathologic changes in mice. METHODS: Five hybridoma cell lines were grown in groups of 20 mice. Fourteen days prior to inoculation with 10(6) hybridoma cells, mice were primed with 0.5 ml of pristane given intraperitoneally. Ascites fluid was collected a maximum of three times by abdominal paracentesis; volume was measured and antibody concentration was determined by ELISA for each sample. RESULTS: Trends differed among cell lines when comparing ascites volumes and antibody concentrations over time from the first to the third tap. Antibody production was greatest at tap 1 for Groups 2B11 and 2C6D9; tap 2 for Group 3C9; and tap 3 for Groups RMK and 3D6. Total antibody production ranged from 422.90 to 996.64 mg; total ascites fluid volume ranged from 74.2 to 115.7 ml; and mean antibody concentration for taps 1, 2, and 3 ranged from 2.50 to 15.03 mg/ml among cell lines. CONCLUSION: Production characteristics were significantly different among hybridoma cell lines. Determination of production characteristics of hybridomas and correlation with clinicopathologic changes in mice may be valuable in making recommendations for managing mice with ascites.

Familial megacecum and colon in the rat: a new model of gastrointestinal neuromuscular dysfunction.

Gastrointestinal motility disorders are of considerable clinical importance in humans and animals. Abnormalities of smooth muscle and the enteric nervous system have been described. We have identified and characterized a new mutant stock of rats that develops severe megacecum and colon with pseudo-obstruction, Familial Megacecum and Colon (FMC). The inheritance pattern of FMC was characterized by selective breeding. Gastrointestinal motility was evaluated radiographically. Complete pathologic evaluations, including ultrastructural examination and staining of colonic segments for acetylcholinesterase, peripherin, vasoactive intestinal peptide, substance P, nitric oxide synthase, and somatostatin, were performed. Spontaneous contractility and contractile force in isolated colonic muscle strips were examined. Familial megacecum and colon is inherited as an autosomal recessive trait. The markedly dilated cecum and proximal portion of the colon are followed by a short, funnel-shaped segment and distal portion of the colon with normal or slightly reduced lumen. Although clinical features and gross anatomic changes of the colon resemble those of Hirschsprung's disease in humans and animals, aganglionosis is not a feature of FMC. An increase in somatostatin staining was observed in dilated regions of bowel. The spontaneous contractile frequency and contractile force were diminished in the affected colon. Familial megacecum and colon is a new mutant, distinct from previously described hereditary and targeted mutant rodent models that develop megacecum and colon as a result of distal colonic dysfunction. The functional or morphologic defect(s) that result in colonic dysfunction in rats with FMC was not determined. The disease may result from an absence or overexpression of a single or group of neurotransmitters or their respective neurons, receptor abnormalities, or defects in the intestinal pacemaker system.

Intestinal plasmacytosis in rabbits: a histologic and ultrastructural study.

A retrospective study revealed intestinal plasmacytosis in 53 of 102 rabbits used in various experimental studies and as controls. The breeds affected included New Zealand white (n = 46), Dutch-belted (n = 6), and Watanabe (n = 1) rabbits. Sex predisposition was not found in any breed. The mean (+/-SD) ages were 3.1 +/- 1.4 years for New Zealand white rabbits, 1.3 +/- 1.1 for Dutch-belted rabbits, and 2 years for the Watanabe rabbit. The severity increased with animal age. The incidence was higher (P < 0.05) in rabbits used in antibody production and cholesterol studies. The lesions were characterized by multifocal to diffuse infiltration of well-differentiated plasma cells in the intestinal mucosa. Electron microscopic examination revealed typical plasma cell morphology of the infiltrating cells. Small intestine and cecum were the major sites affected. In severe cases, colon, rectum, trachea, esophagus, mesenteric lymph node, and spleen were also involved.

Cystic urogenital anomalies in ferrets (Mustela putorius furo).

