Fun facts answers 11-20


Q20: Pneumocystis


1. True/false. Pneumocystis was first discovered in 1909 and was thought to be a stage in the life cycle of Trypanosoma cruzi     TRUE.... Discovered by Chagas... for whom Chagas disease (caused by T cruzi) is named.
2. True/false.  Pneumocystis is only a clinical problem for immune-suppressed animals  FALSE...  Although in most species Pneumocystis is a true opportunist and found in immune deficient animals, normal immune competent rats get interstitial pneumonia.
3. True/false.  Pneumocystis species are not host specific and they affect a wide range of mammalian species.  FALSE.  Pneumocystis as a genus does affect a wide range of mammalian species, but the Pneumocystis spp. are host-specific.
4. The following diagnostic methods for rat pneumocystosis are the most sensitive:

  1. PCR of an oropharyngeal swab or wash
  2. PCR of feces
  3. Serology 10-20 days after the infection
  4. All of the above
  5. None of the above
ANSWER:  5.   Although all of these tests are able to diagnose Pneumocystis, none of them are very sensitive.   Oropharyngeal swabs rarely reveal positives, fecal PCR will only be positive for a short period, and IgG can be detected only from 14d  onwards.  Diagnosis may be confirmed in individual rats by serology or PCR of lung tissue (but not PCR of feces), and colony health screening can be accomplished by sentinel serology or rack PCR.


5. In rats, Pneumocystis was previously known as rat respiratory virus and the species name is/are P. carinii and P wakefieldiae. In mice the species name is P._murina__________
6. Pneumocystis is intra/extra cellular, is classed as an  _Ascomycetous fungus and does/does not respond to antifungal treatments
7. In people, the Pneumocystis species name is P. jirovecii, the infective stage is the _cyst____ and transmission is by the _aerosol_______ route
8. True/false.  In people, Trimethoprim/sulfa is the usual treatment.
9. The course of the disease in rats is acute/chronic and peak  disease occurs _3___wks after infection  (note: in immune competent rats).
10. Pneumocystis can/cannot reliably be cultured in vitro
11. True/false. Pneumocystis cysts can survive freezing at -80C, but are susceptible to most common disinfectants. TRUE
12. The usual stain for identification of Pneumocystis in the lung is Gomori Methenamine Silver (GMS)

References: 
Peter Walzer in J eukaryot microbiol 2013 60(6)
Procop et al in J Clin Microbiol 2004 p.3333-
Menotti et al PLOS 1 8(4) April 2013
Kim et al experimental and therapeutic medicine 2014. 8:442-446
http://www.criver.com/files/pdfs/infectious-agents/rm_ld_r_pneumocystis.aspx (accessed 9/2017)
http://dora.missouri.edu/rats/pneumocystis-carinii-formerly-known-as-rat-respiratory-virus/









