Verification of bacterial killing effects of cage wash time and temperature combinations using standard pericylinder methods. Contemporary Topics 39(4), 09.
Abstract: Automated cage wash equipment is legislated to meet minimum standards for temperature. This study was undertaken to provide the time required, at legislated temperatures, to kill specified vegetative bacteria. Bacteria tested: Salmonella, Pasturella, Staphylococcus. All three were killed at: 2 sec. 82.2C 3 sec. 80 C 4 sec. 77.8C 5 sec. 75.6C All of these times are less that the time of exposure of equipment in most cagewashers or tunnel washers.
Questions: Questions:
1. What is the minimum standard set for water in cagewashers based on a) The Guide b) 9CFR, Parts 1,2,3?
2. What do the following acronyms stand for? a) AOAC b) EPA?
3. If water temperature is adequate in a cage washer, what are other possible causes for inadequate killing of vegetative bacteria? Name
Answers: Answers:
1. a) "Effective disnifectionn can be acheived with wahs and reisne water at 143-180F or more. The traditional 82.2C (180F) temperature requiemtns for rnse water refers to the water in the tank or in the sprayer maniforld" p.44 b) 82.2C (180F)
2. a) Association of Official Analytical Chemists b) Environmental Protection Agency
3. · improper water pressure · improper angling of water jet sprays · inadequate water flow through jets · improper machine warm-up · improper detergent use · improper loading of machine · improper scraping of organic debris from cages/equipment
The influence of husbandry schedules on the number of embryos collected from superovulated mice. Contemporary Topics 39(4), 13.
Abstract: This study assessed the cage cleaning frequency on recovered embryos from superovulated mice (B6C3F1 strain). The results demonstrate that the number of embryos recovered from females housed in cages cleaned 1 or 24 hour prior to mating were not significantly different. However, from cages cleaned 7 days prior to mating significantly fewer embryos were recovered. The authors felt that ammonia buildup in the unclean cage is responsible for this effect, however no data was presented to support this idea.
Questions: Questions:
Male mice compared to female mice generally have significantly higher intracage ammonia concentration buildup?
A. True
B. False
C. Not known due to lack of presented data
At what time point has the observation of reduced embryo collection from superovulated mice (B6C3F1 strain) been observed?
A. Changing Cages one hour prior to mating.
B. Changing Cages 24 hours prior to mating
C. Changing Cages 168 hours prior to mating
D. Not known due to lack of presented data
Answers: No answers provided
Eradication of simian retrovirus type D from a colony of cynomologus, rhesus, and stump-tailed macaques by using serial testing and removal. Contemporary Topics 39(4), 16.
Abstract: Both simian retrovirus D (SRV/D) and SIV are capable of inducing immunosuppressive disease in macaques. However, SRV/D is responsible for most cases of viral-induced immunodeficiency and is more difficult to eliminate from captive macaque colonies. Therefore, successful strategies for eradication of this virus from research facilities using macaques are significant.
This report chronicles an eradication program from a large pharmaceutical company which uses three species of macaques in drug-development protocols. During a certain 1.5 year period, their primate colony exhibited a myriad of clinical illnesses dominated by acute, fulminating anemia. Other conditions noted were lymphadenopathy, splenomegaly, diarrhea, neutropenia, thrombocytopenia, myeloblastic leukemia, multiple myeloma, lymphoma, hepatic abscess, retroperitoneal fibromatosis, and acute ulcerative gingivitis. Virus isolation procedures detected SRV/D in the colony and it was realized that this may be an underlying etiology for many of these illnesses.
