Comparative Medicine
Volume 55, Issue 3 (June 2005)
Behavioral and Clinical Pathology Changes in Koi Carp (Cyprinus carpino) Subjected to Anesthesia and Surgery with and without Intra-Operative Analgesics (pages 221-226)
The authors discuss the increased use of fish in the biomedical research. Surgical procedures have been performed on fish in research settings for a long time and are now being performed regularly on client-owned and display aquarium fish. However, fish surgery have been reviewed just recently and includes procedure for tissue or organ ablation, catheterization for fluid collection or drug delivery. Even if cold blooded animals are exempt from the Animal Welfare Act, institutions receiving PHS funding should extend coverage to all vertebrate animals used in research. Current studies involving fish surgery do not incorporate protocols to provide postoperative analgesia. Although the neuroanatomy of fish has called into question anthropomorphic interpretation of pain perception, it is believed fish do exhibit strong aversion to acute noxious stimuli and subtle behavioral and color changes in response to chronic stimuli. Effects of surgery on fish behavior and physiology that could alter research results are under investigation. In particular, analgesic technique that could alleviate surgical impacts have not been evaluated in fish.
Materials and methods
Pilot study was conducted in conjunction with a fish medicine and surgery course for veterinary students under a protocol approved by NCSU IACUC. Six Koi carp (Two groups of three) were used for exploratory celiotomy. MS-222 used for anesthesia. One group received butorphanol 0.4 mg/kg im and the other group an equal volume of sterile physiologic saline solution im immediately after skin closure. Fish were returned to individual 55 liter water tank after surgery. Thirty fish (Three groups of 10) were used for the actual experiment (exploratory celiotomy). One group received butorphanol 0.4 mg/kg im. The other received ketoprofen 2 mg/kg im and the third group received physiologic saline solution im. MS-222 at 100-200 mg/liter is used for anesthesia. All doses adjusted to equal volume per body weight of fish. Experiment was conducted using standard husbandry practice for housing and under approved protocol by NCSU IACUC. Fish were evaluated for vertical position in the water, caudal fin beat rate, and response to food before and after surgery. Hematocrit, total solids, plasma cortisol, and plasma chemistry panels were compared before and after surgery. Data was analyzed using statistical software.
Result and Discussion
For all fish, surgery resulted in reduced activity, lower position in the water column, decreased feeding intensity at different time point post surgery. Butorphanol treated group didn’t show significant change from pre surgical behaviors. Decreased hematocrit, total solids, phosphorus, total protein, albumin, globulin, potassium, and chloride and increased plasma glucose, aspartate aminotransferase, creatinine kinase and bicarbonate. The only difference is a lower increase in creatinine kinase in ketoprofen treated group. The result showed a mild behavioral sparing effect of butorphanol and reduced muscle damage from the anti inflammatory activity of ketoprofen. No adverse effect of both butorphanol and ketoprofen identified.
Questions:
1. True or False. Interspecies variation due to opiod sensitivity is not influenced possibly by water (body) temperature and has no effect on the therapeutic index of opiods in fish.
2. The animal welfare act (AWA) currently exempts cold-blooded animals from Institutional Animal Care and Use Committee
3. Cortisol production is considered as an equivocal indicator of pain and analgesic effect.
4. Name the Genus and species of Koi Carp
5. The use of Ketoprofen is limited due to its
a. Effects on homeostasis
b. Gastric ulceration
c. Hepato and nephro-toxicity
d. All of the above
6. Butrophanol is a
a. Synthetic Opoid antagonist Class III
b. Synthetic Opoid agonist, Class III
c. Synthetic Opoid agonist. Class IV
d. Synthetic Opoid antagonist, Class IV
7. List few advantages associated with the use of Ketoprofen over Butorphenol?
Answers:
1) False
2) True
3) True
4) Cyprinus carpio
5) D
6) C
7) Less muscle damage, longer duration of action and not a controlled drug
Genetic Characterization of Indian-Origin and Chinese-Origin rhesus Macaques (Macaca mulatta) (pages 227-230)
Chinese origin macaques have been used with increasing frequency over Indian origin macaques because the supply of Indian-origin animals has been discontinued. For some disease models developed in the Indian-origin macaque, Chinese-origin macaques may respond differently, as with experiment infection with SIV as an example. Class I and Class II major histocompatibility loci have been used to distinguish the tow groups of animals from each other but the theory for this paper is that loci that evolve more rapidly than coding regions such as microsatellite (short tandem repeat loci [STR]) and the first hypervariable segment of the mitochondrial DNA genome would be much more effective in differentiating among closely related populations of the same species.
Buffy coats, serum and DNA samples of 132 Indian macaques and 168 Chinese macaques were studied and compared against the reference 835bp fragment form a Barbary ape.
