Microbiological monitoring in individually ventilated cage systems

Housing rodents in IVC racks has many advantages over conventional cages but also presents unique challenges related to health monitoring. The authors review the issues to consider in design of a sentinel program using IVC systems.     

Effects of housing density on nasal pathology of breeding mice housed in individually ventilated cages

The 2011 edition of the Guide for the Care and Use of Laboratory Animals includes new recommendations for the amount of floor space that should be provided to breeding mice. When pairs or trios of continuously breeding mice are housed in shoebox cages, they may have less than this recommended amount of floor space. High housing densities may adversely affect animal health, for example, by compromising air quality inside the cage. Hence, some institutions are carefully reevaluating the microenvironments of breeding cages. The use of individually ventilated cages (IVCs) to house research mice allows for greater control over the quality of the cage microenvironment. The authors evaluated the microenvironments of shoebox cages in an IVC rack system housing breeding and non-breeding Swiss Webster mice. Ammonia concentrations were significantly higher in cages housing breeding trios with two litters. Histopathologic lesions attributable to inhaled irritants such as ammonia were found in mice housed in breeding pairs and trios. The authors conclude that the microenvironments of cages in an IVC rack system housing breeding pairs and trios may be detrimental to animal health.     

Transient hypertension and sustained tachycardia in mice housed individually in metabolism cages

The novel environment of a metabolic cage can be stressful for rodents, but few studies have attempted to quantify this stress-response. Therefore, we determined the effects on mean arterial pressure (MAP) and heart rate (HR), of placing mice of both sexes in metabolism cages for 2 days. After surgical implantation of a carotid artery catheter mice recovered individually in standard cages for 5 days. Mice then spent 2 days in metabolism cages. MAP and HR were monitored in the standard cage on Day 5 and in metabolism cages on Days 6-7. MAP increased by 18+/-3 and 22+/-4 %, while HR increased by 27+/-4 and 27+/-6 %, in males and females, respectively, during the first hours after cage switch. MAP decreased to baseline in the fourth and eighth h following metabolism cage switch in males and females, respectively. However, HR remained significantly elevated in both sexes during the entire two-day period in metabolism cages. Females had lower MAP than males both pre- and post-metabolism cage switch, but there were no sex differences in HR. These results demonstrate sustained changes in cardiovascular function when mice are housed in metabolism cages, which could potentially affect renal function.     

Evaluation of individually ventilated cage systems for laboratory rodents: occupational health aspects

New ventilated caging systems for laboratory animals were compared with conventional caging regarding allergen distribution, ergonomic suitability, cage environment and animal welfare. This paper presents occupational health evaluations. Mice were placed in individually ventilated cage (IVC) systems, a ventilated cabinet, and in cages on open shelves (conventional husbandry). The IVC systems were studied at negative and positive airflow. Aeroallergens were sampled on filters (n = 204, including controls) in undisturbed rooms and during cage changing. Concentrations of mouse urinary allergen (Mus m 1) in filter eluates were measured using sandwich ELISA. An ergonomic evaluation was performed with measurement of traction forces. Staff exposure during cage changing was high in all systems, range 116-4430 ng Mus m 1/m3. In undisturbed animal rooms, allergen levels were orders of magnitude higher when using conventional caging compared with ventilated systems; P < 0.001. At positive pressure both IVCs leaked allergen (median Mus m 1 concentration was < 0.08 ng/m3 at negative, but 6.5 ng/m3 (IVC1) and 0.8 ng/m3 (IVC2S) at positive pressure). The IVC systems had ergonomic disadvantages compared with the conventional husbandry and the ventilated cabinet, for instance with cages in unsuitable working heights. Ventilated husbandry solutions reduce levels of airborne allergen substantially at negative pressure, but are ergonomically less suitable. To prevent allergen exposure during cage changing, we propose that this procedure should be performed under ventilated conditions. Producers and users must cooperate in optimizing animal caging systems for both animals and staff.     

Evaluation of individually ventilated cage systems for laboratory rodents: cage environment and animal health aspects

The use of individually ventilated cage (IVC) systems has become an attractive housing regime of laboratory rodents. The benefits of IVC systems are, reportedly, a high degree of containment combined with relative ease of handling, and a high degree of protection from allergenes. In the present study we tested whether two IVC systems (BioZone VentiRack, IVC1 and Techniplast SealSafe, IVC2S), in which we held mature male NMRI mice, were constructed to maintain a constant differential pressure, positive or negative, during a prolonged period of time. We also measured ammonia (NH3) concentrations after about 2 weeks of use, and CO2 build-up during a 60 min simulated power failure situation. In addition, animal weight development and bite-wound frequency were recorded (Renstr?m et al. 2000). From the present study it is concluded that the IVC1 air handling system provides a more uniform and balanced differential pressure than the IVC2S. Both systems effectively scavenge NH3 when bedding material is not soaked by urine. Although the IVCs are dependent on the continual function of the fans to work properly, it seems unlikely that CO2 concentrations increase to hazardous levels, as a result of a one hour power failure, with the type of cages used in this study. Differences in weight development and bite-wound occurrence were noted between the two IVC systems. Causes for these differences could not be established and need more investigation.     

