Contemporary
Topics in Laboratory Animal Science
Volume 42(4)
Experimental murine Cryptococcal infection results in contamination of bedding with Cryptococcus neoforans 9-12
Summary: Mice with pulmonary cryptococcus neoformans
infection shed the organism and contaminate their bedding. Precautions are
warranted when disposing bedding.
M&M:
Results:
C.
neoformans was cultured only from bedding of those animals inoculated
intratracheally and only on days 5 & 7 post inoculation Discussion:
Questions:
1.
What does CDC consider the biosafely level required for work with Cryptococcus
neoformans?
a. BSL I
b. BSL II
c. BSL III
d. BSL IV
2. Bedding becomes contaminated from mice
infected with Cryptococcus neoformans
a. IP
b. IV
c. IT
d. All the above
Answers:
1. b
2. c
Species-specific assessment of pain in laboratory animals 13-20
Bottomline: Article is a short review of current
literature regarding pain scales and a description of how an IACUC has
implanted a pain scoring system.
Physiology of Pain
·
Chronic pain
occurs when pain is ongoing and associated physiologic adaptation. Therefore
measurable physiologic changes are not always evident and can be very difficult
to treat.
Indications for Managing
Pain Scale
Development
Application of
Pain Scales
Questions
1)
Pain score systems should include all but...
a. Locomotor activity
b. Sleep patterns
c. Anthropomorphic assessment
d. Temperament
Answers
1) c
Postoperative pain management using Fentanyl patches in dogs 21-26
Evaluated the abililty of the fentanyl patch to control pain in a postoperative canine model. The dogs underwent a major abdominal surgery. Serum plasma levels of fentanyl were analyzed at various time points. Animals were subjectively evaluated for postoperative pain using a Simple Descriptive Scale at regular intervals postoperatively. Also recorded heart and respiratory rates, rectal temperature, appetite, and activity. The patch was shown to adequately control pain. Study also showed that increased subcutaneous fat delayed absorption. Fentanyl is a synthetic, Schedule II controlled substance related to the opioid agonist morphine but 75-100 times more potent. Has high affinity for mu opioid receptors in CNS to produce analgesia and sedation. The transdermal administration mimics constant IV infusion and maintains a stable fentanyl plasma concentration to provide analgesia for up to 2 days postoperartively. Application of the patch should occur 12-24 hours pre-op to allow for peak serum levels to be obtained at the time of surgery. The area where the patch was applied was clipped to remove hair. Care was taken not to excoriate the skin as this could alter the delivery rate of the fentanyl. The delivery rate of each patch is directly proportional to the surface area in direct contact with the skin. However, this rate can be affected by a number of physiological factors including blood flow, hydration status, body and skin temperature, fat content, and skin integrity. Regarding the Simple Descriptive Scale for pain assessment, a 0 classification describes an animal that is clinically normal in activity, appetite, and attitude. A 1 denotes an animal in mild pain (e.g. slightly sensitive to touch at the surgical site and may inconsistently cry or whine relative to position or to pressure applied to the surgical site). The 2 classification indicates moderate pain with marked reduction in activity and appetite. The animal constantly cries or whines and reacts adversely when touched. The 3 classifications indicate severe pain; the animal is inactive or recumbent, demonstrates no appetite, constantly cries or whines, and reacts adversely when touched. A 1 classification indicates mild pain and is commensurate with USDA pain category C. Animals in classifications 2 or 3 are considered to be experiencing unacceptable levels of pain. Analgesic levels were maintained until the patches were removed 48 hours postoperatively. At no time during the study did any animals appear to be suffering from moderate (2) to severe (3) postoperative pain. Heart rate and respiratory rate were lowered due to the patch. Patches also caused mild hypothermia. The surgery performed would be expected to produce substantial postoperative pain if left untreated. The patch is not too expensive as the estimated cost is $12-18/patch.Questions:1. What DEA schedule is fentanyl?2. What factors affect fentanyl's transdermal delivery rate?Answers:1. Schedule II2. The delivery rate of each patch is directly proportional to the surface area in direct contact with the skin. However, this rate can be affected by a number of physiological factors including blood flow, hydration status, body and skin temperature, fat content, and skin integrity.