Because of the compromised health of the colony animals, questions about the validity of study results due to the presence of virus, and potential zoonotic disease issues, a three-tiered approach was taken to remove SRV/D from this colony. This included test and removal, strict adherence to procedures employed to prevent fomite transmission, and the purchase of animals from SRV/D-negative sources followed by an extended quarantine testing program. Several important issues to be considered when developing an eradication strategy are which tests will be used to document the presence of virus, cost, test and removal programs vs. separation of infected animals from non-infected animals, and mechanisms of virus transmission. Here, it was shown that testing should be done repeatedly over time and should use both antibody and virus testing (virus isolation or PCR) to detect all infected animals, regardless of the health status of the animal. A test and removal strategy was employed because infected animals were not suitable for research studies and the cost of delayed FDA approval of products (flawed data/repeating studies) would have surpassed those of purchasing supplies associated with decreasing fomite transmission, viral testing, and replacing positive animals. Further, procedures to prevent fomite transmission of virus were based on the knowledge that virus is transmitted between animals by saliva primarily, but also by blood, urine, milk, tears, and vaginal secretions. Finally, for continued success of this program, macaques were obtained from clean sources.
Questions: Questions:
1. Why is it important to test for both antibody and virus for determination of a positive SRV/D?
2. What cell types are infected by SRV/D?
3. What are the genus and species names of Cynomolgus, Rhesus, and Stump-tailed macaques?
Answers: Answers:
1. not all SRV/D infected animals mount a vigorous immune response and those animals which have antibodies may have latent virus in other tissues besides peripheral blood.
2. SRV/D infects T cells (CD4/CD8), B cells, macrophage, epithelial cells, and cells of the choroid plexus
3. Macaca fascicularis (Cyno's), M. mulatta (rhesus), M. arctoides(Stump-tails)
A technique for restraining rodents during hindlimb interventions. Contemporary Topics 39(4), 24.
Abstract: SUMMARY: This article describes a rodent (rat) restraint device that is used to restrain rats during external hindlimb procedures such as ultrasound. The method allows for restraint without anesthesia and was considered acceptable for restraint lasting 20 minutes. The device is composed of a piece of molded thermoplastic forming a tube approximately 50% longer than the body of the rat. Its' design had a slit along the longitudal axis (top) to allow for animal insertion or expansion, and holes in the posterior ventral portion for hindlimb exit/exposure. The front 2/3 of the rat was wrapped (1 wrap) in a small towel prior to insertion. Once placed in the tube, plugs with appropriate tail or respiration holes were placed on each end to close the tube, and velcro straps were used to secure the longitudinal axis. The device also contained padding and a holder with foot rest. According to the article, rats could be placed within the device within 1 minute. Additionally, this study examined weight gain and hindlimb bone mineral content (BMC) as compared to a non-restrained control group. The restrained rats gained slightly less weight than their comparative controls, but this effect was only present during the first 6 weeks of the study (12 weeks of 20 min. daily restraint). There was no effect on BMC. The author's assessment was that this device was useful method of restraint allowing for hindlimb exposure, without anesthesia and being well tolerated by the animal.
Questions: QUESTIONS:
1) What was the purpose of this device?
2) Was there evidence of distress from the restraint, and if so, describe?
Answers: ANSWERS:
1) The purpose was to restrain rodents with the hindlimbs exposed so that procedures such as ultrasound examinations, lasting up to 20 minutes, could be accomplish without anesthesia.
2) Some evidence of distress was noted, evidenced by decreased weight gain during the first 6 weeks. Animals adjusted to this stress by the end of 6 weeks and returned to weight gains equivalent to the controls.
Effect of laboratory acclimation on food and water consumption of pregnant sheep after fetal catheterization. Contemporary Topics 39(4), 28.
Abstract: Two objectives were investigated in this study.
1) to determine the time required by late-gestation, pregnant sheep to stabilize food and water consumption after 6 to 7 hours of shipment by truck.
2) to determine if the period of laboratory acclimation prior to fetal and maternal vascular catheterization would affect post-surgical food and/or water consumption.
No other studies addressing these issues could be located in the literature although it is generally believed that sufficient acclimation periods are required to allow stabilization of animal subjects prior to experimentation. Sufficient acclimation periods should help to decrease environmental variables and help to identify the true effect of experimental variables.
Methods and Materials: Two separate studies were performed. The first was a retrospective, semi-quantitative analysis of food and water consumption. One hundred and twelve sheep were utilized which had been in the laboratory for 2 days (n=20), 3 days (n=31), 4 days (n=11), or 5 or more days (n=50) prior to surgery (fetal and maternal vascular catheterization). After surgery, sheep's food and water consumption were visually estimated by comparing the remaining food and water in each animal's bucket with the amount present the previous day.