Higher heart rate of laboratory mice housed individually vs in pairs

Many studies have shown that housing mice individually over a long period significantly alters their physiology, but in most cases measurement has required human interference and restraint for sampling. Using a radio-telemetry system with implantable transmitters, we recorded heart rate (HR), motor activity (ACT) and body temperature (BT) of freely moving male mice (NMRI) housed either individually or in pairs with an ovarectomized female. Data for each parameter were collected at 5 min intervals for two consecutive 24 h periods. Even after several weeks of habituation to the social conditions, HR was increased in mice housed individually compared with mice housed in pairs, although their measured ACT did not differ. Additionally, BT tended to be reduced in individually-housed mice. When the data were analysed according to different ACT levels, HR was increased in individually-housed mice during phases of low and high, but not intermediate, motor activity. Furthermore, individually-housed mice had more, but shorter, resting bouts, indicating disruption of the normal circadian sleep pattern. Enhanced HR in individually-housed mice does not necessarily indicate stress, but might be an important physiological indicator of discomfort. The fact that individual housing alters basic physiological parameters in laboratory mice highlights the need to control for housing-dependent variation, especially in experiments that are sensitive to changes in these parameters.     

The effects of different types of individually ventilated caging systems on growing male mice

Ventilation rate and turnover rate of dry air vary among different types of ventilation systems used with individually ventilated cages (IVCs) and can affect the well-being of rodents housed in these cages. The authors compared the effects of two types of IVC systems, forced-air IVCs and motor-free IVCs, on 4-week-old C57Bl/6J male mice. The mice were acclimatized to the cages for 8 d and then monitored for 87 d. Their body weights, food and water consumption and preferred resting areas were recorded. Mice that were housed in motor-free IVCs had a significantly greater increase in body weight than those housed in forced-air IVCs, despite having similar food consumption. Mice in forced-air IVCs had greater water consumption than mice in motor-free IVCs. In addition, mice in forced-air IVCs were more frequently located in the front halves of their cages, whereas mice in motor-free IVCs were located with similar frequency in the front and back halves of their cages, perhaps because of the higher ventilation rate or the location of the air inlets and outlets in the rear of the cage. These results suggest that body weight, food and water consumption and intracage location of growing male mice are influenced by the type of ventilation system used in the cages in which the mice are housed.     

Effects of an individually ventilated cage system on the airway integrity of rats (Rattus norvegicus) in a laboratory in Brazil

The ventilation method used in the management of laboratory rats is important in maintaining their health. Rats kept under general diluting ventilation (GDV) are exposed to high levels of pollutants present in the environment (dust, airborne bacteria, etc.) or those pollutants produced by animal metabolism and excretion inside the boxes (e.g. ammonia and carbon dioxide). These pollutants may contribute to respiratory pathologies. An alternative experimental ventilation system for laboratory animal housing using intracage ventilation technology (individually ventilated cage system, IVC) was developed. In this system, ammonia levels decreased and rats exhibited better reproductive performance and a lower incidence of pneumonia than rats maintained under GDV. Using two different levels of air speed (0.03-0.26 m/s: IVC(1); 0.27-0.80 m/s: IVC(2)), the effects of IVC were compared with GDV (control) in Wistar rats in terms of respiratory mucus properties, on the nasal epithelium (as measured by quantitative morphometry) and on the lungs (as determined by the cellular composition obtained by bronchoalveolar lavage). Mucus of the respiratory system was evaluated using the following techniques: rheology (viscoelasticity) by microrheometer, in vitro mucociliary transportability (frog palate) and contact angle (an indicator of adhesivity). Also, membrane transepithelial potential difference was measured as a biomarker of airway integrity. After bedding was changed, ammonia concentrations inside the cages on day 3 were significantly higher for GDV than for IVC(1) and IVC(2). The potential-difference values for IVC(1), IVC(2) and GDV in the epiglottis and in the trachea also showed differences. Although some significant differences were observed across the three groups in counts of some cell types, the intragroup results were highly variable among individuals and inconsistent between sexes. No significant differences in the other parameters were found across groups. These results establish that rats maintained under GDV in relatively unregulated conditions are exposed to factors that can lead to deleterious effects on the ciliated epithelium of the airways, and that these effects can be prevented by the use of IVC.     

