Lab Animal Vet: Answers Q 31-40:

Q35: Immune Deficient Mouse Models

1. List 6 components of the innate immune system

Epithelial barriers

Inflammation stimulated by chemical factors released by injured cells, initiated by cells resident in tissues such as macrophages and dendritic cells.

Phagocytes

macrophages - engulf bacteria and microbes, killing via merging with lysosomes; bacterial binding on surface causes generation of a "respiratory burst" and release of reactive oxygen species.
neutrophils -generate respiratory bursts of oxidizing agents such as hydrogen peroxide. Kill and inhibit bacteria and fungi
Dendritic cells (Langerhans in skin) - phagocytes that also present antigens

Natural killer NK cells - destroys compromised host cells such as virus-infected or tumor cells by recognizing lower than normal levels of MHC I cell surface marker.

Gamma delta T cells respond to molecules common to many microbes, ie acting as 'pattern recognition receptors'.

complement system - alternative pathway

mast cells in connective tissue and mucous membranes - release histamine and recruit neutrophils and macrophages.

eosinophils - secrete free radicals and toxic proteins that kill parasites

basophils - release histamine and work vs parasites

2. In which 2 cells do the innate and adaptive arms of the immune system interact?

Gamma delta T cells - rearrange T cell receptor genes so as to provide T cell memory (adaptive) in addition to acting as pattern recognition receptors in response to molecules present on a wide variety of pathogens (innate).
Dendritic cells - act as phagocytes (innate) and antigen presenting cells (adaptive).

See Wikipedia for a good summary!

3. NOD scid gamma (NSG) mice are a popular mouse strain for engraftment of human immune cells. a. Describe the 3 major components of their immune deficiency

No B cells
No T cells
No NK cells

b. Which populations of immune cells remain?

Monocytes
Neutrophils
Defective dendritic cells and macrophages

c. Why are they likely to live longer than scid mice in a protected environment?

No thymic lymphomas

(See Jax website for a good description)

4. Myd88 mice are commonly used for infectious disease and immunology studies.

a. What does Myd88 stand for,?

Myeloid differentiation primary response gene 88

b. what is the mechanism for their immune deficiency?

The MyD88 protein is a cytoplasmic adaptor molecule essential for signaling of IL1 and Toll-like receptor family. It is essential for cellular responses to IL1, IL18 and bacterial cell wall components (lipopolysaccharide, peptidoglycan and lipopeptide).

c. What clinical problems are most likely to be seen?

Bacterial infections - resulting in morbidity and mortality. See this case report

5. The various immune deficient strain combinations can be hard to memorize: a good start is to consider all the different components so that you can deduce characteristics of the combinations. List the complete name, common name (if applicable) and the main immune deficiencies for the following strains... (NOD scid gamma were already listed in Q1)

a. RAG1 or RAG2

Recombination activating gene 1 or 2.
Essential for V(D)J gene rearrangements needed for Ag receptors in T and B cells
No T or B cells

b. Bg

Beige
Lysosomal trafficking regulator, beige (Lyst superscript bg)
deficient in NK cells
also defective cytotoxic T cell and cytotoxic antibody response to tumor cells, defective bactericidal activity of granulocytes.
Note: Chediak-Higashi model

c. Foxn1 nu

Nude
Forkhead box N1
athymic, no T cells
Single base pair deletion -> frameshift and premature stop codon.

d. Il2rg

Interleukin 2 receptor gamma chain
no NK cells or Peyers patches (or gut intraepithelial lymphocytes)
Reduced T and B cells,

e. Prf1

Perforin 1
Defective cytotoxic T cell and NK cell cytolysis

f. B2m

Beta-2 microglobulin
No MHC class I Ag on their cells
Deficient in CD4- and CD8+ T cells which mediate cyotoxic T cell function

Q34: Treating Abnormal Behaviors

What are some common abnormal behaviors in NHP?

Excessive grooming
Saluting (NHP)
Stereotypies
Floating limb
Self biting or self injury

If a basic behavior loop is stimulus -> internal processing -> behavior -> feedback, which of those steps can most be influenced by veterinary medicine alone (i.e. without behavioral management techniques)? Which can be influenced by behavioral management?

Vet medicine can provide input and measure output of internal processing, and measure output of the behavior. Behavioral management can provide input and get output from every step.

Barbering or hair pulling is a relatively common behavior in multiple species, including humans.

What is thought to be the primary physiologic mechanism,

oxidative stress producing free radicals which are destructive

What is the usual treatment?

