Cheetah, Cheetaahhhhh!
Hi Guys! I'm finally finding a free moment to post some of my research from my Spring semester of my Master's AIP program work! I did a study on cheetah conservation, albeit, not in person! I did a ton of technical research though in efforts to find the key to maintaining cheetah populations both in the zoo and in the wild. It is a very complex subject! Read it if you like and you will learn something about one of our most revered felines! All material is copyrighted and cannot be reproduced or used without my express permission.
African cheetahs in captivity, especially the young cubs,
are particularly susceptible to disease including their biggest threat, the
Feline Coronavirus, which is the deadliest virus these cats can face (O’Brien
et al., 1985). Cheetahs in north central
Namibia, as well as to a lesser extent in eastern central Namibia, are in
addition exposed to such viruses as FPV (Feline Parvovirus), CDV (Canine
Distemper Virus) and FHV (Feline Herpes Virus Type 1) due to their close
proximity to urbanization and feral dogs and cats. The east central Namibian cats are further
away from any civilization and thus have less exposure to these three viral
threats.
Cheetah Major Histocompatibility Complex: The Key to their Future?
N. Lichtenbert MT SV (ASCP)
CPT (NASM)
Environmentally Fashionable/Globally Healthy
_________________________________________________________
Abstract
Cheetahs
have existed for millions of years, yet today find themselves on the brink of
extinction in the wild. Large ranges,
solitude, lack of protection from predators, low birth rate, high infant
mortality and low genetic variability have all led to the demise of this
creature. Recent efforts to conserve
cheetahs have focused on molecular techniques and the determination of the Major
Histocompatibility Complex (MHC) and other allele variability. While various methods of DNA molecular
technology have been used, controversy exists as to whether or not these
findings will lead to better conservation methods in the future. In this paper we will discuss MHC studies
which involve both captive and wild cheetahs.
Results of these findings are presented with further discussion on newer
techniques on the horizon and their potential for leading to greater cheetah
captive breeding and release into the wild. Molecular techniques can be costly
and the vested interest of financial donors would be beneficial to further
studies and positive conservation work.
Lastly we discuss the use of the EcoSpot and it’s use of engaging the
public in this fascinating topic.
__________________________________________________________
Introduction
While
there are 37 species of cats in the Felidae
family of cats, all but housecats
are either endangered or extinct. The
biggest reason for this dramatic decline is the fact that these 36 species of
cat need very large ranges in order to exist.
For example, a single tiger can span a range of up to 400 miles while
cheetahs can wander a range of up to 600 miles wide (O’Brien & Johnson,
2005).
The
African cheetah (Acinonyx jubatus) once
inhabited all of Africa, extending towards the Middle East and into part of the
subcontinent of India (Drake et al., 2004).
Being the last member to survive from the genus Acinonyx, the cheetah has other factors involved
that also lend to further decreases in population. They have exceptionally low-density ranges
due to their being very solitary animals.
Also, their cubs have a very high mortality rate, close to 70%. Various factors go in to this high mortality
rate including maternal neglect, being very susceptible to diseases as well as
cubs having insufficient defenses to protect them from any predators. Lastly, up to 71% of male cheetah sperm has
abnormalities and is not functional. The
ejaculate is more than ten times less concentrated than seen in domestic cats
(O’Brien et al., 1985).
Molecular
studies have shown that cheetahs, both free ranging and captive, show low
genetic variability, probably due to the inbreeding that took place over ten
thousand years ago. Low variability
leads to reproductive abnormalities, greater susceptibility to disease and high
infant mortality (CCF, 2012). Species
with less genetic variation have been shown to have less fitness or ability to
survive when faced with challenges (Wielebnowski, 1996). Hedrick (1996) suggests that there is a
“cheetah paradigm” in play that due to genetic homozygosity, cheetahs are on
the brink of extinction. Possible
theories on how cheetah DNA lost its heterozygosity include a metapopulation
theory where large colonies of cats went extinct with smaller populations of
the cheetahs surviving in remaining patches.
With fewer members to mix with, inbreeding occurred and the bottleneck
effect on the species’ genetics led to low variation (Hedrick, 1996).