The sedative and behavioral effects of Nalbuphine in dogs 27-31
Summary:
In this study, the authors examine the usefulness of nalbuphine as a premedicant for dogs. Nalbuphine is a mixed agonist-antagonist opioid that has analgesic properties comparable to that of morphine in humans. Nalbuphine has several positive traits when compared to other opioids in that it tends to cause less severe respiratory depression than morphine, especially at higher doses; it is less expensive; and it is currently not a controlled drug in the U.S. Although previous studies had shown minimal cardiovascular and respiratory side effects in dogs administered nalbuphine, little was known with respect to its sedative effects in dogs.
To determine whether nalbuphine would be suitable for use alone, the authors conducted a pilot study looking at 5 doses of nalbuphine: 0.5, 1, 2, 4 and 6 mg/kg administered subcutaneously. The initial study showed that nalbuphine alone had little sedative effects and these effects did not increase with increasing dosage. Based on the pilot study, the authors opted to use nalbuphine in combination with other drugs in order to determine its usefulness as a premedication in dogs undergoing routine surgeries.
In the actual study, twenty-four dogs undergoing routine ovariectomy or castration were randomly assigned to one of four premedication groups: nalbuphine, 0.5 mg/kg and xylazine 0.5 mg/kg [NX]; xylazine 0.5 mg/kg [X]; nalbuphine, 0.5 mg/kg and acepromazine, 0.05 mg/kg [NA]; and acepromazine, 0.05 mg/kg [A]. All drugs were administered subcutaneously in the interscapular region and all dogs received glycopyrrolate at 0.01 mg/kg SC. The dogs were then evaluated for sedation by an investigator unaware of the treatment groups using a simple descriptive scale (SDS) and a visual analogue scale (VAS). The dogs were evaluated both inside and outside of the kennel at 10, 20 and 30 minutes post-administration. Heart rates and respiratory rates were also measured at these timepoints. The dogs’ behavior was evaluated following the last sedation timepoint. Three distinct timepoints were used to evaluate behavior: placement of the dog on the table; prepping of the leg for catheter placement; and placement of the catheter. The following are some of the behavioral scoring parameters that were assessed: struggling, panting, shivering, leg withdrawal, orienting (appropriate focus on leg during prepping or catheterization) and biting. The behaviors were scored as 1 (little), 2 (moderate) or 3 (extreme). The expired halothane concentrations were also measured and dogs were assessed using an SDS during the recovery period.
The results from the sedation portion of the study revealed significantly higher SDS and VAS scores (e.g., increased sedation) for the NX and X groups when compared to A at the 30 minute timepoint. Interestingly, there were no significant differences between the NX and X groups and NA and A groups which supports the premise that nalbuphine has weak sedative properties.
With respect to the behavioral assessments, the NX group had significantly lower scores on leg withdrawal and orienting when compared to the X group but no other significant differences were detected between groups. There were no significant differences between groups with respect to expired halothane concentration or recovery assessments.
The authors conclude that nalbuphine alone has minimal sedative effects that do not increase with increasing doses. Theoretically, this may be due to the partial agonist-antagonist properties of nalbuphine (mu agonist, possible antagonist and kappa agonist) - mu and kappa receptor activation are reported to cause sedation as seen with butorphanol another mixed agonist-antagonist. Nalbuphine alone also did not diminish adverse behaviors associated with distress and discomfort and therefore negating its usefulness as a premedicant when used alone. When used in conjunction with xylazine, nalbuphine improved the antianxiety and sedative properties of xylazine and the author concludes that additional studies to optimize dose ratios would be useful.