The second study was a prospective, quantitative analysis of food and water consumption utilizing 20 sheep. Ten animals were acclimated for 2 days and 10 animals were acclimated for >= 7 days prior to fetal and maternal vascular catheterization. Food and water consumption was determined by weighing food and water buckets every 24 hours (prior to cleaning and refilling the buckets). Evaporative water loss was estimated through use of a water bucket placed in the room with the animals. Animals were weighed at the beginning of the study. Their weights were used to normalize food and water consumption rates to help compare between animals.
Anesthetic protocol for both studies: premed = IM atropine 30 prior to induction. induction = IV Na thiamylal or Na pentobarbital. maintenance = 0.5% - 2.0% halothane
Surgical procedure for both studies: 'a maternal femoral artery and vein were catheterized with polyvinyl tubing, the abdominal wall and uterus were incised to expose the fetal hindlimbs or forelimbs, and polyvinyl catheters were placed in femoral or brachial arteries. Some fetuses also received femoral vein or brachial-cephalic vein catheters and/or urinary bladder catheterizationè..Analgesics were not administered after surgery." Intra-amniotic, prophylactic antibiotics were given to the fetus. "[E]ach ewe received 1 L lactated Ringer's solution with 5% dextrose by slow intravenous drip during the surgery. At 2 h post-surgery, each ewe received an additional liter of Ringer's solution without dextrose intravenously, and 500 mL lactated Ringer's solution was infused into the amniotic compartment to replace any amniotic fluid lost during surgery. Intravenous and intra-amniotic fluids were not included in the daily water consumption."
Results: When normalized to consumption per 24hr, food and water intake did not differ significantly for the first 5 days after shipping. Therefore it was concluded that regional ground transportation of sheep for 6 to 7 h does not significantly disrupt food and water intake by most late-gestation pregnant sheep. Also, in both experiments (semi-quantitative and quantitative analysis of food and water consumption), post-surgery recovery in food and water consumption did not differ significantly as a duration of acclimation prior to surgery. Therefore it was concluded that 2 d to >=7 d acclimation periods are equivalent with respect to the animal's well-being after surgery and fetal catheterization.
Note: Although not statistically significant, it was observed that food and water consumption did decrease for the first 2 to 4 days after surgery. This may be an indication that this is the most painful period after surgery.
Questions: Questions:
1. What is the purpose of an acclimation period prior to an experimental procedure?
2. T/F Food and water consumption is often considered a reliable indicator of an animal's health and well being.
Answers: Answers:
1. Acclimation periods help to decrease environmental variables and help to identify the true effect of experimental variables.
2. True
Zinc responsive dermatopathy in goats: two field cases. Contemporary Topics 39(4), 32.
Abstract: Two adult female goats used for antibody production presented with symmetrical alopecia, rough hair coat, hyperkeratosis, wt. loss and unthriftiness. Diet was alfalfa which is a legume high in calcium. Calcium is known to block zinc uptake. All goats were housed outdoors with free access to coastal Bermuda hay supplemented with grain conc.
Case 1: Goat experienced tooth loss due to old age and had diet changed to alfalfa/rice/Hematinic plus gruel. Hair loss developed ~ 2 weeks after diet change. Blood work, fecal analysis and skin scrape were WNL. The goat was treated with antibiotic ointment, Animax and miconazole cream as well as shampooed with benzyl peroxide. 4-6 weeks after diet change, hyperkeratosis and symmetrical alopecia developed. The goat was treated by reducing alfalfa, discontinuing the Hematinic and supplementation with zinc. Clinical signs resolved.
Case 2: 1 year later another goat developed similar clinical signs. This goat was still eating coastal hay supplemented with grain but her diet was also changed to alfalfa gruel. Alopecia subsequently developed. Serum zinc (normal) and skin biopsy was performed. Goat was treated and clinical signs resolved.
