1. Identification of MH Class IIβ alleles
conferring resistance/susceptibility to
Flavobacterium psychrophilum in
Oncorhynchus mykiss families
Masters Thesis Defense
Calvin Kellendonk
Dec 4th 2014
4. Major Histocompatibility Genes
• Encode cell surface receptors that present peptides
• Part of the adaptive immune system
• Highly polymorphic
• In humans:
– 3,284 alleles for class I (Gonzalez-Galarza, 2011)
– 1,411 alleles for class II (Gonzalez-Galarza, 2011)
– Most polymorphic protein-encoding gene
5. Genomic Organization
• Are divided into class I and
class II
• In humans they are located on
the same chromosome
• In teleost fish, the classes are
located on separate chromosomes
and therefore the “complex” part
of MHC is dropped
6. MH Class II
• Presents peptides from extracellular pathogens
to helper T cells
cell membrane
β2 α2
β1 α1
#ofaminoacids
(Murphy, 2012)
10. Bacterial Cold Water Disease
• Causative agent = Flavobacterium psychrophilum
• Infections occur in cultured salmonids
• Infection can result in skin lesions, fin rot, abdominal
distension and death
11. Effect on aquaculture industry
• Total aquaculture production of Canada valued
around $ 1 billion in 2012
– Trout production valued at $36.7 million
– Majority of rainbow trout produced in Ontario and
Quebec
– Trout production in Ontario valued at $18.3 million
• Mortality rates between 20-30% typically
observed in aquaculture facilities, although
sometimes up to 80%
http://www.dfo-mpo.gc.ca/stats/aqua/aqua12-eng.htm
12. Prevention and Treatment
• No commercially licensed vaccine
• Surface disinfection with iodophor treatments
• Disinfect equipment with formalin or chlorine
• In USA, two approved antibiotics
– Florfenicol (aka Aquaflor)
– Oxytetracycline dihydrate (aka Ferramycin)
– Both delivered orally via medicated feed
13. Selective Breeding
• Breed fish with desirable traits
• Resistance to BCWD moderately heritable and
not adversely correlated with growth
• Examine candidate genes for involvement in
resistance
14. Why should we care about MH genes
in Fish?
• MH receptors present peptides and are part of the
adaptive immune system
• Certain MH alleles might confer greater
susceptibility or greater resistance to a specific
pathogen
– Previous research has identified specific MH class IIβ
alleles associated with better performance against a
bacterial pathogen
15. Hypothesis
• A relationship exists between MH class IIβ
alleles individual fish carry and susceptibility
or resistance to BCWD.
16. Dec 2012 Experimental Infection Trial
Figure 1.1 Survival plot for rainbow trout
infected i.p with F. psychrophilum strain 101 Figure provided by L. Al-Hussinee
Low mortality
High mortality
17. Sampling
• 108 fish sampled (mix of survivors and morts)
• Caudal fin collected post trial and stored in
RNAlater
• Extract DNA and PCR amplify
Exon 1 Intron Exon 2
Leader
peptide
Β1 domain
primer
primer
19. Figure 3.1. Alignment of Oncorhynchus mykiss MH class IIβ exon 2 nucleotide sequences. Solid grey
boxes represent the nucleotide sequences and colored lines within them indicate a nucleotide difference
from the consensus sequence. The DAB*1001 exon 2 region is from GenBank (Accession number:
KF528072) and represents the longest partial sequence of that allele in the database. Sequences obtained
from this research included introns and were trimmed to show the complete exon 2 region.
20. Figure 3.2 Alignment of Oncorhynchus mykiss MH class IIβ exon 2 amino acid sequences deduced from
nucleotide sequences. Dots above the consensus sequence represent residues known to interact with the
peptide in human class II receptor. Red boxes indicate unique peptide binding amino acid residues with
different physical properties.
22. Family Number
3 4 6 7 9 10
Allele
DAB*401 - - - - 0.21 -
DAB*801 0.56 0.11 0.61 0.33 0.47 0.50
DAB*1001 0.56 1.00 0.94 1.00 0.89 0.89
DAB*1101 0.56 0.16 0.06 - 0.16 0.50
DAB*1201 0.50 - - - - -
Table 3.1 MH Class IIβ allele occurrence among six families of Oncoryhnchus mykiss. A total of 19 individuals were
sequenced for families four and nine, while 18 individuals were sequenced for the other four families. The numbers shown
are the proportion of individuals containing that allele. For example, a value of 0.56 represents 56% of the sampled fish
containing that allele.
23. Families
3 4 6 7 9 10
MH Class IIβ Genotype
DAB*1001/DAB*1001 - 0.74 0.39 0.67 0.26 -
DAB*1001/DAB*1101 0.22 0.16 - - 0.11 0.44
DAB*0801/DAB*0801 - - - - 0.11 0.06
DAB*1001/DAB*0801 - 0.05 0.56 0.33 0.21 0.39
DAB*1101/DAB*0801 0.22 - 0.06 - - -
DAB*1201/DAB*1001 0.17 - - - - -
DAB*1201/DAB*0801 0.17 - - - - -
DAB*0401/DAB*1001 - - - - 0.05 -
DAB*0801/1001/1101 0.11 0.05 - - 0.05 0.11
DAB*0401/0801/1001 - - - - 0.16 -
DAB*1001/1101/1201 0.06 - - - - -
DAB*0401/0801/1001/1101 - - - - 0.05 -
Table 3.2MH Class IIβ genotype frequencies among the six families of Oncorhynchus mykiss. A total of 19 individuals
were sequenced for families four and nine, meanwhile 18 individuals were sequenced for the other four families. Frequency
was calculated using the formula f=x/n, where x is the number of times the genotype was observed and n is the number of
individuals.