Questions:
Answers:
Comparison of ketamine versus combination of ketamine and mehetomidine in injectable anesthetic protocols: chemical immobilization in macaques and tissue reaction in rats 32-37
This paper assessed anesthetic protocols of ketamine used alone (K), or
ketamine used in combination with medetomidine (KM) in nonhuman primates (NHPs)
and rats. Young male macaques undergoing routine quarantine exams,
phlebotomy, and tuberculin (TB) testing were assessed for muscle relaxation,
anesthetic depth, and ease of recovery. Response to TB injection, femoral
venipuncture, toe pinch, and spontaneous movement were used to assess
anesthetic depth.
As expected, ketamine alone provided very little skeletal muscle
relaxation. Deeper and longer anesthesia was achieved with KM.
Authors found statistically significant differences between KM and K in NHP
parmaters of TB injection reaction and spontaneous movement.
The other two
parameters (response to toe pinch and femoral venipuncture) did not indicate
statistically significant differences. Some animals had anesthesia
reversed with atipamezole, which significantly reduced the length of anesthesia
by 40%.
Rats were used to evaluate tissue inflammation and muscle necrosis from
these anesthetic agents. Rats were either given ketamine alone or a lower
dosage of ketamine with medetomidine. It was determined that rats given
the combination exhibited markedly less damage to muscle at injection sites.
Questions:
1. Give the
scientific name for the pig-tailed macaque and the rhesus macaque.
2. Name the
agent which reverses medetomidine.
3. What agent
does yohimbine reverse?
4. Where, how
much, and how is tuberculin injected when conducting TB testing in NHPs?
5. What is the
minimum amount of time to TB test imported NHPs?
6. Which
designates a NEGATIVE reaction to a TB test?
a. no
swelling
d. red, with mild swelling
b. redness, no
swelling e.
redness, with lid droop
c. bruise, no
swelling
f. eye is closed
Answers:
1. Macaca
nemistrina; Macaca mulatta
2.
Antipamezole
3. Xylazine
4. Intradermal
injection of 0.1 ml tuberculin (1500 U Mammalian Old Tuberculin) in upper
eyelid, using 27 gauge needle on a 1 cc syringe.
5. Three
times, with 2 weeks between each TB test.
6. a, b, and c
Effect of pair-housing on operant behavior task performance by rhesus monkeys 38-41
Pair housing for non human primates has been well documented to play an important role in their psychological well being. This study evaluated the effects of social housing on performance scores on a standard battery of operant tasks. Short term memory and attention (delayed matching to sample, DMTS), motivation (progressive ratio, PR), color and position discrimination (conditioned position responding, CPR) and learning (incremental repeated acquisition, IRA) were evaluated using food reinforcement techniques. Sixteen, individually housed, colony bred males ranging in age from 2.5-5.5 years were split into two groups, with eight animals singly housed (controls), and the remaining eight monkeys pair housed. The transition from single to pair housing was implemented over a 2 month period. All animals had been perfroming the operant test battery (OTB), outlined above, for at least 6 months prior to commencement of the experiment. Behavioral assessments were conducted 5 days a week, with the DMTS task presented on one day and the PR, CPR and IRA tasks the following day. Therefore each task was conducted two to three times weekly. Results for the two experimental groups were compared for 2 months, following establishment of the pairs. For the CPR and IRA tasks, performance remained unchanged for both groups. The number of completed trials for the PR and DMTS tasks increased only in the singly housed animals. Overall, the pair housed monkeys performed more poorly on average. Given the known benefits to pair housing non human primates, such as reduced incidence of stereotypical and/or self injurious behaviors and decreased disease susceptibility, one would also have to consider the possibility of inhibition and thus poorer performance of a subordinate animal in a pair housed situation, or decreased motivation for food reinforcement, affecting PR and DMTS results. Complex brain functions may be evaluated through operant task perfromance of pair housed of monkeys, however the housing conditions may affect certain test parameters. Questions:1.Define operant conditioning. How does it differ from instrumental conditioning ?Answers:1. Operant conditioning is learning in which a specific stimulus elicits a particular response because the response produces a desirable outcome. Instrumental conditioning occurs only after the subject performs a previously designated act. The classic example is the pressing of a bar to obtain the reward (food).