Mt-DNA results: There were multiple polymorphic sites unique to Indian and Chinese rhesus macaques. Ind1, Ind2 are the 2 halotypes for Indian macaques. ChiE, ChiW1, ChiW2, Chi3 are the 4 halotpyes for the Chinese rhesus macaques.
STR Results: three STR loci showed the highest frequency of diversity between Indian and Chinese origin animals. D1548 occurred in Indian animals only, not Chines origin animals. The other two loci, D18s547 and DXs2506 exhibited difference at specific alleles as well, but they were found with varying frequency in both popoluations of animals. For example in Indian rhesus 79% of all the alleles demonstrate the 155 and 166bp alleles in the D18s537 locus, while only 13% of the alleles in Chinese rhesus macaques exhibit this.
Questions:
1. What is the genus and species:
Barbary ape
Rhesus macaque
Taiwanese macaque
Japanese macaque
2. What is a) MHC, b) mtDNA, c) Micorsatellite loci or STR, d) allele??
3. Why is it important to distinguish between Indian and Chinese macaques??
Answers
.Barbary ape=M. cyclopis
Rhesus macaque=M. mulatta
Taiwanes macaque= M. cyclopis
Japanese macaque= M. fuscata
2. a) MCH= major histocompatibility complex, a group of linked loci that codes for cell-surface histocompatibility antigens and is the principal determinant of tissue type and transpalnt compatibility. These cell surface markers are vital elements in cell-cell recognition, and are the proteins which determine the level and tyhpe of immune response.
b) mtDNA= mitochondrial DNA. Extranuclear DNA that is located in the mitochondria. There is little change in mtDNA from generation to generation.
c) A microsatellite is a short block of DNA sequence (or short tandem repeat sequences), often less than 150 base pairs long, that is repeated many times within the genome of an organism. Many repeats tend to be concentrated at the same locus. In a microsatellite, the repeated sequence is very simple, consisting of two, three or four nucleotides (di-, tri-, and tetranucleotide repeats respectively), and can be repeated 10 to 100 times. Classically, the first microsatellites identified were dinucleotide repeats often called "Ka-Ka" repeats or "CACACACACA....". These are highly frequent in human and other genomes, and present every few thousand basepairs. Variability or dynamism of a dinucleotide repeat is typically a function of size: repeats larger than 10-15 repeats and without interruptions, tend to be polymorphic. Larger repeats often are more polymorphic. The number of repeats at a particular locus is hypervariable or highly polymorphic between individuals of the same species. It is for this reason that microsatellite sequences can be used for genetic fingerprinting. Most loci of the genome, even non-coding parts, would be too similar to allow individuals to be reliably distinguished. They are often less useful in evolutionary studies since they are unstable and prone to mutating to different sizes. The hypervariability arises because the repeated simple sequences cause a high frequency of loss or insertion of additional repeats by confusing the DNA replication machinery. Self-complementary sequences may aid this process.
d) An allele is any one of a series of two or more different genes that may occupy the same locus on a specific chromosome.
3.) Itis important to distinguish between to the two species because the Chinese macaque may not display the same phenotype as the Indian macaque, and therefore may not respond the same way when used as a model of human disease.
Squamous Cell Carcinomas of the Skin at Ear Tag Sites in Aged FVB/N Mice (pages 231-235)
The authors researched the development of cutaneous squamous cell carcinomas (SCC’s) that arose adjacent to nickel-copper alloy ear tags used for identification of incipient congenic FVB/N mice that contained the human BCL6 transgene (n=68), their littermate controls, and wild-type FVB/N. Some of these mice had been involved in previous studies involving ENU (n=47). During the median observation period of 25 months (n=160) 14 (8.8%) developed SCC’s in the tagged (Rt) ear and 0 mice developed SCC’s in the opposite ear. There was no statistically significant difference among the groups of mice in terms of SCC incidence.
The authors note that while a variety of other tumor types have been previously attributed to metallic devices this is the first report describing SCC’s associated with ear tags.
Interestingly, 9 additional SCC’s were noted at sites other then the ear tagged site. Those included 3 [lower esophagus (one of which metastasized to the lungs, liver, pancreas and pelvis)], 1 (oral), 5 (skin other sites). At death the 14 mice with SCC’s at the ear tag site ranged in age from 331 to 921 days with a median age at death of 693 days. Histologically, the majority (64%) of SCC’s were considered high grade malignancies. The authors emphasize, the FVB/N mouse strain is known to be particularly susceptible to the development of squamous cancers and the chronic presence of the metal (especially nickel/copper) is likely to have contributed to the high incidence of SCC’s at the ear tag site in this population.