Q19: Rabbit Bacterial Infections

From WikiHow
  1. E coli.  True or False?
    1. E coli can be identified in normal rabbit feces and E coli overgrowth often results secondary to another infection TRUE
    2. Enteropathogenic strains of E coli can be diagnosed by serotyping and biotyping  in addition to PCR. TRUE
    3. PCR identification relies upon the abc gene that codes for intimin FALSE - the gene name is eae (ie ecoli attaching and effacing)
    4. Disease manifestation is highly dependent upon serotype: O15:H is highly pathogenic whereas O103:H2 and O123 are not. FALSE.   O:15H and O103:H2 are the 2 highly pathogenic strains. And in case you forgot,  O= somatic antigen; H = flagellar antigen; K=capsular antigen.
    5. Pathogenic strains typically exhibit attaching and effacing pathology.TRUE
  2. Treponema paraluiscuniculi
    1. Was formerly known as__Treponema cuniculi___
    2. Is closely related to T pallidum the cause of human _SYPHILIS__ (disease)
    3. Disease is usually transmitted during_BREEDING _
    4. The  tpr_ genes are primarily responsible for virulence.  (Treponema pallidum repeat - it is a repeating gene) 
    5. Diagnosis can be confirmed from a microscopic sample stained with__a silver stain such as WARTHIN STARRY.  They can also be visualized on a dark field exam of a wet mount_
  3. T paraluiscuniculi (True or false?)
    1. Antibodies to T pallidum and T paraluiscuniculi cross react TRUE
    2. Antibodies to one organism provides protection against the other FALSE (but they are protective against the homologous species)
    3. The lesions are generally restricted to mucocutaneous junctions and regional lymph nodes and can persist for many months TRUE
    4. Following infection, seroconversion follows within 3 weeks FALSE.  Lesions appear 3-6 weeks following infection, and seroconversion follow 5-6 weeks after that.
    5. Positive serology in the absence of lesions indicates complete recovery from the disease. FALSE. The infection can be latent in the lymph nodes.
  4. Tyzzers Disease 
    1. is caused by _Clostridium piliforme__ formerly known as Bacillus piliformis_
    2. Most infections are subclinical/severe. Subclinical
    3. During an outbreak,  there is typically high mortality in __WEANLINGS_ (age group)
    4. The most consistent organ infected is the liver; cecum/intestine; heart (pick one)CECUM/INTESTINE
    5. Infection is generally via ingestion of SPORES______
    6. True or false?  Latent infections exist, and clinical disease can result from stress TRUE
    7. _GERBILS_ (another species) are highly susceptible to Tyzzers disease and have been used to diagnose latent infections
    8. True or false? PCR diagnosis is complicated by cross reaction with non-pathogenic bacteria. TRUE
  5. Enterotoxemia in rabbits 
    1. Is caused by the Clostridial organism _C.spiroforme__________
    2. The toxin responsible for disease is neutralized by antiserum to _ iota (toxin) from this organism C perfringens__________
    3. Enterotoxemia is most likely to follow changes in _feed, antibiotic treatment, weaning, illness, parturition/lactation_.  Mortality is highest in  weanlings 4-6 weeks_ 
  6. The most common bacterial infection in rabbits is pasteurellosis_ caused by P. multocida__
    1. The most common clinical form of this disease is _rhinitis (AKA snuffles)____________
    2. This organism is also a primary cause of _abscesses, otitis media, pneumonia, conjunctivitis, septicemia, genital disease in rabbits
    3. Serotypes A__ and _D_  are most commonly associated with disease in rabbits
    4. Infection is usually acute/chronic CHRONIC
    5. Humoral immunity protects against _SEVERE DISEASE_ but not _RHINITIS__
    6. Culture diagnosis based on colony morphology can be accomplished by incorporation of CLINDAMYCIN , which suppresses most other likely flora,  into the growth medium
    7. A single serological test is sufficient to assure negative status. FALSE -rabbits may be seronegative in the early stages of disease.
Reference: the 'blue book' "The Laboratory Rabbit, Guinea Pig, Hamster, and Other Rodents" Elsevier 2012.

Q18: Bats.... Answers


Picture from Pinterest
1. Classification:
  • Which order (from the Greek 'hand' and 'wing' do bats belong to? _Chiroptera________  
  • There are over 1300 known species of bats. Big Brown Bats and Little Brown Bats are common in the United States. What is the genus and species of big and little brown bats? 
  • Big brown bat: Eptesicus fuscus (Eptesicus is derived from the Greek, meaning "house flyer")
  • Little brown bat: Myotis lucifugus (Myotis derived from Greek, meaning "mouse-ear")
  • The big and little brown bats are part of the bat family_ vespertilionidae__ (vespertilio means "bird of evening" in Latin)
2.  Bats in North America are a reservoir species for rabies, and have surpassed dogs as the major source of human rabies cases in America.  Although raccoons are the most common wild species diagnosed with rabies, most of the human rabies in the US (21 out of 23 non-transplant cases) were caused by the bat rabies serovariant despite the fact that most of the victims did not report being bitten by or exposed to a bat.
    • {ref: Arch Virol. 2013 Apr;158(4):809-20.  Epub 2012 Dec 4.Unique characteristics of bat rabies viruses in big brown bats (Eptesicus fuscus).Davis AD1, Gordy PA, Bowen RA.}
  • Rabies virus belongs to the family Rhabdoviridae__ and genus Lyssavirus__ 
    • {ref: CDC https://www.cdc.gov/rabies/transmission/virus.html}
  • All personnel working with bats or bat tissues should be vaccinated against rabies. Initial rabies vaccination consists of single/multiple (pick one) doses. 
    • MULTIPLE
  • True or false?  Following potential exposure, vaccinated individuals do not need post exposure prophylaxis. 
    • FALSE.  Pre-exposure vaccination reduces the course of post-exposure treatment but it doesn't eliminate it. Current CDC recommendations post exposure are for 4 doses of vaccine plus immune globulin for non-vaccinated individuals, and 2 doses of vaccination no immune globulin for vaccinated individuals.
    • ref: {CDC  https://www.cdc.gov/vaccines/hcp/vis/vis-statements/rabies.pdf }
  • True or false? Post exposure prophylaxis may consist of more than one vaccine dose and immune globulin. 
    • TRUE.  See above.
  • True or false?  The incubation period for rabies in bats may exceed 13 weeks. 
    • TRUE.    {Moore, G. J., and H. Raymond. 1970. Prolonged incubation period of rabies in a naturally infected bat Eptesicus fuscus (Beauvois). The Journal of Wildlife Diseases 6:167–168}. More recently it is reported that although bats usually die of rabies 3-8 weeks after exposure, they can actually recover after being infected.  The exact epidemiology of rabies in bats is not reported, in part because there are several serotypes and susceptibilities in different species of bats.  (see references above).                    
3. Biology
  • Although some species of bats may suck blood, eat fish or small mammals, or eat nectar or pollen, the vast majority of bat species feed on insects.  
    • Vespertilionidae are insect-eating bats.
  • Bats are nocturnal/diurnal/crepuscular and sleep in roosts (which can contain 100's of bats)
  • True/false  They live many times longer than other mammals of the same size 
    • TRUE.  Some bats live up to 40 years- the big brown bat weighs only ~23g but can live up to 20 years.  {http://animaldiversity.org/accounts/Eptesicus_fuscus/}
  • The gestation period is __2 months for the big brown bat__ and newborn bats are precocious/altricial. {http://animaldiversity.org/accounts/Eptesicus_fuscus/}
  • True/false  Some bats hibernate and migrate seasonally 
    • TRUE.  Eptesicus hibernates. 