Some observations on the pharmacological properties of ivermectin during treatment of a mite infestation in mice 42-45
Summary: Authors, using pooled
serum samples from mice exposed to drinking water containing 32 mg/L
ivermectin, were able to establish some kinetic data on
peak serum concentration, time to peak concentration and decay after treatment
was discontinued. Ivermectin medicated water used for three ten day
periods, with
a week of no treatment between each period, was correlated with a
reduction or elimination of fur mites evaluated by looking for eggs or
mites in the pelage.
Using the criteria above to designate infested mice,
there were not positive animals detected at the end of the second 10 day water
treatment cycle. While these
authors did not experience any toxicity associated with the medicated water,
they acknowledge that the literature support the fact that even lower doses
have been
toxic to certain strains and/or transgenic mice.
Questions:
1. The life cycles for the murine fur mites are direct/indirect (circle one)?
2. The neurotransmitter blocked by
the appropriate use of avermectins (ivermectin) for ectoparasite control, is
___________________________________.
A.
norepinephrine
C. dopamine
B.
acetylcholine
D. gamma- aminobutyric acid
3. These
investigators provided ivermectin at 32 milligrams/liter of drinking water for
ectoparasite control. Assuming
that these mice consumed 150 mL/kg/24
hours, what would be the 24hour ivermectin intake in a 28 gram mouse?
_____________________________________
4. T/F.
Morphologically, Radfordia affinis, and Myobia musculi, are quite
similar. One way they can be distinguished is
by the two claws on the second digit in Radfordia
affinis.
5. If one were asked to
identify the three fur mites commonly found on rats and mice, the
"squatty" specimen, nearly
as wide as it is long, would be identified as
____________________________ (Radfordia
affinis; Myobia musculi or
Myocoptes musculinus.)
6. Infestation with fur mites is
termed: _________________.
Answers
1. Murine fur mite life cycles
are direct.
2. The neurotransmitter blocked by
the appropriate use of avermectins (ivermectin) for ectoparasite control, is
gamma-aminobutyric acid.
3. These investigators provided
ivermectin at 32 milligrams/liter of drinking water for ectoparasite
control. Assuming that these mice consumed 150mL/kg/24 hours, what would
be the 24hour ivermectin intake in a 28 gram mouse?
32 mg
X 150 mL
X 0.027 Kg = 0.13
mg/24 hr.
1000mL Kg-24 hr
4. True. Radfordia
affinis and Myobia musculi can be distinguished by the two (1 pair)
of claws on the second digit of Radfordia affinis but only one claw on
the second digit of Myobia musculi.
