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.

Infections transmitted by large and small laboratory animals.

Although zoonotic spread of infectious disease continues to occur in laboratory animals used in biomedical research, reported outbreaks have been minimized with the advent of rigorous veterinary and husbandry procedures, the use of commercially reared animals, and the institution of appropriate personnel health programs. Maintaining animals in modern facilities with appropriate safeguards against introduction of vermin and biologic vectors is also important in preventing zoonotic disease in personnel. Nevertheless, established zoonotic agents, newly discovered microorganisms, or new animal species not previously recognized as carriers of zoonotic microorganisms are encountered, and the potential for spread of infectious disease from animals to humans still exists. Active dialogue between veterinarians and physicians regarding the potential of zoonotic disease, the species of animals that are involved, and the methods of diagnosis, is an indispensable component of a successful preventive health program involving personnel who deal with laboratory animals.

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.

Evaluation of isolator caging systems for protection of mice against challenge with mouse hepatitis virus.

Two isolator caging systems were evaluated against challenge with MHV-Y, an enterotropic strain of mouse hepatitis virus. The systems were similar in that they both used an identical shoebox cage equipped with a polycarbonate filter top incorporating a Reemay filter. They differed in that one system supplied HEPA-filtered air through a grommet in the filter lid so that the cage was pressurized slightly. A rack holding 60 cages (30 front and back) was utilized. Thirty cages without filter tops housed one mouse each that had been infected orally with 19,000 ID50 of MHV-Y and an uninfected cagemate. The remaining 30 cages, each housing 2 uninfected mice were divided into 3 groups of 10 cages. Group I cages (controls) had no filter top; Group II cages were equipped with filter tops; and Group III were equipped with filter tops and intracage HEPA-filtered air. The cages housing uninfected mice were interspersed between, above, below and behind cages housing infected mice. The uninfected mice were maintained in contact with the MHV-Y infected mice for 8 weeks. Transmission of MHV-Y was determined serologically by indirect ELISA. All mice housed within the Group I cages (control) seroconverted to MHV, while only 4 mice (2 cages) seroconverted in Group II, and no mice seroconverted in Group III.

The effects of intracage ventilation on microenvironmental conditions in filter-top cages.

Filter-top cages, while effective in reducing cross contamination by particulate material including microbes, can also cause accumulation of the waste gases carbon dioxide and ammonia as well as increased intracage relative humidity. A prototype system which provided each cage with 23 air changes per hour through a nozzle inserted in the filter lid was evaluated. The ventilated cageing system was effective in reducing intracage carbon dioxide, ammonia and relative humidity levels. Mean weekly carbon dioxide levels were 2900 ppm lower, ammonia levels 240 ppm lower and intracage relative humidity levels 8% lower in the ventilated cages than in unventilated controls.

Utilization of cholestyramine resin as a preventive treatment for antibiotic (clindamycin) induced enterotoxaemia in the rabbit.

Cholestyramine, an ion exchange resin shown to bind bacterial toxins, was utilized to treat rabbits with antibiotic induced enterotoxaemia. Three groups of 6 rabbits were administered 30 mg/kg clindamycin phosphate intravenously on day 1. One group was untreated; 2 groups were treated daily by gavage with 2 g cholestyramine in 20 ml water until day 21, starting on either day 1 or 3. Daily body weights, faecal output, faecal occult blood, food and water consumption, and body temperatures were determined. Four of 6 rabbits in the untreated group either died or were moribund and euthanased. There were no deaths in either treatment groups. Dramatic decreases in food consumption (86%), water consumption (62%), and faecal output (89%) were noted within 3 days after clindamycin administration in all groups. These parameters remained depressed throughout the study. There was no clear trend in body weight changes, body temperature, or faecal occult blood test results. Cholestyramine was effective in eliminating mortality associated with the intravenous administration of clindamycin and is recommended to prevent the development of enterotoxaemia when pyrogen testing or administering antibiotics known to induce the syndrome in rabbits.

Probable transmission of Chlamydia psittaci from a macaw to a cat.

A 5-year-old Siamese cat developed unilateral mucopurulent ocular discharge and conjunctivitis 1 month after the introduction of a macaw into the household. Despite treatment with antimicrobial ophthalmic ointment, the conjunctivitis became bilateral and other systemic signs developed. Intracellular inclusions consistent with a Chlamydia psittaci infection were detected in conjunctival epithelial cells stained with a fluorescein-labeled monoclonal antibody. Chlamydia psittaci was isolated from samples obtained by conjunctival scraping. Subsequently, C psittaci was recovered in samples obtained from the feces of the bird. The cat and the bird were successfully treated with doxycycline. Historic and epidemiologic findings supported the theory of orthozoonotic transmission of C psittaci from the bird to the cat.

Estradiol-17 beta-secreting adrenocortical tumor in a ferret.

Severe generalized alopecia and marked vulvar enlargement were observed in a 5-year-old spayed ferret with high serum estradiol concentrations. A neoplastic left adrenal gland was removed. Staining of the neoplastic cells for estradiol was demonstrated by use of immunohistochemistry. Clinical findings in this ferret were typical of adrenal-associated endocrinopathy, a syndrome characterized by increased secretion of adrenocortical hormones by hyperplastic or neoplastic adrenal glands.

A comparison of rodent caging systems based on microenvironmental parameters.