4. Bats use a unique method to locate their prey_echolocation_  This, together with study in the wild for evidence of _climate change__constitute 2 major areas of research using bats.

5. Over 200 viruses have been detected in bats and there is some evidence that some bats can remain asymptomatic while harboring viruses that can infect humans.  In  addition to rabies virus, bats are increasingly being linked with emerging human diseases.  
  • True/false  In addition to rabies, bats are suspected to have acted as reservoirs, either directly, via fomites/vectors (such as partially eaten fruit), or via intermediate hosts such as carnivores (e.g. ferrets, raccoon dogs) perissodactyls (e.g. horses) or artiodactyls (e.g. pigs and camels)  for the following diseases:
    • Rabies-related viruses such as Australian bat lyssavirus
    • Paramyxoviruses such as Hendra virus and Nipah virus
    • Coronaviruses such as SARS/MERS
    • Filoviruses such as Ebola virus and Marburg virus
    • Alphaviruses such as Venezuelan equine encephalitis virus 
    • ALL TRUE
      • {Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 110(1): 1-22, February 2015
      Bats and zoonotic viruses: can we confidently link bats with emerging deadly viruses?
      Ricardo Moratelli, Charles H Calisher}{Bats as ‘special’ reservoirs for emerging zoonotic pathogens Cara E. Brook and Andrew P. Dobson. Trends in Microbiology March 2015, Vol. 23, No. 3 179}
6. True/false. Wild caught big brown bats may be infested with mites, ticks, bugs and various endoparasites and should be treated for parasites on arrival in the laboratory. 
    • TRUE. We have clinically identified both helminths and macronyssid mites from locally caught big brown bats.
    •  {ref: J Parasitol. 2007 Jun;93(3):518-30.Ectoparasites in an urban population of big brown bats (Eptesicus fuscus) in Colorado.Pearce RD1, O'Shea TJ.}.
  • True/False  Ectoparasites have been implicated in the transmission of white nose syndrome (Geomyces destructans), which is causing rapid destruction of small brown bat populations in North America. 
  • General reference for bat husbandry and care: 
  • "Bats in captivity" Ed. Susan M Barnard. Vol 1 Biological and Medical Aspects. Logos Press 2009.




Q17: Coat Color Answers



Considering the following...

A. Nonagouti locus, a
B. Tyrosinase-related Protein 1 locus, Tyrp
C. Tyrosinase locus, Tyr


1. Which is responsible for:
- black/brown color?           B
- albinism                            C
- agouti coat                        A


2. Regarding the nonagouti gene,                
- which is dominant, agouti or nonagouti?      Non agouti
- what is the banding pattern of colors along the hair for agouti?   Black/yellow/black
- what are the colors and banding pattern in a cinnamon mouse?   Brown/yellow/brown
- is a DBA mouse agouti?   No.  Stands for dilute brown nonagouti.


3. Regarding nude mice (OK I know they don't grow a proper haircoat, I digress)
- What is the gene/allele symbol for a nude mouse? Expand the gene symbol into words...Foxn1__nu  Forkhead box N1   (note the nu is superscripted - I can't show that so I put an underscore first)
- What is the developmental abnormality that results in the immune deficiency in nude mice?
They are athymic
- Do they have normal B and NK cells?
Yes
- What happens to their immune deficiency as they age?
They get "leaky" - may develop extrathymic T cells


4. What color is this mouse?
- a/a Tyrp1b/Tyrp1b Tyrc/Tyrc 
 (please note the alleles should be superscripted, but I can't do that easily on here)
It's albino - the Tyr_c /Tyr_c  overrides any other coat color genes.