Anti oxidants particularly N-Acetylcysteine

Why does it have a wide safety margin?

Benzodiazepines are sometimes suggested for treatment for behavioral problems

How do benzodiazepines work in the brain?
They readily cross the blood brain barrier and act on the inhibitory neurotransmitter GABA (gamma aminobutyric acid)
True or false: benzodiazepines are useful for a wide range of problem behaviors
False. It makes many conditions worse, and causes disinhibition. Drug tolerance readily develops and withdrawal is unpleasant and anxiogenic.
For what kind of conditions would they be appropriate to use?
Management of known stress triggers such as transportation or cage move - and immediate treatment of acute crises.
When should they never be used?
Fear aggression.

5. Self-injurious behavior

What conditions of rearing predispose an animal to develop self-injurious behavior?

Abnormal rearing particularly isolation rearing and early weaning

What is thought to be the neurochemical deficit in self-injurious behavior? Serotonin deficit

Which amino acid has proven useful in treating abnormal behaviors due to abnormal rearing? tryptophan

What category of drugs with a similar downstream mechanism to Q3 effects is often useful for self injurious behavior? SSRI's (selective serotonin reuptake inhibitors)

Which member of this drug category takes up to a month to work, has off-target effects and has withdrawal effects? Prozac/fluoxetine

Which member of this drug category is not useful >8 weeks but has no withdrawal effects? Buspirone

Which alpha 2A adrenergic agonist has proven useful in the most severe cases of self injurious behavior in NHP? Guanfacine

What are some advantages of this drug? It shows minimal tolerance and sedation, it allows pulse dosing and has post-treatment efficacy.

What are some disadvantages? It can be very expensive depending on whether you buy the formulation for ADHD (expensive) or blood pressure (cheap), it requires very large doses compared to the human dose, and it has effects on blood pressure.

Which drug could you use occasionally prior to a known trigger for self injurious behavior to help prevent it? Midazolam.

6. General concept: What effect on behavior would you expect on withdrawing drug treatment therapy? Exacerbation of undesirable behavior.

Some references:

Risk factors for stereotypic behavior and self-biting

Physiology and neurochemistry of self injurious behavior

Q33: CRISPR-Cas9

1. In what categories of organism was the CRISPR-Cas9 system discovered?

Bacteria and archaea(single celled organisms with no nucleus)

2. What was its "native" purpose?

Microbial cellular immunity vs. viruses

Back in 1993, palindromic repeats ( In a palindromic repeat, the sequence of nucleotides is the same in both directions) of DNA containing fragments of other genes were found in prokaryotes. Many years later it was discovered that the CRISPR sequences were the bacterial version of the immune system vs. viruses (or bacteriophages). CRISPR has an 'CRISPR ASsociated' Cas9 nuclease (an enzyme that cleaves the chains of nucleotides in nucleic acids into smaller units) that creates a double stranded break in the viral DNA and inactivates it. The nuclease is targeted to a particular part of the virus by a Guide RNA, and part of the destroyed virus is then saved within the palindromic repeats as the bacterial immune system memory, so it is primed to destroy future attacks by the same virus.

3. What does CRISPR Cas9 stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR associated endonuclease protein

4. If we say that transgenic mice are created by inserting genetic material into the male pronucleus of the oocyte by microinjection, and gene-targeted (knockout) mice are created by inserting genetically modified ES cells into the blastocyst by microinjection, how are CRISPR-Cas9 mice created?

CRISPR-Cas9 mice are created by causing a point mutation in the developing embryo. The resulting organism thus has a precisely targeted mutation and does not require further breeding or manipulation to obtain that result.

How is this better than conventional methods such as transgenesis or gene-targeted knock-ins and knockouts? Well both of those methods were limited in their species applicability, were highly technical and resulted in low percentage success rate and a long timeline. With transgenic mice, because the genetic material is injected into the pronucleus of the unfertilized egg(oocyte), you end up with random insertion of genetic material at multiple sites along the chromosome, so each injection created a different "version" of the inserted genetic material. Further, the resulting mouse doesn't have the corresponding male half of the chromosome so the mice are hemizygous. Targeted mutations in contrast do provide insertion or deletion of genes at a site targeted by homologous recombination, but the process requires manipulation of ES cells and injecting the modified cells into a blastocyst. The result is a chimeric animal and multiple subsequent generations of breeding are required to determine if the gene went germline, and then to standardize the gene on an inbred background because the genotypes that were easily manipulated were rarely the desired genetic background.
In contrast CRISPR Cas9 is applied directly to an embryo and results in a precisely targeted point genetic modification.
5. True/False: CRISPR Cas9 has been used to make genetic modifications in plants, multiple animal species and human genetic material.
TRUE

6. From which organism is the most commonly-used type of Cas9?

The most common are the Cas9 nucleases modified from the Streptococcus pyogenes (spCas9). However others are available, such as the Cpf1 (CRISPR from Prevotella and Francisella), which provides a staggered DS DNA cut better for homology-directed repair, or SaCas9 from Staph aureus, which is much smaller than SpCas9 so it can be packaged in an Adeno-Associated Viral Vector for delivery into the cell.