Cheetahs
exhibit a low level of genetic variability, especially at the MHC loci. MHC, or the Major Histocompatibility Complex,
are glycoproteins found on the surface of cells, and are present in all
vertebrates. They are responsible for
self identity while also modulating the host’s response to emerging pathogens
(Castro-Prieto, A., Wachter, B., & Sommer, S., 2011). In order for an immune response to proceed,
the body must be able to identify itself by means of an antigen presenting cell
(APC) and be able to identify the antigenic threat to the body system, both of
which the MHC molecule is involved in. MHC
Class I and II have a variety of important and overlapping roles. Major Histocompatibility Complex Class I proteins
are responsible for the presentation of intracellular antigens such as viruses
while MHC Class II proteins are responsible for the presentation of
extracellular antigens such as bacteria. Class I gene products are responsible for
helping T cells to recognize themselves while also recognizing and attacking
the viral antigen. Class II gene
products augment the production of B cell antibodies used to eliminate the
antigen invader. There is also a Class
III MHC protein which is found in the body’s complement system but this
molecule will not be discussed in this review as it is not involved in the
studies presented (O’Brien et al., 1985).
It is through environmental and immunological pressures that MHC alleles
develop in response to a mammal’s surroundings.
MHC genes are the one area of mammalian systems that exhibit the process
of natural selection, where the species most able to adapt are the ones that
survive (Parham, 1999).
Molecular
studies have recently been used to gauge the variation of Cheetah genetics
using various methods. Since it has been
somewhat controversial as to whether their low genetic variability at the MHC
loci has truly led to the demise of the cheetah, it may be questionable as to
the value of molecular studies in captive breeding programs and further down
the road release of these cats back into the wild (Drake et al., 2004). With this being said, it is worth examining two
of the previously performed studies, their results and new molecular techniques
coming up in the future which may prove to be more useful to the long-term
conservation of the cheetah species.
This review will describe two recent studies while describing some of
the technology used and its findings.
Then more current molecular techniques will be described which may lead
to larger, more valuable studies in the future.
Lastly,
an EcoSpot and the use of video channels such as YouTube and Vimeo are examined
in efforts to engage the public in this issue.
Only in engaging the public are we able to find the financial donors and
the man-power of volunteers needed to continue the work of the scientists.
_________________________________________________________
Location
Study 1 – A study of cheetah MHC diversity
in the world’s largest free-ranging popultion (Castro-Pietro, A., Wachter, B.
& Sommer, S., 2011)
Methods
In this
study, 149 wild cheetahs and 28 captive cheetahs had their blood drawn. This blood was analyzed by DNA methods to
determine MHC variability. DNA was
extracted, isolated and purified using a manufactured kit. Primers and oligos are genetic sequence
pieces which are used either to start the transcription of the genetic material
or to incorporate into the new piece of DNA respectively. These primers and oligos were designed and
obtained for PCR (Polymerase Chain Reaction) thermal cycling which expands the
DNA sequence to be studied through a cycle of heating (disruption) and cooling
(annealing). This product is then
further purified for further analysis (Castro-Pietro, A., Wachter, B. &
Sommer, S., 2011).
Observation
Description
MHC
Genotyping was then performed on the samples, which gives scientists the direct
sequence of each strand in question.
Alleles, which are genetic coding sections that code for specific
protein products, were then delineated and compared for the MHC Class I and the
MHC Class II-DRB locations (Castro-Pietro, A., Wachter, B. & Sommer, S.,
2011).
Data
Analysis
Through
the use of software, MHC Class I and II sites were compared and calculated for
their differences and similarities.
Further work was done using statistics, including a t-test and using a
Bayesian inference approach to relate species together phylogenetically. Further sequence comparisons were performed
using the MHC I and II DRB genomic sequences from domestic cats (Castro-Pietro,
A., Wachter, B. & Sommer, S., 2011).
Sequences for such work are widely available on the GenBank, part of the
NCBI website, and comparisons can be performed using alignment tools also
located on the NCBI website. The NCBI
website is a conglomerate of the molecular scientific community which, for
example, has a listing of all the patented genetic sequences used in testing
methods for such organisms as Chlamydia
trachomatis or Mycoplasma hominii. The GenBank section of NCBI is a database
of genetic sequences for all species which can be used in numerous molecular
studies including cloning, sequencing and for the selection of base pair
sequences which can be used in testing.