The impact of reduced frequency of cage changes on the health of mice housed in ventilated cages

Our purpose in this investigation was to determine if we could reduce cage changing frequency without adversely affecting the health of mice. We housed mice at three different cage changing frequencies: 7, 14, and 21 days, each at three different cage ventilation rates: 30, 60 and 100 air changes per hour (ACH), for a total of nine experimental conditions. For each condition, we evaluated the health of 12 breeding pairs and 12 breeding trios of C57BL/6J mice for 7 months. Health was assessed by breeding performance, weanling weight and growth, plasma corticosterone levels, immune function, and histological examination of selected organs. Over a period of 4 months, we monitored the cage microenvironment for ammonia and carbon dioxide concentrations, relative humidity, and temperature one day prior to changing the cage. The relative humidity, carbon dioxide concentrations, and temperature of the cages at all conditions were within acceptable levels. Ammonia concentrations remained below 25 ppm (parts per million) in most cages, but, even at higher concentrations, did not adversely affect the health of mice. Frequency of cage changing had only one significant effect; pup mortality with pair matings was greater at the cage changing frequency of 7 days compared with 14 or 21 days. In addition, pup mortality with pair matings was higher at 30 ACH compared with other ventilation rates. In conclusion, under the conditions of this study, cage changes once every 14 days and ventilation rates of 60 ACH provide optimum conditions for animal health and practical husbandry.     

A behavioral comparison of New Zealand White rabbits (Oryctolagus cuniculus) housed individually or in pairs in conventional laboratory cages

Despite their gregarious nature, rabbits used for research are often housed individually due to concerns about aggression and disease transmission. However, conventional laboratory cages restrict movement, and rabbits housed singly in these cages often perform abnormal behaviors, an indication of compromised welfare. Pairing rabbits in double-sized cages could potentially improve welfare by providing both increased space and social stimulation. We compared the behavior of female New Zealand White rabbits (Oryctolagus cuniculus) housed either individually (N=4) in cages measuring $\text{61}\text{cm}\text{×76}\text{cm}\text{×41}$ cm or in non-littermate pairs (four pairs) in double-wide cages measuring $\text{122}\text{cm}\text{×76}\text{cm}\text{×41}$ cm. The rabbits were kept under a reversed photoperiod (lights on 22:00–12:00 h). Each rabbit was observed five times per week for 5 months, using 15-min focal animal samples taken between 08:00–09:00, 12:00–13:00, and 16:00–17:00 h. Data were analyzed using a repeated measures General Linear Model (GLM). Over the 5 months, individually housed rabbits showed an increase in the proportion of the total behavioral time budget spent engaged in abnormal behaviors (digging, floor chewing, bar biting), from 0.25 to 1.77%, while pairs remained unchanged at 0.95% (treatment×time interaction, F_1,24=4.60; P≤0.0422). Paired rabbits engaged in more locomotor behavior (F_1,6=16.49; P≤0.0066) than individual rabbits (average proportions of time budget: 2.71 and 0.70% for paired and individual rabbits, respectively), which may be important because caged rabbits are susceptible to osteoporosis and other bone abnormalities due to the restricted ability to move. Time spent feeding and body weights of dominant and subordinate rabbits in a pair did not differ, indicating that food competition was not a problem, and paired rabbits were often observed in physical contact (26.7% of data records) although the size of the cages allowed physical separation. Aggression between pairmates did not increase significantly during the study. However, one pair did have to be separated at the end of the study due to bite wounds from persistent aggression. Thus, although methods for decreasing injurious aggression require further investigation, the beneficial effects of pair housing in decreasing abnormal behaviors and increasing locomotion suggest that pair housing should be considered as an alternative to individual housing for caged laboratory rabbits.     

The influence of the location of a nest box in an individually ventilated cage on the preference of mice to use it

The improvement of housing conditions for mice by using environmental enrichment materials is of high concern for the scientific community. Plastic, autoclavable nest boxes are commercially available and ready to use for specific cases such as in individually ventilated cages, metabolic cages, or during toxicological studies. The aim of this study was to see if the location of the nest box within the cage could influence the mice to prefer and use it. Located on the cage floor or hung from the cage lid, a nest box (MPLEX, Otto Environmental, Milwaukee, Wisconsin), enriched the cages. The study concluded that the location of the nest boxes in the individually ventilated cage plays a significant role in the mice preferring to use it or to avoid it. It is also important to use environmental enrichment items that provide animals with the possibility of expressing their preferences and manipulating them in a way to cope better with their environmental conditions.     