5. What color is this mouse?

- a+/a Tyrp1B/ Tyrp1b Tyr C/Tyrc

It's agouti.  The agouti locus if not modified by a mutation is usually shown as a+.  If modified by a mutation suck as A_w, then it is usually shown as a capital A


6. True or false?
- FVB and BALB/c (and other common albino mice) are albino because of a point mutation from a shared ancestor    TRUE
- Mouse albinism is caused by disruption of the first step in the production of melanin  TRUE
- The suppression of one gene by another is called epistasis.  TRUE


7. Which of these gene/alleles are responsible for coat color dilution?
- Myo5a_d
- Mlph_ln
- Lyst_bg
- Kitl_sl
- Kit_W
ALL OF  THEM.

8. In Q7, what are the names of the dilution alleles?
Myo5a is myostatin - a dilution gene
Mlph_ln is leaden
Lyst_bg is beige
Kitl_sl is steel
Kit_W is dominant white spotting (lethal if homozygous)
Q 16: Photoshop Answers – Chris Zink

1. When making a new file, you should set the resolution to:

a) 72 dpi
b) 240 dpi
c) 300 dpi
d) 600 dpi

 Answer: c

2. When working on an image in Photoshop, every time you add something to your image (lines, letters, new images to make composites, etc.) you should open a new layer and perform that action on the new layer. The main reason for doing this is so that:

Answer: You retain maximal flexibility in changing your image later. Working in layers allows you the flexibility to change or delete each element of your image.

3. True or false: Photoshop works on a vector system, in which the program memorizes the directions of various lines that are drawn when an image is being made. This prevents the problem of pixellation. 

Answer: F. Photoshop works on a pixel system, in which each pixel has an assigned color, shade, etc. Illustrator is a vector-based system. Thus if you blow up an image in photoshop you may end up with a pixelated image (right) with a low resolution original.





4. Which of the following Photoshop modifications can you ethically make to a photographic image of data that you plan to publish in a scientific journal?

a) Selective cropping
b) Whole-image brightening
c) Cloning
d) Changing resolution without resampling
e) White balancing
f) Changing tint
g) Whole-image sharpening
h) Changing resolution with resampling

Answer: a, b, d, e, g

5. True or False: Photoshop is an excellent program for creating and editing images

Answer: F – it is great for editing images (especially photographs), but if you want to create an original image, Illustrator is your best bet.


6. The best tool to white balance an image is:

a) Brightness Contrast too;
b) Levels tool
c) Curves tool
d) Tint tool

Answer C



7. When adding text to label images in Photoshop, you should generally use:

A) A serif font
B) A sans serif font

Answer: B) A sans serif font



8. True or false? When modifying photographs in Photoshop that you plan to submit to a journal for publication, you should change your image to its final resolution first, so that all of your changes are performed in that resolution. 

Answer: F. Changing resolution should be the very last thing you do before submitting your images to a journal. 


9. True or false: When modifying images in Photoshop, always record all of the steps you are taking, so that you can go back if needed

Answer: F. If you are using Layers, Photoshop records all of the steps you are taking anyway, so if you save your file as a .tiff file while working on it, the computer will save your image in layers so that you will always have access to what those steps were. 


10. True or false:  When you want to save an image that you have been modifying in Photoshop, it doesn’t matter whether you save it as a .tiff or a .psd file – in both cases the image will be saved in layers. 

Answer: T

 _________________________________________________________________________________


Q15: Powerpoint do's and don'ts

Here were my hints - now filled in with some content!   

Preparation

  • Yes you should practice.  Not only for delivery, but also for timing.   In general it is better to go short and allow time for questions rather than having too much material and rushing through - most people overestimate the amount of new information that can be absorbed in a lecture anyway so you don't need to tell them everything you know.  Leave that for questions. Take it slowly and explain things. And if you're nervous about speaking in front of people - practice in front of people.  Start that talk and do the first 5-10 minutes for as many people as you can until you can do it in your sleep! There's a lot of truth in the old adage "fake it until you make it."
  • Personally I generally get by with 1 slide per minute of talk.  YMMV.
  • If you get the chance, go up to the podium  before everything starts, get used to being up there. Figure out any controls you may need to know so that it's not a further source of stress when you give your talk.
  • Make sure everything is working well before it's time to get started.   How many talks have you turned up for and waited while someone tries to figure out how to get the computer to sync with the overhead?   Worse still, finds out they don't have the right cable to connect and there's a wait while someone scrambles to find one in a different building (mac users take note - no-one ever provides that adapter you need).  
  • This is especially true for videos or sound clips.   Check and double-check in the room using the equipment that you will be using.
General appearance of the slide
You've probably heard this before:  
  • Don't use white on black/blue/red whatever.  It's difficult to read, especially if it's not particularly dark in the lecture room or it's a small screen 
  • Don't use one of those potted templates - we've all seen them before and it makes your talk look just like all the others.  
  • Keep it clean - fancy backgrounds are distracting and get old fast.
  • The audience should be able to read EVERYTHING on the slide.   Have you heard someone say "I know you can't read this but...."  How much did you get out of that?  Usually because fonts are too small or there is too much info on slide
  • Sans Serif fonts are easier to read. 
Components you put on the slide
  • Don't use animated gifs. They are sooooo annoying!
  • Don't use clip art unless it adds to the content (I make exceptions for this, but it has to be relevant clip art).
  • Added extras that don't increase comprehension take away from your talk.  Don't use 3-D, multiple fancy swoop-ins or swoop-outs, difficult-to-read fonts, 
  • Be consistent from slide to slide.   Multiple fonts or sizes just look messy. Ditto different colors, layout, transitions etc.
  • Don't  have more than 4 (or at most 5) bullets. 
  • Don't have more than 4 (or at most 5) words per bullet 
  • Don't use acronyms unless you have to.  If you have to, then explain them or spell them out the first time.
  • Graphs should be formatted for the talk so the audience can see what you're talking about. Axes should be labeled,  important parts highlighted, etc.
  • DO tell your audience what you are going to talk about (OUTLINE)
  • DO summarize your talk at the end
  • DO arrange your talk in a logical fashion.   
    • Think about a story: set the scene, build the tension, provide the resolution and the closure.  
    • Think about a paper: what is the background? how is it leading to the questions you have asked? How did you go about asking those questions?  What were the results? What did you deduce from the results? What will you do next?
What you talk about and how you talk about it