7. What 2 functional components constitute the CRISPR-Cas9 system?

A Guide RNA that targets the part of the genome you want to edit, and a Cas9 endonuclease, an enzyme that creates a double stranded break in the DNA at that site.

8. True of false. The CRISPR Cas9 system relies upon the host cell-repair system to function.
TRUE. The simplest use of CRISPR-Cas9 is to allow the cell's own repair mechanism to repair the break using non- homologous end-joining, thus inactivating the gene due to a frameshift mutation.

9. True or false: CRISPR Cas9 can be used to knock out genes, to knock in genes and to study the genome wide function of genes and non-genomic DNA through interference.
TRUE.

knockouts are the simplest type of CRISPR Cas9 operation. What else can it be used for? CRISPR Cas9 can be used to "knock in" genes by inducing cells to repair the break by homology- directed repair. In this case by providing copies of homologous DNA for the cell to use as it repairs the break.

CRISPRi is the CRISPR equivalent to RNA interference (RNAi). In this case the Cas9 is mutated so that it binds to its target but does not cause a break. However the binding of this inactivated Cas9 to the DNA now blocks the cell's transcription machinery from accessing that DNA, silencing gene expression. Because CRISPRi has higher efficiency and lower off-target effects than RNAi, it is now replacing RNAi in large scale genome screening to determine the function of individual genes.

10. True or false: to date CRISPR Cas9 improves upon existing methods for gene modification in all areas.
FALSE. CRISPR still has problems with off-target effects see this reference (although less so than its predecessors, Zinc fingers and TALENS) and to date the number of available PAM (protospacer adjacent motif)sequences is limited. What are PAMs? The PAMs are species-specific sequences that are part of the guide RNA. They are necessary to allow the CRISPR complex to distinguish non-CRISPR sequences from those same sequences that are contained within the CRISPR itself (ie originally, those that were incorporated as a result of a prior exposure of the bacterium to the invading virus). I'm not explaining this very well - for a really nice explanation try this short video

For more information:
A Guide for Beginners
The Heroes of CRISPR
Is CRISPR ready for treating human disease?
CRISPR review 2018

DISCLAIMER: I am not a molecular biologist. Please contact me if there is information I can correct... :)

Q32: Ferrets

1. Unbelievably, ferrets have been domesticated for more than 2000 years. They belong to the subfamily Mustelinae which contains 5 subgenera - name the subgenera and their common names.
Family: Mustelidae
Subfamily: Mustelinae
Genus: Mustela
Subgenera: Mustela (weasels): Lutreola (European mink); Vison (American mink), Putorius (ferrets); Grammogale (S American weasels).
Ferret: Mustela putorius furo

2. The ferret is being used to replace the cat in which (general) types of experiment? - according to the BLUE BOOK, neuroendocrinology (eg https://www.ncbi.nlm.nih.gov/pubmed/17459108 ), neuroanatomy (eg https://www.ncbi.nlm.nih.gov/pubmed/18986853,) cardiology, due to relatively common occurrence of dilated cardiomyopathy, arrhythmias and valvular disease (https://www.ncbi.nlm.nih.gov/pubmed/19131034 ).

3. What special light considerations are needed to maintain successful breeding in ferrets in the lab?
For breeding and lactating Jills, 16h of light daily, and a winter season of 14h dark for at least 6 weeks. Disruption of the usual photoperiod can cause inappetance and subsequent pregnancy toxaemia.

4. What features of the gastrointestinal tract are reflective of the fact that they are strict carnivores?
Monogastric stomach, no cecum, very short GI tract resulting in fast GI transit time (as short as 3h).

5. What features of their reproductive cycle make them good research subjects for study of the HPA axis?
Estrus is readily detected by vulvar swelling facilitating the study of the effect of sex hormones on the adrenal hypophysis- pituitary- hypothalamic feedback loop.