Microbiological contamination of laboratory mice and rats in Korea from 1999 to 2003.

To survey the microbiological contamination of laboratory mice and rats in Korea during a 5-year period, we monitored animals housed in mouse and rat facilities with either barrier or conventional systems. At barrier and conventional mouse facilities, the most important pathogen identified was mouse hepatitis virus (MHV), while Mycoplasma pulmonis was the most important pathogen at conventional rat facilities. Interestingly, hantavirus was recovered from both barrier and conventional mouse facilities. The most common protozoon identified was Tritrichomonas muris in mouse facilities and Entamoeba muris in rat facilities. In addition, we found that the microbiological contamination of mice and rats in conventional facilities was severe. These results suggest that conventional facilities should be renovated and monitored regularly to decrease microbiological contamination. We also propose that hantavirus should be monitored in Korea as an important mouse pathogen.     

Microbiological processes in a high-temperature oil field

Thermophilic sulfate-reducing bacteria (SRB) oxidizing lactate, butyrate, and C12-C16 n-alkanes of oil at a temperature of 90 degrees C were isolated from samples of water and oil originating from oil reservoirs of the White Tiger high-temperature oil field (Vietnam). At the same time, no thermophiles were detected in the injected seawater, which contained mesophilic microorganisms and was the site of low-temperature processes of sulfate reduction and methanogenesis. Thermophilic SRB were also found in samples of liquid taken from various engineering reservoirs used for oil storage, treatment, and transportation. These samples also contained mesophilic SRB, methanogens, aerobic oil-oxidizing bacteria, and heterotrophs. Rates of bacterial production of hydrogen sulfide varied from 0.11-2069.63 at 30 degrees C and from 1.18-173.86 at 70 degrees C micrograms S/(1 day); and those of methane production, varied from 58.4-100 629.8 nl CH4/(1 day) (at 30 degrees C). The sulfur isotopic compositions of sulfates contained in reservoir waters and of hydrogen sulfide of the accompanying gas indicate that bacterial sulfate reduction might be effective in the depth of the oil field.     

Environmental factors influencing methanogenesis in a shallow anoxic aquifer: a field and laboratory study

Summary The environmental factors influencing methanogenesis in a shallow anoxic aquifer were probed in a combined field and laboratory study. Field data collected over a year revealed that in situ rates of methane production were depressed in winter and elevated in summer. Over the same period, ground water pH values ranged from 6.0 to 7.8 while temperatures varied from 7–22°C. In situ methanogenesis was severely inhibited at temperatures < 13°C or by pH values < 7. The influence of these factors on microbial methane formation from both endogenous and exogenous substrates were tested in aquifer slurries adjusted to pH 5–9 and incubated at temperatures ranging from 5–45°C. Temperature optima for methane production from endogenous substrates varied as a function of pH, but the pH optimum was 8 at all temperatures. Optimal conditions for acetoclastic methanogenesis were found at pH 8 and 35°C. An analysis of variance revealed that pH, temperature, and a pH-temperature interaction are all significant variables influencing aquifer methanogenesis. In addition transient sulfate accumulations were also found to limit methane production in some areas. A comparison of field and laboratory methane production patterns suggest that pH, temperature, and sulfate accumultations are important, but not the only environmental variables influencing the mineralization of organic matter in shallow aquifers.     

Ten-year long monitoring of laboratory mouse and rat colonies in French facilities: a retrospective study

From 1988 to 1997, a total of 69 mouse colonies and 36 rat colonies were examined for the presence of antibodies to 14 indigenous viruses of mice and rats. Among mouse viruses, high positivity rates were observed with mouse hepatitis virus (MHV), Theiler's encephalomyelitis virus (THEMV), minute virus of mice (MVM), Sendai virus and pneumonia virus of mice (PVM); the prevalence rates were high in rats with Khilam's rat virus (KRV), THEMV, Toolan's H-1 virus, Sendai virus, Parker's rat coronavirus (RCV/SDA) and PVM. During the last decade, the prevalence of some agents such as MHV, Sendai virus, THEMV, PVM and MVM has apparently decreased although they were still present in 1997 (except for PVM). Another point is the constant increase of colonies found free of viruses through this decade, demonstrating the efforts of the French research community to increase the quality of hygiene in laboratory animals.     