Questions: Questions:
1. What are normal zinc values for goats?
2. What is hyperkeratosis?
A. hypertrophy of the corneous layer of the skin, aka acanthokeratodermia.
B. persistence of the nuclei of the keratinocytes into the stratum corneum (horny layer) of the skin.
C. dissolution of the intercellular bridges in the prickle-cell layer of the epidermis.
3. What is parakeratosis?
A. hypertrophy of the corneous layer of the skin, aka acanthokeratodermia.
B. persistence of the nuclei of the keratinocytes into the stratum corneum (horny layer) of the skin.
C. dissolution of the intercellular bridges in the prickle-cell layer of the epidermis.
4. What is acantholysis?
A. hypertrophy of the corneous layer of the skin, aka acanthokeratodermia.
B. persistence of the nuclei of the keratinocytes into the stratum corneum (horny layer) of the skin.
C. dissolution of the intercellular bridges in the prickle-cell layer of the epidermis.
Answers: Answers
1. 0.65-2.50 micrograms/L
2. A
3. B, parakeratosis is normal in the epithelium of the true mucous membrane of the mouth and vagina.
4. C, mechanism of formation of the intraepidermal vesicles in phemphigus vulgaris.
Caerulein-induced pancreatitis in Wistar rats. Contemporary Topics 39(4), 36.
Abstract: This paper describes a model of caerulien-induced reversible pancreatitis by using implantable osmotic infusion pumps in conscious unrestrained Wistar rats. The technique reliably induced mild edematous pancreatitis verified by histology. This model has the advantages of unrestrained animals, reduced animal handling and acclimation, and reduced cost.
Questions: Questions:
1. A study that uses unrestrained animals, reduces animal handling and acclimation, and reduces cost is an example of reduction, refinement or replacement?
2. Explain the mechanism of the osmotic pump.
Answers: Answers:
1. Refinement.
2. See the first full paragragh on the right side of page 36.
Metastatic large intestinal adenocarcinoma in two rhesus macaques (Macaca mulatta). Contemporary Topics 39(4), 40.
Abstract: Spontaneous tumors of the large intestine are rare in nonhuman primates, although they are common in humans. Tumors of the large intestine of rhesus monkeys are usually adenocarcinomas, and they arise from colonic epithelium. Some will invade the wall of the colon and form annular,stenotic areas (“napkin-ring� constriction of the colon) while others manifest as irregular thickening of the mucosal surface.
Colonic adenocarcinomas typically do not metastasize although this case report describes metastatic adenocarcinomas of the large intestine of two female rhesus monkeys from a breeding program. “Monkey 1� was13 yrs. old, was repeatedly negative for tuberculosis, and was herpes B antibody negative. “Monkey 2� was 19 yrs. old, repeatedly negative for tuberculosis, and was herpes B antibody positive. Monkey 2 had been ovariectomized at 12 yrs. of age due to the presence of a leiomyoma.
Both monkeys presented with depression and anorexia. In addition, monkey 1 had not defecated for 4 days, and monkey 2 had been vomiting for 2 days. Physical examination of both monkeys revealed pale mucous membs.,delayed capillary refill times, palpable gas in the intestines, and low body temps. Monkey 1 had a firm midabdominal mass that was freely moveable. Contrast radiography (barium enema) suggested a constriction of the proximal large intestine. Ultrasonography confirmed an intestinal mass near the cecocolic junction. Chest x-rays of both monkeys were normal.
Exploratory laparotomies were performed on both monkeys. In monkey 1 a firm mass encompassed the entire ileocecalcolonic junction and the adjacent intestinal serosa was inflamed. Intestinal resection and anastomosis was performed. Also, two enlarged mesenteric lymph nodes were removed. In monkey 2 a fibrous, annular ring constricted the descending colon. In addition,the right medial hepatic lobe had an oblong, pale nodule. Monkey 2 was euthanized while under anesthesia. Monkey 1 was euthanized 2 months after the laparotomy.Both monkeys received complete necropsies.