6. Which organism commonly causes acute abdominal distension with dyspnoea and cyanosis in weanlings subjected to sudden dietary changes or overeating?
(toxin production by )Clostridium perfringens Type A (previously known as Clostridium welchii) causes death exclusively in weanlings. Treatment after the fact is unrewarding, therefore prevention is paramount.

7. Which organism is widespread in ferrets, often asymptomatic, but can cause gastritis and peptic ulcers? How would you treat it? Helicobacter mustelae. Triple therapy with amoxicillin, metronidazole and bismuth (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC171790/ ) or ranitidine bismuth and clarithromycin (http://agris.fao.org/agris-search/search.do?recordID=US2000002937 )

8. Which organism is commonly associated with proliferative bowel disease? Lawsonia intracellularis. It has been isolated from proliferative enteropathy in swine, horses, hamsters, deer and ostriches. (https://www.sciencedirect.com/science/article/pii/S0378113596012643 )

9. What is the most serious viral disease of ferrets causing almost 100% mortality? Describe the phases of this disease. Canine distemper caused by a paramyxovirus genus Morbillivirus
Catarrhal phase 7-10 days postinfection (anorexia pyrexia photosensitivity, nasal discharge, rach +/- hardpad)
CNS phase: (ataxia tremors paralysis) -> death in 12-16 days (ferret strain) to 35 days (canine strain)

10. What viral disease would you suspect if the principal signs were hypergammaglobulinemia and chronic weight loss? What is the most consistent histological finding with this disease? Is there a vaccine available?Aleutian disease. periportal lymphocytic infiltrates in the liver. No vaccine due to immune-mediated pathogenesis.

11. Which disease is a zoonosis and has been used extensively to model the disease in humans?
Influenza caused by an orthomyxovirus.

12. What clinical chemistry abnormalities are found in 70% or more of ferrets with pregnancy toxemia? According to https://www.ncbi.nlm.nih.gov/pubmed/10480641anemia (4 of 8 jills tested), hypoproteinemia (5 of 7), azotemia (7 of 7), hypocalcemia (5 of 6), hyperbilirubinemia (3 of 3), and high liver enzyme activities (6 of 6).
What percentage of ferrets with this condition will be anemic? 50% see above.

13. What is the treatment for hyperestrogenism?
As this condition, resulting in bone marrow suppression, is caused by prolonged estrus due to failure to mate, treatments are supportive (eg blood transfusion, antibiotics) and aimed at taking the ferret out of estrus (eg HCG injection, spay). See https://www.all-about-ferrets.com/wp-content/uploads/hyperestrogenism_in_ferrets.pdf

14. Which 4 types of cancer account for most ferret neoplasms?
pancreatic islet cell tumors, adrenocortical cell tumors, lymphoma and skin cancers

15. In aging ferrets, what common condition presents with weight loss, lethargy and anorexia?
Cardiomyopathy

Q 31: Unusual Rodents

1. Match the following rodents with their suborders:

Suborders:

Sciuromorpha : squirrels, prairie dogs
Hystricomorpha: guinea pigs, degus, chinchillas, mole rats,
Myomorpha: mice, hamsters, gerbils, voles
Castorimorpha: pocket gophers, kangaroo rats

Rodents

Gambian pouched rat

2. Why is there a ban on import of exotic African rodents for the pet trade? Which of the rodents was involved in the incident that resulted in this ban?

This ban was put in place in 2003 as a result of Prairie dogs transmitting monkeypox to humans - they got it from being cohoused with Gambian pouched rats. http://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0004130 Gambian pouched rats are used to clear mines in eg Mozambique - they are nocturnal so have poor eyesight and excellent sense of smell. They are trained to detect TNT in landmines - this takes around a year of training- they use clicker training and banana rewards - they are too light to set off the mines . https://www.youtube.com/watch?v=L0swUc492hU

3. What is the genus and species of the kangaroo rats?
Dipodomys spectabilis and D. merriami. Kangaroo rats often leap a distance of 6 feet...!https://www.youtube.com/watch?v=gORCCxPG_cU

List 3 uses in research and indicate what features of the kangaroo rat make them useful for these models.
a. Renal physiology - they are adapted for water conservation, their urine is 5x more concentrated than humans
b. Disuse osteoporosis and effects of space walking or microgravity. Their hindlimbs show decreased mineral content in captivity.
c. Decompression sickness. https://www.ncbi.nlm.nih.gov/pubmed/734799 They get gas bubbles in between tendon bundles of the tail similar to limb bends seen in humans subjected to overly fast decompression.