Results
The cheetahs studied, which were
sampled from all over the country of Namibia, did exhibit all the documented
alleles that had previously been observed except for one. Yet, the number of alleles that were observed
was still much lower than that found for most other mammals. This study did confirm the low level of
variability at the MHC Class II locus.
It was however found that variation was highest in the ABS, the antigen
binding sites of the MHC molecule. This
area of genetic material is the most crucial in terms of being able to launch
an immunologic response against an emerging pathogen in the area.
Location
Study
2 – A Study in 88 Cheetahs to Determine MHC Variation Based on Differential
Pathogen Exposure compring the cheetahs of the North Central region and those
cheetahs of the East Central region Namibian Farmlands (Castro-Pietro, A.,
Wachter, B., Melzheimer, J., Thalwitzer, S., Hofer, H. & Sommer, S., 2012).
Methods
Serological studies using antibody detection were used to
determine the exposure of cheetahs of the north central region and compared
them to the cheetahs in the east central region of Namibia first. Viral exposure was observed to be higher in
the north central region where there was increased civilization and feral cats
and dogs. Feral cats and dogs increased
the likelihood of cheetahs being exposed to pathogens such as CDV (Canine
Distemper Virus), FCoV (Feline Coronavirus), FPV (Feline Parvovirus) and FHV1
(Feline Herpes Virus Type 1). While the
wild cats exhibited exposure to these viruses, they did not show signs and
symptoms of disease (Castro-Pietro, A., Wachter, B., Melzheimer, J.,
Thalwitzer, S., Hofer, H. & Sommer, S., 2012).
Observation
Description
Based with this background knowledge then, studies were
performed to determine MHC I and II variation using molecular (DNA) techniques
such as SSCP (Single-Strand Conformation Polymorphism) and sequencing. In the SSCP method, single-stranded DNA is
moved electrophoretically through a polyacrylamide gel matrix. Based on structure and size, which can be
different even if there is one base pair change, different strands will move
different distances through the gel.
This technique is most often used to detect polymorphisms at a single
locus with the ability to compare different individuals who are placed in
different lanes (Davidson College, 2003).
Data
Analysis
Once SSCP was performed, these DNA sequences were then
isolated with their allele sequences being determined using computer software.
Results
Results showed that the cheetahs from the north central
region of Namibia exhibited more variability at the MHC Class I loci when
compared against their neighbors in the east central region, which was located
more in the countryside. There was however no detected variation in terms of
the MHC Class II loci (Castro-Prieto et al., 2012). While results show that there is definitely
an exchange of genetic material between the two population locations, the
cheetahs do not have the variation needed at the Class I location, possibly leading
to more vulnerability to certain viral pathogens.
__________________________________________
Discussion
In
Study 1, while low genetic variability is found in both wild and captive
cheetahs, it appears that captive cheetahs prove to be more susceptible to
disease or lack of adaptation to the environment. This may be due to less than favorable
husbandry situations which lead to stress and lowered immunocompetence. Also, due to the cheetah’s solitary nature,
their disease transmission in the wild is lowered as they are less able to come
into contact with one another in order to spread diseases (Castro-Prieto, A.,
Wachter, B. & Sommer, S., 2011).
The
differentiation noted in Study 2,
comparing the cheetahs in the north central region and cats in the east central
region for the Class I molecule does imply that the greater exposure to various
viruses has led to greater differentiation at the MHC Class I loci. This makes sense and the information may be
useful in future management and conservation programs (Castro-Prieto et al.,
2012).
While
the information provided in these two studies is useful and confirms some
previous knowledge about cheetah genetics, results were also somewhat
ambiguous, still leaving a lot of questions to be answered. It is still not known whether or not the lack
of genetic variability at the MHC complex is directly leading to cheetah
decline. Exact relationships have not
yet been determined as to why captive cheetahs are so susceptible to diseases,
namely the Feline Coronavirus. Therefore,
more genetic studies will need to be completed.