Necropsy results:
Monkey 1: the primary tumor was a large, poorly delineated,luminal mass at the cecocolonic junction. The mesenteric lymph nodes were irregularly enlarged and effaced by the tumor mass. Monkey 2: the primary tumor was a narrow fibrous, annular constriction of the descending colon. The liver had multiple, discrete,slightly raised nodules up to 8cm in diameter. In both monkeys the lumen of the bowel was reduced but not obliterated.
Histologically, the tumors were classified as adenocarcinomas of the undifferentiated (monkey 1) and tubular (monkey 2)types. A detailed description of the histologic appearance of both the undifferentiated and tubular adenocarcinomas and associated pictures are given in the journal. Metastases were confirmed in mesenteric lymph node and lung of monkey 1 and in the liver of monkey 2.
Spontaneous adenocarcinomas in the colon are common inhumans and often arise from adenomatous polyps. Again, this condition is rarein NHP. In the rare reports of this condition in NHPs, there has been metastasis to regional lymph nodes in 85% of cases, to lung in 24%, and to pancreas and adrenal gland in 15%.
The clinical course of these two monkeys differs from thatin previously reported cases of adenocarcinomas of the large bowel. Typically, symptoms of anorexia, weight loss, diarrhea, and changes in piolosis have been described.
The apparently low prevalence of adenocarcinomas of the large intestine in NHPs vs. humans suggests a difference in genetic and environmental risk factors between humans and non-human primates. Furthermore,the authors report that this condition in rhesus macaques appears to have more to do with environmental risk factors than with a genetic predisposition.
Questions: Questions:
1. The article states that a colony of cotton-top tamarin were reported to have had a high prevalence of colonic adenocarcinomas
conjunction with chronic ulcerative colitis. These tumors had no pretumor stage. How does thisdiffer from colonic adenocarcinomas in man?
2. What is the genus and species of the cotton-top tamarin?
Answers: Answers:
1. Colonic adenocarcinomas in man often arise from adenomatous polyps.
2. Sanguinus oedipus oedipus
Endocarditis and meningitis caused by Streptococcus suis after cardiac surgery in a sheep. Contemporary Topics 39(4), 43.
Abstract: This summarizes a case report involving the demise of a ewe due to meningitis and endocarditis following the implantation and subsequent infection of a cardiac device.
The goal of the study was to evaluate the biocompatibility of a cardiac device in a 38-week-old female Dorset-cross sheep. A left thoracotomy was performed to expose the heart, cardiopulmonary bypass was instituted by conventional ventricular fibrillation, and extracorporeal circulatory support was established for maintenance of anesthesia. A carbon-coated annuloplasty ring was implanted in the mitral position. Surgery and recovery went well and the animal was administered IV gentamicin and IM ceftiofur perioperatively. Ceftiofur was continued for 7 days post surgery. Analgesics were also given perioperatively and postoperatively.
15 days post-surgery, the sheep became anorexic, weak, ataxic and febrile with labored breathing. A CBC revealed a mild leukocytosis with a neutrophilia. Hypokalemia was determined by serum chemistry.
Fluid treatment was initiated with gentamicin and ceftiofur which improved the condition, but the sheep became depressed, anorexic and recumbent, but normothermic 48 hours following the termination of fluid administration. Repeat bloodwork showed a slightly elevated WBC count, due to a marked neutrophilia. Blood was submitted for culture and the animal was given analgesics for 2 days. With no sign of improvement, the animal was euthanatized.
At necropsy, gross lesions were limited to the heart and brain. Heart lesions included vegetative endocarditis of the mitral and tricuspid valves. Brain lesions included meningitis adjacent to the ventral diencephalon and a white exudate from the infundibular stalk.
Histological findings included necrotic areas containing colonies of gram positive coccoid bacteria associated with the vegetative lesions in the heart and similar necrotic lesions of the lateral ventricles also containing gram positive cocci within the meninges. Thrombosis occurred within one area of the kidney.
Streptococcus suis type II was cultured from these areas. The isolate was resistant to cephalosporins, gentamicin, erythromycin, lincomycin, and penicillin. It had an intermediate susceptibility to ampicillin and was susceptible to chloramphenicol. High level aminoglycoside resistance testing showed the organism was susceptible to higher levels of vancomycin, streptomycin and gentamicin.