Four different mouse caging systems were evaluated for microenvironmental temperature, carbon dioxide, relative humidity (RH) and ammonia levels during a 7-day testing period. All caging systems evaluated had polycarbonate bases and consisted of either a molded polyester (MP) filter lid, one of two different polycarbonate filter lids, or no filter lid which served as a control. At 50% macroenvironmental RH (study I), all systems maintained an intracage temperature of 75.5 degrees F +/- 0.5 degrees. Both polycarbonate systems averaged greater than 2200 ppm of carbon dioxide more than the MP system and the controls. When compared with RH in the control cages, RH levels averaged over 20% and 5 to 8% RH greater in the polycarbonate filter lid systems and the MP system, respectively. There were no appreciable ammonia levels in either the MP or control systems. In the polycarbonate filter lid systems, ammonia levels were detectable on day 4 and were greater than 200 ppm by day 6. At 20% macroenvironmental RH (study II), there was a proportional 15 to 30% RH decrease from study I levels. Ammonia levels were undetectable in any system until day 7 and averaged only 17 ppm in one of the polycarbonate systems. Minimal differences were observed in studies III, IV and V when pine shavings were used instead of hardwood chips, a CD-1 stock instead of a DBA/2J strain, and different grades of filter inserts in the polycarbonate systems, respectively.

A comparison of ketamine/xylazine and ketamine/xylazine/acepromazine anesthesia in the rabbit.

Parenteral anesthetic combinations such as ketamine and xylazine have become the agents of choice for anesthesia in the rabbit, because they are effective, easily administered and inexpensive. A number of recent reports have recommended including acepromazine in this combination, but a critical evaluation of this combination in the rabbit has not been reported. Five adult New Zealand white rabbits were anesthetized intramuscularly with ketamine (35 mg/kg) and xylazine (5 mg/kg) with or without acepromazine (0.75 mg/kg). The study was conducted in a double blind fashion, where each rabbit was administered both combinations at a minimum of 7 day intervals. Physiologic parameters were evaluated including heart rate, respiratory rate, central arterial blood pressure, pedal, palpebral and postural reflex activity. The duration of general anesthesia, estimated by the time elapsed between the loss and return of the palpebral reflex, was greater (means = 99 +/- 20 minutes) when acepromazine was employed in the combination compared to (means = 77 +/- 5 minutes) when ketamine/xylazine were used alone. Mean central arterial blood pressure reached a lower level when acepromazine was utilized (means = 46 +/- 8 mm/Hg) than when it was not (means = 57 +/- 12 mm/Hg.). The addition of acepromazine in a ketamine/xylazine combination resulted in a 28% longer period of anesthesia, a 19% lower mean central arterial blood pressure and a 32% longer recovery of postural reflexes. The ketamine/xylazine/acepromazine combination is a useful regimen for normovolemic animals when anesthetic duration greater than that produced by ketamine/xylazine alone is required.

Reversal of ketamine/xylazine anesthesia in the rabbit with yohimbine.

Ketamine and xylazine used in combination have been shown to be effective, easily administered, cost efficient agents for surgical anesthesia in the rabbit. The effect of xylazine on the central nervous system has been shown to be mediated through alpha-2 adrenergic receptors. Yohimbine, an alpha-2 adrenergic antagonist has been shown to reverse xylazine induced depression and partially antagonize ketamine in other species. We evaluated the antagonistic effect of yohimbine on ketamine/xylazine anesthesia in the rabbit. Six New Zealand White rabbits were anesthetized with intramuscular ketamine (50 mg/kg) and xylazine (10 mg/kg) to establish baseline parameters including respiratory rate, heart rate, and palpebral, pedal and postural reflex activity. Fourteen days later each rabbit was subjected to the same anesthetic regimen followed 30 minutes later by the intravenous administration of yohimbine (0.2 mg/kg). The duration of anesthesia estimated by the time elapsed between the loss and return of the palpebral reflex was reduced in the yohimbine treated trial (means = 29.7 +/- 1.9 minutes) compared to the control trial (means = 67.0 +/- 13.5 minutes). The palpebral reflex returned within 5 minutes following yohimbine treatment. Our results indicated that yohimbine is an effective antagonist of ketamine/xylazine anesthesia in the rabbit. Yohimbine decreases anesthetic duration after intravenous administration and also may aid in the control of undesirable anesthetic effects and overdosage.

Rederivation of MHV and MEV antibody positive mice by cross-fostering and use of the microisolator caging system.

This study established the feasibility of rederiving numerous mouse hepatitis virus (MHV) and mouse encephalomyelitis virus (MEV) antibody positive strains of mice using cross fostering techniques and a new caging system, thus permitting introduction of virus antibody free mice into a barrier facility. Serologic status of dams within the nucleus breeding colony was determined, and all mice within the breeding colony were housed in individual Microisolator cages. Specific pathogen free (SPF) foster mothers purchased from a commercial source were determined to have no detectable serum antibody to 11 murine viruses including MEV and MHV. Pups delivered naturally from time pregnant dams were cross fostered onto the SPF foster dams. The procedure of cross fostering was conducted within a positive flow, HEPA-filtered, mass air displacement unit within 24 hours of parturition. The virus status of pups from 49 litters was monitored serologically at weaning and again at 6 weeks of age. All cross fostered litters were serologically negative for antibody to mouse hepatitis virus. Seven of 29 litters were negative for MEV antibody titer using this cross fostering technique. Those litters negative serologically to both MHV and MEV (at 3 and 6 weeks) were transferred to a barrier facility and held in isolation. All rederived mice transferred to the barrier facility remained negative for MHV and MEV when sampled at 12 weeks of age.