5. If one were asked to
identify the three fur mites commonly found on rats and mice, the
"squatty" specimen, nearly as wide as it is long, would be identified
as Myocoptes musculinus
6.. Infestation with fur mites is termed: acariasis
Evaluation of coinfection with pinworms (Aspicularis tetraptera, Dentostomella translucida, and Syphacia obvelata) in gerbils and mice 46-48
This article describes coinfection with pinworms in Mongolian gerbils (Meriones unguiculatus) and mice (Mus musculus). 10 female adult gerbils used were purchased from a pet store which were previously obtained from a farm supplier where animals were reared free-range in the rural area of Mage, State of Rio de Janeiro, Brazil. 20 female Swiss Webster mice were obtained from closed breeding colonies from an institutional animal facility in Brazil. Screening showed that gerbils were infected with Dentostoella translucida and mice were infected with either Syphacia obvelata or Aspiculuris tetraptera or both nematodes. Gerbils, assigned group A, were infected with S. obvelata and A. tetraptera eggs by being fed bread crumbs contaminated with viable eggs obtained from mice. 10 mice, assigned to group B, after being sedated, were infected with embryonated eggs obtained from gravid female nematodes from gerbils using adapted tracheal aspiration-type catheter. 10 mice, assigned group C, were infected with D. translucida. Gerbils and mice were kept in separate cages, but cohabitation of gerbils and mice has been reported. Gerbils were positive for S. obvelata or D. translucida or both, but were negative for A. tetraptera on day 12 and 28 postingestion. Group B mice were positive for S. obvelata and A. tetraptera eggs 29 days post infection, but negative for D. translucida. The testing dates were chosen in light of the helminth species' life cycles. Mice of group C were negative for all three nematodes. Necropsies between 30 and 35 days post infection showed that gerbils harbored D. translucida, S. obvelata and A. tetraptera, mice from group B were positive for two pinworms and negative for D. translucida. Group C mice were negative for pinworm eggs. This study shows that gerbils are highly susceptible to S. obvelata and are susceptible to but are poor hosts for A. tetraptera. In addition, this study showed that mice are resistant to D. translucida from gerbils. Questions:1) Gerbils are highly susceptible to the following: A Aspiculuris tetraptera B Dentostomella translucida C Syphacia obvelata2) Mice are resistant to the following : A Aspiculuris tetraptera B Dentostomella translucida C Syphacia obvelata3) The prepatent period for Dentostomella translucida is: A 7 days B 21 days C 29 daysAnswers:1 C2 B3 C
Inoculation of Staphylococcus xylosus in SJL/J mice to determine pathogenicity 49-52
In 1997 there was an outbreak of spontaneous necrotic tail Lesions in a colony of naive SJL/J mice at the National Institute of Neurological Disorders and Stroke, Department of Health and Human Services, National Institute of Health. Staphylococcus xylosus was isolated from the lesions in these mice. S. xylosus is a gram positive, coagulase-negative Coccoid bacterium of the family Micrococcacae. It has been considered a nonpathogenic skin and mucous membrane commensal organism and has rarely been implicated in infection. In order to determine whether S. xylosus was a causative factor in the tail lesions of the SJL/J mice, SPF female SJL/J mice were inoculated with this agent. Oral cavity swab samples were obtained from all mice and were cultured to detect the presence of the S. xylosus organism. Only mice determined to be S. xylosus-negative were used in the inoculation groups. The suture material was impregnated with the concentrated suspension of bacteria for the experimental groups and in sterile water for the control group. A 27-gauge needle with swaged on 4-0 silk suture was inserted into deep tissue of the tail and tied in a surgical square-knot. Tail lesions developed in 30 of the 35 experimentally infected mice inoculated with S. xylosus. Lesions did not develop in the control group, which remained negative upon culture at the oral and tail site locations. 9 mice were treated with SulfaTrim antibiotic (200mg sulfamethoxazole and 40 mg trimethoprim per 5ml) at 15mg/kg per os for a 28-d administration. The tail lesions had resolved in all mice allocated to a treatment phase. The tail lesions that developed in the inoculated mice exhibited focal to multifocal erythema, hyperemia, edema, ulcerative dermatitis, and scar tissue formation. All of the lesions were located on the dorsal aspect of the tail. The histopathologic changes ranged from moderate edema with hyperkeratosis and full-thickness epidermal hyperplasia to more severe lesions, which included ulcerative focally extensive chronic-active suppurative dermatitis with multifocal hemorrhage and serocellular crust formation. Gram-positive coccoid bacteria were observed on a Gram stain. The results of this study indicate that S. xylosus may be a pathogenic organism in SJL/J mice.Questions:1) The SJL/J mice are characterized by: a. aggression in males b. susceptibility to experimental autoimmune encephalomyelitis (EAE) c. high incidence of reticulum cell sarcomas d. b and c e. all of the above2) Which antibiotic was successful in treating the tail lesion? a. Penicillin b. Enrofloxacin c. Sulfamethoxazole-Trimethoprim d. AmikacinAnswers:1) e
2) c
An alternative method of chronic cerebrospinal fluid collection via the cisterna magna in conscious rhesus monkeys 53-59
Models of chronic cerebrospinal fluid collection have been established for nonhuman primates where cannulas were placed into the lateral or 4th ventricles or catheters into the cerebral or spinal subarachnoid space. These models have proved successful and reliable but unfortunately require invasive techniques to pass through the skull or require a laminectomy to enter the subarachnoid space, involve the use of expensive and highly specialized stereotaxic equipment for the precise placement of the implants, and may require exteriorized hardware which is cumbersome to maintain and unaesthetic.