Single or multiple semispherical to bilobulated fluid-filled cystic structures of variable size were observed on the dorsal aspects of the urinary bladder of four male and two female ferrets (Mustela putorius furo). All ferrets had been neutered. On physical examination, the cysts were palpated as caudal abdominal masses. Three of the six ferrets presented with dysuria, and two ferrets had signs compatible with endocrine dysfunction. Adrenal cortical hyperplasia or neoplasia were observed in all of the five ferrets examined. Sex hormones assayed in one of the six ferrets revealed elevated levels of serum estrodiol. The posterior aspect of the cysts was located on and/or attached to the trigone or neck of the bladder, with variable intraluminal communication with the bladder and/or the urethra. The anterior aspect of the cysts projected dorsally or dorsocranially into the caudal abdomen. The cysts were thin walled and contained urinelike fluid (n = 5) or viscous yellow fluid (n = 1). Histologically, the cyst walls were composed of three layers, epithelium, muscle, and serosa, with fibrovascular stroma between layers. The epithelium consisted of simple to stratified transitional, columnar, or squamous epithelial cells. The muscular layer consisted of intermittent bundles and/or single to double layers of continuous to discontinuous smooth muscle. The serosal layer consisted of loose fibrous stroma covered by flattened mesothelial cells. The cystic anomalies in these ferrets were most likely derived from the urogenital glands/ducts or other remnants.

Evaluation of hollow fiber bioreactors as an alternative to murine ascites production for small scale monoclonal antibody production.

The objective of this study was to compare monoclonal antibody production in hollow fiber bioreactor systems and murine ascites to determine the feasibility of the bioreactor system as a potential alternative to the use of mice. Three hybridoma cell lines were grown in each of three different hollow fiber bioreactor systems and in groups of 20 mice. Mice were primed with 0.5 ml pristane intraperitoneally 14 days prior to inoculation of 1X10(6) hybridoma cells. Each mouse was tapped a maximum of three times for collection of ascites. Ascites volumes and daily clinical observations were recorded. Bioreactors were harvested three times weekly for 65 day and were monitored by cell counts, cell viability and media glucose consumption. Time and materials logs were maintained. The total quantity of monoclonal antibody produced in 20 mice versus the mean production for the three different bioreactors in 65 days was as follows: cell line 2B11, 455 mg vs. 168 mg; cell line 3C9, 446 mg vs. 565 mg; and cell line RMK, 997 mg vs. 1023 mg. Mean monoclonal antibody concentration ranged from 4.07 to 8.37 mg/ml in murine ascites, and from 0.71 to 11.10 mg/ml in hollow fiber bioreactor system. Although time and material costs were generally greater for the bioreactors, these results suggest that hollow fiber bioreactor system merit further investigations as potentially viable in vitro alternatives to the use of mice for small scale (< 1 g) monoclonal antibody production.

Detection of Clostridium difficile toxins from the small intestine and cecum of rabbits with naturally acquired enterotoxemia.

Four specific-pathogen-free rabbits with anorexia died peracutely; decreased fecal output, nasal exudate, and labored breathing were the only other clinical abnormalities observed in two of the rabbits before death. The animals, three juveniles and one adult, were on a standard polyclonal antibody production regimen and had received immunizations approximately 2 weeks before presentation. External examination revealed distended abdomen and perineal fecal staining. At necropsy the small intestine was distended with fluid, and the cecum was distended with chyme. The small intestines and cecum had marked serosal hyperemia. Anaerobic bacterial culture techniques were used to isolate Clostridium difficile from the small intestine (3/4) and cecum (2/4). In all cases C. difficile toxin B was detected at high titers (10(2) to > 10(5)) in the small intestine by cytotoxicity assay with HeLa 229 cell culture. In two of the four rabbits C. difficile was isolated, and cytotoxin titers were detected at 10(1) and 10(4) in the cecum of affected rabbits. Toxin B was neutralized with C. sordellii antiserum but not C. spiroforme antiserum. In addition, toxin A was detected in each of the cytotoxin B-positive samples by a commercial toxin A enzyme immunosorbent assay. In vitro production of toxins A and B was detected from each culture isolate after incubation in chopped meat broth. These cases are noteworthy because spontaneous (nonantibiotic-associated) C. difficile enterotoxemia has not been previously reported in rabbits. Also the toxins of clostridial organisms are usually documented in the cecum, not the small intestine, of rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)