Image result for squirrels hibernate

4. Which of the rodents listed in Q1 hibernate?
UPDATE: Got some excellent new information from Frank van Breukelen who works on hibernation. I'm quoting some of his response here...
"I work on hibernation in tenrecs and the hibernation community is somewhat confused as to what is hibernation and what's not... Sciuromorpha contains marmots, ground squirrels, dormice, etc that are all excellent hibernators. In fact, I would say ground squirrels are the best hibernators. Black-tailed prairie dogs are not very good at hibernating but white-tailed prairie dogs are okayish (see work by Hank Harlow; ~5 day torpor bouts but not everyone goes torpid in the lab). Back in the day, Fritz Geiser was bringing in a large number of Australian marsupial species to the lab and found quite a few didn't hibernate. Later, he used iButtons to track Tb and it turns out that most of them hibernate in the wild. Soooooooo, I have a hard time wrapping my head around what hibernates and what doesn't especially when work was done in the lab. A couple of years ago, Sandy Martin and I wrote a mini-review talking about a continuum of hibernation e.g. some are better than others and there's a good variety of patterns."
Thanks for the more accurate information Frank!
ps I don't feel too bad because there is obviously some debate about what constitutes "true" hibernation - Julie

5. What are the genus and species of the 2 deer mice?
Peromyscus maniculatus, P. leukopus

List 2 major N American zoonotic diseases for which they are a vector.
Hantaviruses, Lyme disease, https://www.cdc.gov/hantavirus/rodents/index.html note cotton rats and rice rats also carry hantavirus and grey squirrels are another reservoir host for Lyme disease https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2592199/.

Bonus question: how many other zoonotic diseases can you name that they can be a vector for?
Trypanosomiasis, toxoplasma, tularemia, Cryptococcus, Giardia, Leptospirosis, Hymenolepis, Rocky mountain spotted fever, chlamydiosis, Q fever, E adn W encephalitis, plague, coccidioidomycosis (!)

short tailed cane mouse

6. Which rodent (genus species, common name) is the natural host for Venezuelan hemorrhagic fever?
Short tailed Cane mice (Zygodontomys brevicauda ) are the reservoir host. VHF is caused by Guanarito virus which is an arenavirus.

Cotton rat

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7. How do you collect blood from the cotton rat? (hint:it's not the usual site)

Subzygomatic sinus.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314528/
Cotton rats were so-named because they make their nests out of cotton and destroy cotton crops. Sigmodon means 'S-tooth' Hispidus means "rough dirty hairy" which describes their rough coats - might be useful for id purposes. in LatinNative to S. USA central and S America.

marsh rice rat

8. Two of these rodents are used to study dental disease. Which rodents and which diseases are they used for? the role of diet in periodontal disease was discovered in the 1950's in rice rats (Oryzomys palustris) they get a rapid progression to destructive periodontal disease starting as early as 16d and accelerated by sugary diet but retarded by a diet low in carbohydrates. (. 1979 Aug; 96(2): 643–646.) Hamsters were used to discover the role of Streptococcus mutans in caries production - serendipitously golden hamsters were found to have caries whereas albino hamsters did not. The disease was found to be transmissable from the golden hamster to the albino hamster by putting feces from the golden hamster into the albino hamsters' cage. These days models are designed to investigate the role of various organisms in eg humanized mice. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2649707/) From a disease/unusual rodent standpoint, degus, suffer from dental disease, malocclusion and elodontomas, and prairie dogs get elodontomas - benign expansile masses of the odontogenic epithelium of the maxillary incisors.

multimammate rat

9. Which rodent is the model for Zollinger Ellison syndrome? What is Zollinger Ellison syndrome?
Mastomys natalensis, or multimammate mouse/rat. ZE syndrome is a condition in which a gastrin-secreting tumor or hyperplasia of the islet cells in the pancreas causes overproduction of gastric acid, resulting in recurrent peptic ulcers. In Mastomys the hypergastrinemia is caused by gastric carcinoid.

10. Which rodent is carnivorous?
https://www.wired.com/2012/02/mouse-howls-like-a-wolf-bites-like-a-tiger/
Grasshopper mouse Onchomys torridus or leukogaster. They mostly eat insects, especially grasshoppers, beetles, crickets, and scorpions, but also stalk, kill, and eat other small rodents such as kangaroo rats, white footed mice and voles (From Encyclopaedia Brittanica). Same sex animals will kill each other in captivity.

11. Identify these rodents (common name, genus and species) - as a lot of them look similar- I've provided some hints!