Questions:
1. What metals are typically present in common identification ear tags?
2. What is the definition of incipient?
3. What tumor type is associated with the FVB/N strain of mouse?
Answers:
1. Nickel-Copper alloy
2. Beginning to exist or appear
3.Squamous Cell Carcinoma
Green fluorescent protein (GFP)-expressing tumor model derived from a spontaneous osteosarcoma in a vascular endothelial growth factor (VEGF)-GFP transgenic mouse (pages 236-243)
A spontaneous green fluorescent protein (GFP) expressing osteosarcoma occurred on in a congenic vascular endothelial growth factor (VEGF) transgenic mouse. This tumor model Os-P0107 is significant because VEGF _expression was can be used to identify early recurrence of osteosarcoma in clinical patients. The tumor was highly transplantable and the latent period was 8 days. The take rate was 100% during the first five generations. The tumor had a high metastatic potential. All 17 mice developed lung mets, 5 mice developed lymph node mets, 2 mice developed skeletal mets. All tumor cells strongly expressed GFP allowing visualization of the tumor cells in the dorsal skin fold chambers and of lung micrometastasis. Real time tumor angiogenesis, tumor growth, and tumor call migration can be visualized by the GFP fluorescence.
Questions:
1. VEG mediates ______, ______and ______.
2. The highly transplantable, highly metastatic, osteosarcoma, GCF expressing tumor model is named ______.
3. What is the latency time of this tumor?
4. What is the most common site of metastasis for this tumor?
Answers:
1. tumor angiogenesis, growth, metastasis
2. Os-P0107
3. 8 days
4. lungs
Validation of an Enzyme-linked immunosorbert assay kit using herpesvirus papio2 (HVP2) antigen for detection of Herpesvirus simiae (B virus) infection in rhesus monkeys (pages 244-248)
Serologic testing for Herpes B in macaques is problematic due to the biohazardous nature of Herpes B antigens used in the commercial ELISAs available. This article summarized the comparison of a Herpesvirus papio 2 based ELISA to the commercially available test system. After testing 447 samples from 290 rhesus monkeys, statistical evaluation showed a high degree of correlation (weighted kappa coefficient 0.94) with a 95% confidence interval of 0.91 to 0.97 between the two tests indicating the HVP2 ELISA a sensitive and reliable assay for in-house testing of rhesus monkeys.
Herpes B (also known as cercopithecine herpesvirus 1, herpesvirus simiae, and monkey B virus) is an alpha-herpes virus that is endemic in macaques. While generally producing localized and self-limiting herpetic lesions and latency in its natural host, infection is highly pathogenic for humans (with a mortality rate of 60-70% in the absence of treatment). This zoonotic concern has lead to the goal of establishing specific pathogen free colonies for use in biomedical research, thus creating the need for in house testing mechanisms.
Herpesvirus papio 2 is an alpha-herpes virus endemic in baboons that is genetically and antigenically similar to Herpes B but does not have the associated biohazard concerns. Developing a testing system using Herpesvirus papio 2 would eliminate the biohazard concerns of working with B virus antigens in the test system.
Questions:
T or F
1) Herpes B virus is endemic in rhesus macaques but not Japanese macaques.
2) Herpesvirus Papio 2 causes pathogenic infection in humans with a 60-70% mortality.
Answers:
1) F ( endemic in all macaques)
2) F ( Herpes B causes pathogenic infection with 60-70% mortality.
Circadian Temperature Rhythm of Laboratory Swine (pages 249-255)
Mammalian circadian rhythm (the biological clock) is based in the hypothalamic suprachiasmatic nuclei, this is reset daily by such environmental synchronizers as photoperiod, sleep/wake cycles, and daily routines. Core body temperature, cortisol, and melatonin are reliable biomarkers of circadian rhythm in humans and swine. Circadian temperature rhythm (CTR) profiles were monitored using an ear implanted probe for 5 1/2 - 9 1/2 consecutive days. Eight of the nine (89%) pigs had CTR with a mean period (± standard error) of 23.6 (0.5) hours, amplitude of 0.18 (0.02)° C (half of the distance between the highest and lowest value within the period), mesor of 38.7 (0.24)° C (rhythm-adjusted mean), and acrophase at 19:44 hours (timing of the cosine maximum). A noninvasive tympanic membrane temperature sensor was fashioned from the YSI 400 series skin surface thermistor probe (Steri-probe) for connection to a portable physiologic data logger. Ear-based instrumentation acute stress was reflected in attenuated CTR amplitude during the first 3 days after instrumentation. To determine the effect of procedural stress on CTR, each subject’s time series were separated into two subsets: the first 3 days after instrumentation and subsequent days of analysis.