  • You might want to introduce yourself and tell the audience what you do. It's easy and it may help you settle in for your talk.
  • Explain what you're going to talk about and what you hope to they will get out of the talk.
  • Don't RUSH
  • Don't READ YOUR SLIDES!   The audience can read too... (unless your fonts are too small!) Not to mention they will get/bored/fall asleep, lose interest if you just read your slides. Most people read in a monotone - very soporific... Instead use your slides as 'reminders" for what you are going to talk about.  If you haven't done this before and have a panic attack about not having something to read,  practice with a script beforehand until you kinda know it.  Then practice giving the talk without the script.   When you get up there - have the script if you must but don't refer to it unless you get lost...
  • Make EYE CONTACT with your audience. I'm not going to suggest you imagine them naked though...
  • If you have complicated graphs or graphics, WALK YOUR AUDIENCE THROUGH THEM. Even the most complicated graphics can be explained if you start at the beginning and build them point by point (this is a really good use for powerpoint animation where you add the next layer as you talk about it). 
  • Tell the audience if you'll take questions during the talk or at the end.  Be warned, you can get waylaid if you have lots of questions during the talk, so you need to build in extra time and have a good way of cutting off discussion if you're going to take questions during the talk. It's probably best to take questions at the end if you're new to this.

What accessories you use

  • I'm thinking laser pointers here.  Don't you hate it when someone waves one around constantly?  I'm as guilty of this as anyone - right before a recent talk I was given a fancy green laser pointer to use.   Unbeknownst to me, it apparently turned on every time you picked it up then turned off again.  And the 'on' button was not very user-friendly.   I ended up giving up and putting it down after the first few slides, but in the interim I spent more time trying to get it to work than talking to my audience!  Next time I'll practice first  😏
Time management

  • This bears repeating.  Practice. Know how long your talk takes.  Build in time for questions.  If for some reason you get behind or don't have as much time as expected, know which slides you will cut out rather than trying to rush through them all.

So how many did you think of?    Please add more thoughts that I didn't mention....

ANSWERS Q 14: NAKED MOLE RAT


Naked Mole Rat


  1.   Genus and species is__Heterocephalus glaber_  and this means literally "different-headed, hairless"  - and in case you were wondering, the Family is Bathyergidae.
  2.  The are classified in the order rodentia but are actually not rats or mice, they are most closely related to guinea pigs and porcupines and chinchillas.
  3.  Native to East Africa (the "Horn")  they live entirely in subterranean tunnels
  4.  Tunnels can reach (10 feet/ 10 yards/ 2km/ 2 miles) in length
  5. In captivity a housing system must consist of tunnels, toilet chamber, food supply area and communal nesting area.
  6.  In captivity they must be kept warmer than other lab animals because they are poikilothermic - temperatures of 82-87 F and a humidity of ~50%
  7. Why would water bottles be contraindicated? they don't drink water, getting their hydration from fresh food
  8. This is a colony animal with a unique social structure like bees, what is this structure called? eusocial
  9. A breeding colony can consist of up to (10/30/100/300) animals but contains only _one__  breeding female and 1-3 breeding males
  10. Gestation period is approx 70 days.  Why is it approximate?  Determining the exact time of copulation not necessarily that easy...
  11. Litter size is 1x/ 2x / 3x/ that of  outbred rodents.  This is open to debate but the reference below suggests an average litter size of 11-12 which is not dissimilar to outbred mice. 
  12. In captivity, limited  space may result in cessation of breeding 
  13. The majority of the non-breeding mole rats are (male/female)
  14. Animals should not be removed from the colony for more than a few minutes because they will not be accepted back into the colony, and may well be killed.
  15. Serious aggression between soldiers in the colony can result when the queen dies
  16. After the first 2 weeks of age, the diet of pre-weaning pups consists of cecotrophs from colony members
  17.  Sexing naked mole rats is accomplished by the female has a horizontal red line between the genital mound and the anus
  18. Their life span is up to 30 years in captivity
  19. They lack substance P, a neurotransmitter  and as a result are insensitive to dermal pain
  20. Their unique social structure is the stimulus for research on effect of social status on sex differentation/neuroplasticity
References: 
Husbandry standards for keeping naked mole rats in captivity: David Wood and Raymond 
Mendez
Poster on the Naked Mole Rat presented at National AALAS by Alison Weller, U Toronto Mississauga, ON
The long gestation of the small naked mole rat studied with ultrasound biomicroscopy and 3D-ultrasonography, Roellig et al, March 2011 PLOS one.
ANSWERS Q 13: ON NUTRITION