Telemetric monitoring of blood pressure in freely moving mice: a preliminary study

This paper describes for the first time the possibility for recording the systolic pressure (SP), diastolic pressure (DP), and the mean arterial pressure (MAP) as well as the heart rate (HR) and locomotor activity (LA) in freely moving mice, using a commercially available telemetry and data acquisition system. The system comprises a new, small radio-telemetry transmitter implanted in the peritoneal cavity, a receiver board placed underneath the home cage, a multiplexer and a computer-based data acquisition system. The signals from the receiver were consolidated by the multiplexer and were stored and analysed by the computer. The telemetered pressure signals (absolute pressure) were corrected automatically for changes in atmospheric pressure measured by an ambient pressure monitor. The effects of implantation on animal behaviour, and, after the animals had recovered, the effects of handling on the SP, DP, MAP and HR were examined. The radio-telemetry system for recording the SP, DP, MAP and HR provides an accurate and reliable method for monitoring the direct effects of handling on SP, DP, MAP and HR. In addition, by using this new blood pressure (BP) transmitter, we maintain that BP measurements in freely moving mice are more efficient, reliable, and less labour-intensive than the measurement techniques described in the literature thus far.     

Efficacy of three microbiological monitoring methods in a ventilated cage rack

The use of individually ventilated caging (IVC) to house mice presents new challenges for effective microbiological monitoring. Methods that exploit the characteristics of IVC have been developed, but to the authors' knowledge, their efficacy has not been systematically investigated. Air exhausted from the IVC rack can be monitored, using sentinels housed in cages that receive rack exhaust air as their supply air, or using filters placed on the exhaust air port. To aid laboratory animal personnel in making informed decisions about effective methods for microbiological monitoring of mice in IVC, the efficacy of air monitoring methods was compared with that of contact and soiled bedding sentinel monitoring. Mice were infected with mouse hepatitis virus (MHV), mouse parvovirus (MPV), murine rotavirus (agent of epizootic diarrhea of mice [EDIM]), Sendai virus (SV), or Helicobacter spp. All agents were detected using contact sentinels. Mouse hepatitis virus was effectively detected in air and soiled bedding sentinels, and SV was detected in air sentinels only. Mouse parvovirus and Helicobacter spp. were transmitted in soiled bedding, but the efficacy of transfer was dependent on the frequency and dilution of soiled bedding transferred. Results were similar when the IVC rack was operated under positive or negative air pressure. Filters were more effective at detecting MHV and SV than they were at detecting MPV. Exposure of sentinels or filters to exhaust air was effective at detecting several infectious agents, and use of these methods could increase the efficacy of microbiological monitoring programs, especially if used with soiled bedding sentinels. In contemporary mouse colonies, a multi-faceted approach to microbiological monitoring is recommended.     

Telemetry tag effects on juvenile lingcod Ophiodon elongatus movement: a laboratory and field study

This study tested the behavioural effects of tagging subyearling and yearling lingcod Ophiodon elongatus with acoustic telemetry tags in laboratory tanks and in the natural environment (Puget Sound, WA). In the laboratory, tagged individuals showed less movement and feeding behaviour soon after tagging than untagged controls. The effect dissipated after c. 1 week, presumably as the tagged O. elongatus recovered from surgery or adjusted to the presence of the tags. This dissipation enabled a field study that compared early‐tagged individuals with a long recovery period after tagging to recently‐tagged individuals with a short recovery period after tagging. Consistent with findings from the laboratory experiment, recently tagged individuals showed less movement away from three release sites in Puget Sound than early‐tagged individuals. Together, the laboratory and field results provide evidence of temporary tag effects on actual movement in the natural environment and provide a method for testing tag effects in the field. This study suggests that subyearling and yearling O. elongatus should be held for a recovery period before release. If holding after tagging is not an option, then movement data collected during the first week should be interpreted cautiously.     

Growth in field and laboratory populations of Wood mice (Apodemus sylvaticus)

Intrinsic growth rate and asymptotic body weight parameters were estimated for two laboratory and three field populations of the Wood mouse, Apodemus sylvaticus , using a logistic model fitted by an ordinary least squares method. The data upon which the growth equations were based were differences in body weight for individual animals between sampling occasions. Suckling mice in one of the laboratory studies suffered high mortalities and poor body growth which was related to disturbance to the mothers and their young during examination. Growth in weaned mice appeared normal and was compared with growth in autumn and winter populations of wild mice with poor and good natural seed supplies. Males tended to have higher growth rates, and to a lesser extent, higher body weight asymptotes than females in all groups. Growth rates were highest in the autumn field population with a good seed supply; differences between the other groups were less marked. These results are discussed and it is suggested that, despite the approximations inherent in the method, the method will be a useful tool for studying the the productivity of wild populations of small mammals.