Cases of endocarditis and meningitis have been reported in humans and swine.
Implant associated infections are important complications and are reported in this article to be characterized by an enhanced pathogenicity of the infecting bacteria, resistance of the organism to host defenses, a protracted indolent course of infection, and some level of antibiotic resistance frequently necessitating removal of the device. The asymptomatic period during recovery after surgery and the subsequent fluctuating clinical course during therapy highlight the features of implanted device-associated infections.
Possible ways the device may have been infected are:
Resident bacterial contamination at the time of implantation or cross contamination from swine housed in an adjacent room and sharing surgical facilities (not at the same time).
Stress may have altered normal GI barriers permitting S. suis to become bloodborne. (S. suis is a commensal of GI tract in ruminants)
Bacteria may have had increased resistance to antibiotics and host defenses by adhering to the fibrin coating on the device and colonizing the thrombus.
- S. suis is killed readily by heat and most common disinfectants including quartenary ammonium products.
- S. suis is a worldwide pathogen of swine and causes outbreaks of meningitis, arthritis, pneumonia, endocarditis, abortions and abscessations. It has also been known to cause pneumonia, meningitis or abscessation in horses, cattle, sheep and cats.
- S. suis is a potential zoonotic agent capable of causing hearing loss, meningitis, endocarditis, arthritis and death. It is sometimes maintained in a carrier state in the tonsils of swine and can be shed during periods of stress, predisposing humans to infections through contact with the swine.
Questions: Questions:
1) True or False: S. suis is a zoonotic agent and can cause severe symptoms such as fatal endocarditis and meningitis, but does not appear to have any detrimental affects on swine.
2) Which of the following is NOT reported to characterize device related infections?
a) Decreased antibiotic resistance
b) Increased bacterial pathogenicity
c) Protracted indolent clinical course
d) Neutrophilia
3) What does indolent mean?
a) slow growing
b) undetectable
c) waxing and waning
d) overwhelming
Answers: Answers:
1) False --S. suis can cause pathology in swine
2) A -- Characterized by increased antibiotic resistance
3) A -- In Bailliere's Comprehensive Veterinary Dictionary (Blood and Studdert) it is defined as "causing little pain; slow growing"
Quantitative competitive PCR. Contemporary Topics 39(4), 47.
Abstract: A particular DNA concentration in a sample can be measured by using a technique called quantitative, competitive polymerase chain reaction (PCR). A different size but otherwise identical competitor DNA is synthesized displaying the same primer biding sites as the target DNA. PCR is then initiated in the presence of a constant amount of target DNA (sample) and different amounts of competitor DNA (e.g. serial dilution). The amount of PCR product for both target and competitor DNA is then visualized by ethidium bromide staining of an agarose gel and measured densitrometrically. Plotting the signal as the log of the standard/unknown against log standard results in a straight line and the intercept of this line with the x-axis is identical with the concentration of DNA in the sample. The same technique can be used to measure the amount of RNA in a sample. To accomplish this RNA is first reverse transcribed into cDNA and then processed just like DNA. This test is then called competitive RT-PCR, RT for reverse transcriptase.
Questions: 1 What does RT-PCR stands for?
a) Reverse Transcriptase Polymerase Chain Reaction
b) Recreational Traffic Polymerase Chain Reaction
c) Reverse Transcriptase Poly Clonal Response
d) Reverse Temperature Polyclonal Response
2 What is Quantitative Competitive PCR used for?
a) To find specific viral DNA
b) To find RNA viruses
c) For quantitation of specific DNA in any given sample
d) For quantitation of specific RNA in any given sample
3 Against which standard is a cDNA target sequence measured
a) against a competitor DNA fragment of different size
b) against a competitor cDNA fragment of equal size
c) against a competitor cDNA fragment of different size
d) against a commercially available cDNA standard of variable size
Answers: 1a, 2c, 3d,1a, 2c, 3d