1. Rodent diets are often sterilized by either autoclaving or irradiation.

Which isotope is most commonly used for irradiation?

a. Fluorine-18

b. Cobalt -60

c. Iodine-131

d. Palladium-103

e. Strontium -89

Hyperchlorinator





 2. Water is often (i) acidified or (ii) hyperchlorinated.   What is the usual pH of (i) acidified or (ii) hyperchlorinated water?

a. (i) pH 1.5-2.0  (ii) pH 5.8-6.0

b  (i) pH  2.5-3.0 (ii) pH 5.0-5.6

c. (i) pH 2.5 – 3.0) (ii) pH 5.8-6.0

d  (i) pH 3.0-3.5 (ii) pH 5.0-5.6)



3. Which diet below is most accurately described as follows: prepared using high heat and high pressure steam, porous, cooked, and low density.



a. pelleted

b. extruded

c. purified

d. coleted

e (b and d)

f (a and c)





4. Which commonly-used dietary ingredients are most likely to contain these undesirable substances?

·      Nitrosamines?  Meat and fishmeal

·      Phytoestrogens? Soybean meal, alfalfa

·      Fusarium vomitoxin Moldy wheat and corn

·      Pesticides (choose the highest possibility)
    wheat > corn > oats > barley >soy

5. Regarding fatty acids and fats
a. Which 2 fatty acids are essential for rodents?
linoleic and alpha linoleic
b. What is a good vegetable-derived source of essential fatty acids? Soy
c. What is the usual fat % recommended for rodent diets? 4%

6. Regarding amino acids
a. What is a limiting amino acid?
The amino acid that is exhausted first in protein synthesis
b. What is the limiting amino acid for swine production? lysine
c. Can too much of an amino acid be toxic?
Yes - some amino acids such as methionine can be toxic if overdosed.






7. Testing
Match the description/test (a-d) with the dietary component(1-4)

Soxhlet extractor
a. Kjeldahl or Dumas assay (2)
b. Soxhlet apparatus (1)
c. Ether extraction (1)
d. nitrogen-free extract (3)
e. surface plasmon resonance (4) - used for biotin

 (1) Fat
 (2) protein
 (3) carbohydrate
 (4) none of these

8.  What is the difference between a fixed formula diet and a constant nutrition diet?
Fixed formula indicates the dietary ingredients and their percentage remains the same.  Advantages are that the 'other' substances in the diet (everything other than fat cho protein and fiber) remain constant.  Disadvantage is that the precise percentage of fat, cho, protein, fiber may vary slightly.
Constant nutrition indicates the proximate analysis (ie % of fat, carbohydrate, protein, fiber) remain the same but the ingredients can differ. Advantage is that the fat, cho, protein, and fiber are always the same.  Disadvantage is that the dietary ingredients may vary from batch to batch and thus introduce substances in the diet that can affect research.

9.  What does the Maillard reaction do to the diet? The maillard reaction refers to browning caused by a reaction between a reducing sugar and amino acid.


10.     a. Put the following in order from largest to smallest
· 
·      gross energy >  apparent digestible energy >    metabolizable energy

Alfalfa
          b.  True or false? 
·      I calorie is the energy required to raise 1g of water by 1degF FALSE - it is the energy required to raise 1g water by 1 deg celsius
·      I joule = 4.184 calories FALSE - 1 cal=4.184 joules 
·      Another name for a Calorie is a kilocalorie  - TRUE - note the capital letter

·      Proximate analysis is the division of diet into 6 categories TRUE =water, crude protein, crude fat, crude fiber, nitrogen-free extract and ash.