24. Family Number
3 4 6 7 9 10
Alleles/
Individual
Two
Three - -
Four - - - - -
Table 3.3 Number of MH class II β alleles found per individual in Oncoryhnchus mykiss families.
26. Alleles Percent of Mortalities
n= 62
Percent of Survivors
n= 48
DAB*0801 44 42
DAB*1001 85 94
DAB*1101 29 23
DAB*1201 10 6
DAB*0401 5 2
Table 3.4 Single allele prevalence in mortalities and survivors. Alleles are represented in the table rows and the
percent of each found in the two groups are represented in the columns.
31. Allele Hazard Ratio p-value
DAB*0401 2.728 0.395
DAB*0801 0.872 0.753
DAB*1001 0.368 0.232
DAB*1101 1.318 0.549
DAB*1201 1.113 0.898
Table 3.8 Calculated Hazard Ratios for MH Class IIβ alleles using Logistic Regression.
32. Allele Hazard Ratio p-value
DAB*1001/DAB*1001 0.877 0.848
DAB*1001/DAB*1101 1.528 0.592
DAB*0801/DAB*0801 1.667 0.708
DAB*1001/DAB*0801 0.722 0.649
DAB*1201/DAB*1001 1.667 0.708
DAB*1201/DAB*0801 1.667 0.708
DAB*0401/DAB*0801 1.20 0.763
Table 3.9 Calculated Hazard Ratios for MH Class IIβ genotypes using Logistic Regression.
33. Summary
• High nucleotide variability in exon 2
– Translated to amino acid variability
– Changes altering the binding properties of the groove
• Observed 5 alleles among the 6 families
• Most resistant family 7 had DAB*0801 and DAB*1001
• DAB*1001/DAB*0801
– Found more in survivors
– Lowest HR in both regressions
34. Conclusions
• No strong evidence for an association between
MH class IIβ alleles/genotype and resistance
or susceptibility to BCWD
• Although not statistically significant,
DAB*1001/DAB*0801 had the lowest HR,
suggesting association with resistance
35. Alternative Possibilities
• MH class I role
• Combined MH class I and II role
– Uni Grimholt and Atlantic salmon
• Innate immunity
36. • Other genes
– Resistance could be controlled:
• by a couple loci with large effects
OR
• many loci with small effects
37. Future Directions
• Genotype for MH class I
– Analyze individually and in combination with
class II genotype
• Investigate innate immunity
– RNA and protein expression of immune genes
– Functional assays (Respiratory burst)
– Complement
38. Recommendations for Lyndon
• Genotype other families on the farm for MH
– Overall diversity of MH genes on the farm
– If low, reevaluate the breeding program
• Examine the heritability of resistance to
BCWD for the population of fish at the farm
39. Acknowledgements
Dr. Brian Dixon
Dr. John Lumsden
Dr. Heidi Swanson
Dixon Lab
Lumsden Lab
Lowia Al-Hussinee
Lyndon Hatcheries Inc.
Central Animal Facility
University of Guelph
After adaptive point: there are a few notable features of MH genes, the first being that they are highly polymorphic. ..human ex.
Second notable feature is there genomic organization :
Humans have 3 main class I genes and 6 main class II genes. Gene number is variable and not known for all fish species.
My project focuses on class I. Here is a schematic diagram of the receptor: consisting of alpha and beta chain. Beside it is a figure where the polymorphism exists within the receptor. X –axis residue number, y- axis # of different amino acids found there. I want to point out the beta 1 domain. Its where most of the amino acid polymorphism exists.
This is a different representation of the receptor. Mention ribbon structure and link dots with areas of high amino acid variability
If amino acids in the peptide binding groove differ between receptors, there binding properties might differ as well. Therefore receptors vary in there ability to bind specific peptides. And some receptors might bind certain peptides better than others.
Gram –ve bacteria that is thought to be a facultative intracellular microbe.
Right now this is the # 1 concern for Ontario rainbow trout farms.
To give some context, what is the effect of this disease on the aquaculture industry. Talk about mortality rates. Even if fish survive the carcass is not suitable.
Licensed procedures vary between countries. Bacterial resistance to antibiotics can develop. SO alternate method of control is needed
Next step is looking at genetic variation and resistance, can do QTL’s or look at candidate genes, I did the latter.
So why are MH genes good candidate genes for looking at resistance?
In brook charr with furunculosis
In order to do this, several families of rainbow trout from lyndon hatcheries Inc where experimental infection with F. Psych. This figure shows the mortality results from the Dec 2012 trial that I focused on.
From that trial I sampled 18 fish per family. PCR amplify this region of MH class II beta, clone and then do all the steps to get it ready for sequencing.
Nucleotide variability produces some amino acid variability. Dots/arrows above residue position indicate residues known to bind antigen in human receptor. Highlight and go through some of the amino acid changes that have different physical properties.
Families 9 + 3 had the highest allelic diversity, each possessing 4 different alleles.
8 “two” allele genotypes observed. DAB 1001/0801 found in 5 of the 6 families.
Not a big difference between the groups. 1001 high prevalnce and found in slightly more survivors. 0401 had the lowest prevalnce.
Genotypes towards the top were most commonly obeserved. Again, no big difference between the groups.
To identify alleles conferring resistance or susceptibility, performed a survivor analysis and calculated HR’s by two different methods
First, calculated using a Cox regression, takes into account survivor status (dead/alive) and time until death. Describe hazard ratio meaning.
In addition to the Cox regression, performed a logistic regression. Only takes binary information of survivor status (dead/alive). Again 1001 had the lowest hazard ration and 0401 had the highest.