Thirty-six rhesus monkeys were pre-sedated with ketamine hydrochloride, intubated with a cuffed endotracheal tube, placed on Isoflurane, in sternal recumbancy, and the neck flexed. The hair was clipped and aseptic techniques were used to prepare the surgical site. A # 15 scalpel blade was used to make a 3 to 5 cm incision beginning at the external occipital protuberance followed by blunt dissection of the occipitalis m., midline of the rhomboideus capitis m., splenius capitis m., right and left medial parts of the biventor cervics portion of the semispenalis capitis m., right and left rectus capitus posterior minor m., a fatty layer and through the Atlantooccipital Membrane into the Cisterna Magna. An incision was made with a 1 to 1.5 inch 16 to 18 gauge needle and enlarged with a fine-tipped hemostat almost large enough to accommodate 1 cm of 3.5-french silicone catheter (opened at the end and on both sides) with a tapered-collar which fits inside the Cisterna Magna against the Atlantooccipital membrane and a fixed silicone collar adhered to the outside of the Atlantooccipital membrane with a tissue adhesive to prevent movement of the catheter further into the Cisterna Magna. This catheter was 12 inches in length and was tunneled under the skin to a titanium port located between the scapula and sutured in place from which cerebrospinal fluid (CSF) could be collected with a 22 gauge ¾ inch Huber needle. The port was checked to insure the catheter was patent and CFS could be withdrawn from the port. A sterile irrigating fluid (PSS) was used to clear the line and check for leaks. The musculature was also sutured in place with an absorbable suture material. The animals received two injections of buprenorphine the day of the surgery (and another one the following day) and one injection of ampicillin.
Once the catheter was in place, a protocol was established to maintain CSF flow by drawing samples three times a week and eventually reducing the number to twice a week. After this two-week period collars were placed back on these animals and CSF samples were collected in a standard primate chair. CSF samples were collected using standard aseptic technique using Huber needles. During a two-week post-surgical period animals were anesthetized for CSF collection. CSF samples were centrifuged and the sediment was cultured twice weekly and if bacterial growth was noted another culture was taken. The supernate was used for clinical analysis. When animals had three positive cultures for bacterial contamination, the catheter and port were removed and the animals were placed on antibiotics. Follow-up cultures found the animals negative. The supernate collected by this new catheter technique had more narrow ranges for the cerebrospinal chemistries and the alkaline phosphatase levels were lower than previous published levels.
This study was found to produce a reliable method of chronic CSF collection in rhesus monkeys and is a proven alternative to more invasive and complicated methods. This system provides a minimally invasive approach to collecting multiple CSF samples from conscious rhesus monkeys. A low incidence of bacterial contamination was noted (5.3%) with relative patent flow rates from 0.47 to 0.98 ml/min in rhesus monkeys under 8 kg with a success rate of 76%. Problems were observed in animals weighing more than 8.0 kgs and there are future plans for improved model development in these larger rhesus monkeys.
Questions:
1. What type of needle was used to collect CSF from the titanium port?
2. What cerebrospinal chemistry value was found different than published values?
3. What size rhesus monkey had problems with this alternate CSF collection technique?
Answers:
1. 22 gauge ¾ inch Huber needle
2. Alkaline Phosphatase
3. 8 kg