·      Crude protein is 16% Nitrogen TRUE.  Remember - multiplying nitrogen by 6.25 = crude protein

·      Alfalfa causes autoluminescence and can interfere with imaging studies TRUE

·     All of these are phytoestrogens: daidzein, genistein, coumestrol, lignans TRUE
·      Diet hardness consists of stiffness (puncture resistance) and abrasion resistance:  a texturometer measures stiffness. TRUE

 Reference: The Mouse in Biomedical Research (Blue Book)  Vol 3 Chapter 10 p 321-


Answers Q 12: Statistics

1. What is a reasonably accurate method of estimating sample size for an experiment? Which of these would you need to do this estimation?
            An estimate of variability
            Power
            p-value
            useful effect size 


Power analysis is the usual method for estimating sample size.  All the above values are needed. By convention power is usually set at 80% and a commonly used p value is 0.05.  The useful effect size is the effect that would have clinical significance - a significant effect that makes no real difference is not useful.  For example, a 10% reduction in neonatal deaths in NHP would be clinically significant, whereas a 10% reduction in hair loss would not.  The variability (standard deviation) may be estimated based on preliminary data, or you can take your largest likely value, subtract your smallest likely value and divide by 6.  If you want to be especially conservative divide by 4 or 5.

A "seat of the pants" estimate can be obtained using the resource equation estimate 'E' where E = total number of animals minus total # of groups and E should fall between 10 and 20.


2. What is the difference between the following observational studies?
            Cohort
            Cross sectional
            Case control

Cohort: identify a group or groups and follow them forward over time
Cross sectional: analyze a group at a single time point
Case control: identify a group and compare its history with that of controls

3. Which of the study types in Q2 would best describe this study…
In a colony consisting of rhesus and pigtail macaques, look at the historical records and sort them into diarrhea and no-diarrhea groups.  Determine the proportion of rhesus and pigtails in each group.

Case control.

4.  What is the dependent variable in Q3?   (hint – another term for dependent variable is outcome measure)
The dependent variable is the species of macaque. This can sometimes be confusing.  One way to look at this and evaluate what the groups are (in this case diarrhea or no-diarrhea) - this is the independent variable.  What you are measuring (proportion of rhesus vs pigtails) is the thus the dependent variable.

5. True or false: if statistical evaluation of your study results in you rejecting the null hypothesis, you can deduce that there is a meaningful effect of your treatment.

False.  Although it is commonly assumed that rejection of the null hypothesis means there is an effect, statistically speaking all you can say is that a result as extreme or more extreme than this only had x% likelihood (5% if p = 0.05) of occurring by chance.

6. True or false: a high (ie larger #) p value reduces the chance of false negatives but increases the chance of false positives.
True.  A p value of 0.05 means that the you will get a result like this by chance 5% of the time. If you increase the value to 0.1 now you get a result like this by chance 10% of the time even though there is no difference, so more false positives.  A false positive is a Type 1 error so you could say increasing the p value increases type 1 error.  (And vice versa - decreasing the p value increases false negatives or type 2 errors).
If your brain gets wrapped in circles thinking about this, you might remember it by
- increasing the p value is a positive movement (more false positives),
- decreasing the p value is a negative movement (more false negatives).

7.  You calculate the confidence interval (CI) for an experiment comparing the means of 2 samples using a p value of 0.05:  
  •  The CI does not span zero and 
  • The 2 values of the confidence interval are far apart
How would you interpret these 2 findings ?

 First consider this: 
The confidence interval (C.I.)  is just a fancy name for a range of possible values 
It is an estimate of the difference between 2 means in the actual population based on your experiment.
Any value within this range could be the true difference between the 2 means in the population.  
A value of zero means that there is no difference between the 2 means.

 Accordingly, if your confidence interval is e.g. -0.5 to  +0.5, one of the possibilities is 0, ie there is no difference between the 2 populations, so you cannot reject the null hypothesis.
In our case the C.I. does not span zero, so you reject the null hypothesis.

 a wide confidence interval means that there is a wide range of possible values so the reproducibility (precision) of the data is small.  In effect, your sample size is too small and you may need to redo the experiment with more subjects so as to get a narrower confidence interval.

8. Which of these are considered to be continuous data?
  • a. degree of liver fibrosis
  • b. liver enzyme values
  • c. fish species
  • d. # of dogs in each US State
  • e. number of male students in a classroom
(a), b, d.
The definition of continuous data is that they are infinitely variable or approximately so.  If the numbers are sufficiently large (as in d) then the data are considered continuous even though only whole numbers are available.  'a' could either be continuous if the fibrosis was measured in percentages, or discrete, if lumped into larger ranges such as 0-10%, 11-20% etc.  The data in  'c' and 'e' would both offer the opportunity for only a small range of values so are discrete.

9. Which of these statistical tests would generally be used on continuous data?
  • a.  ANOVA
  • b. Shapiro-Wilk Normality test
  • c. Students’ t test
  • d. Mann Whitney
  • e. Kruskal Wallis
ANOVA, Shapiro-Wilk, t test are all tests used on continuous data, ie parametric tests.
Shapiro-Wilk is a test for normality on continous data.   Parametric tests require the data to meet certain prerequisites, one of which is that it must be normally distributed.   As a bonus - what other factors for the data would be needed to use parametric tests?



10. If you are comparing the means of 4 samples with one dependent variable (data are continuous), which statistical test are you most likely to use?

One way ANOVA.  One-way ANOVA determines if there is a difference between any of the groups when comparing one measurement (dependent variable) (e.g. weight) between multiple groups (eg dogs on different diets).

References


I'm not a statistician and anything other than the basics leaves me scratching my head.  However a good understanding of the basics is enormously helpful for designing and analyzing simple clinical experiments and interacting with statisticians for more complicated ones.  Thus these references are not comprehensive tomes designed to get you through graduate level statistics - for that consult a statistician!   However I have found these useful to help with understanding - many of them use a lot of examples, which I always find helps clarify unfamiliar concepts




Answers to Q11: What's New with Rodent Quarantine?


 Pasteurella pneumotropica is fairly ubiquitous in laboratory mouse colonies, including ours.  How do I know? I test our racks, and all incoming mice from other institutions and 70-80% test positive. Do I do anything about it?  Not currently, but I am considering treating all mice in quarantine to avoid adding to the current incidence.  Antibiotic treatment alone has been successful for us and for others(1).


Bordetella hinzii in rodents, Southeast Asia.Jiyipong T, - Emerging Infect. Dis. (2013)
Bordetella hinzii (2-4)is a relatively new player on the scene, although it was previously identified as B avium.  It is an opportunistic pathogen in people and a primary pathogen in mice, causing rhinitis and tracheitis. I believe it to be a relatively new arrival, despite the fact that we didn’t test for it previously, because our facility rack testing has not identified it in resident colonies.  I don’t want to add a primary pathogen so I have been rederiving mice in quarantine.  For us, antibiotics alone did not work, but we have just had a case where antibiotics plus cross fostering was successful.

From slideshare.net
Campylobacter spp.   We’ve seen this 4 or 5 times now. The problem for us is we can’t readily speciate the Campylobacter to determine if it’s C jejuni, a human pathogen.  However in every case so far, the mice tested negative 2 weeks after the positive test, so we merely extend quarantine until they test negative.

Klebsiella oxytoca.   An opportunist – we don’t see it as often as Pasteurella but it’s not infrequent.  We don’t exclude, and we don’t see the kind of problems we see with Pasteurella (see previous blog post), however in some cases it can cause pathology, such as otitis media (5).  Currently we don't intervene.

Pneumocystis pneumonia
Pneumocystis carinii (rats).  Formerly called rat respiratory virus, a primary pathogen in rats, causing lymphohistiocytic interstitial pneumonia (6).  We just eradicated this from our rat colonies, so this is definitely an undesirable and we look for other uncontaminated sources.  We are investigating cross fostering as a means to eradicate from incoming animals.

Nude mouse with C bovis
Corynebacterium bovis.   A common opportunistic pathogen of immune deficient mice(7). We have it. We attempt to eradicate it from some investigators’ colonies with varying degrees of success.  We test all incoming mice with known immune deficiencies and have successfully rederived mice using antibiotic treatment plus c-section, although most often we look for other sources.
References
1. Elimination of Pasteurella pneumotropica from a mouse barrier facility by using a modified enrofloxacin treatment regime.  Towne et al. J Am Assoc Lab Anim Sci. 201453(5) 517-22.

2. Detection and pathogenesis of Bordetella hinzii in four commonly used mouse strains.  Kenneth Henderson et al.  Charles River.

3. Study of Bordetella hinzii isolate from a laboratory mouse. Hayashimotoet al. Comp Med 2008 (58) 5, 440-446.

4. Bordetella pseudohinzii as a confounding organism in murine models of pulmonary disease . Clark et al Comp Med 2016 (66) 5, 361-366.

5. Gram-negative pathogen Klebsiella oxytoca is associated with spontaneous chronic otitis media in Toll-like receptor 4-deficient C3H/HeJ mice. Carol J. Macarthur et al. Acta Oto-Laryngologica (128), 2008.


6. Pneumocystis carinii infection causes lung lesions historically attributed to rat respiratory virus. Robert S Livingston et al.Comp Med 2011 61 (1) 45-52.

 7. Corynebacterium bovis: Epizootiologic Features and Environmental Contamination in an Enzootically Infected Rodent Room  Holly N Burr et al. J Am Assoc Lab Anim Sci 2012.51(2) 189-198.

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