With newer technology being developed every year, better studies will be
developed, hopefully providing information that will be more useful towards
cheetah conservation work.
Genotyping sequences by RSCA or Reference
strand-mediated conformational analysis is a relatively newer molecular method
which uses a 384-well plate, may prove to be better suited to large-scale
studies which will help to determine a species’ fitness and to correlate
diversity at a given loci. This
technique is more rapid, repeatable and less subject to DNA contamination in
comparison to other techniques being used such as PCR. This technique is robust and also accurate
(Drake et al., 2004). Future studies
will hopefully incorporate these newer techniques providing more detailed, more
wide-scaled information that can show a truer picture
Reflection
It has been proposed
that founding breeders should take a strong interest in working to incorporate
as many different alleles as possible in any given captive species
population. While this idea sounds very
logical and ideal, it does not bear in mind the facts hindering this including
financial and political systems at work (Miller & Hedrick, 1991). Perhaps genetic studies involving both
captive and wild cheetahs will prove to be useful as it may be necessary to use
different management and conservation techniques for each.
Both these studies have confirmed the fact that both
captive and wild cheetahs have low variability at the MCH complex. However, the importance of this fact still is
unclear. While both populations exhibit
the same lack of variability, the captive cheetahs appear to exhibit a higher
susceptibility to disease, most likely due to their being in close proximity to
one another and the ease of disease transferability.
Will more MHC alleles be discerned and found to have
significance? Will the MHC Class I and
II molecule knowledge prove to not be worth anything at all? Only the future work of scientists will shed
light on this topic down the road.
Conclusion
Castro-Prieto, A., Wachter, B., &
Sommer, S. (2011) suggest that the amount of genetic variability needed to
sustain wild populations in the future is still questionable. However, molecular genetic work may prove to
be very valuable in terms of selection of partnering strategies. Captive
breeding strategies should never exempt the use of other techniques commonly
used including pedigree considerations for long-term breeding and
re-establishment of the cheetah species (Miller, P.S. & Hedrick, P.W.,
1991).
Molecular techniques such as cloning,
sequencing, the use of fluorescent-labelled references, SSCP, PCR are all
useful in determining the sequence of, the variability of and the correlation
of particular genetic sequences that may be involved in the longevity of a
species. Studies to this date have been
smaller and may be affected by smaller sample sizes, research costs and the
lack of technical skill needed to perform these types of studies.
Eco
Spot
The use of public video
channels such as YouTube and Vimeo has recently proven to be an effective way
to communicate with the public. Not only
are they easy for the average person to understand, they are stimulating, easy
to re-watch and they are viewable on many different platforms including
computers, smartphones, iPads, tablets, etc.
They also have the ability to be shared at many different locations
including Facebook, Twitter, Google+, etc.
By sharing videos in these many locations, one is more easily able to
reach a wider audience.
An example of video being used to promote a conservation
topic can be seen at the www.savetigersnow.org
website which had an extensive campaign to save tigers last year. The video was created to be exciting and
engaging, creating an interest in the viewer to become more involved in the
campaign.
For this particular research presented in this paper, we
will be working to create a series of a couple of videos to present some of the
topics involving the Cheetah and a couple of the DNA laboratory methods that
were presented. In explaining why
studying the cheetah’s DNA may help cheetahs to be genetically stronger and
better able to survive while also demonstrating their role in the web of life,
it is hoped that the public’s understanding of these topics will provide a
greater motivation for getting involved in cheetah conservation.
Through
the use of an iPhone and the software Microsoft Moviemaker, video footage will
be cut and edited to create the final product.
These videos will then be incorporated into the Environmentally
Fashionable/Globally Healthy Network which works to promote the education of
the general public, namely adults, in the subjects of environmental issues,
environmentally friendly clothing and fitness/wellness. It is with great hope that they will be
well-received and that they serve the purpose of teaching.
References
Castro-Prieto, A.,
Wachter, B., Sommer, S. (2011). Cheetah Paradigm Revisited: MHC Diversity in
the World’s Largest Free-Ranging Population.
Mol. Biol. Evol. 28 (4).
1455-1468.
Castro-Prieto, A.,
Wachter, B., Melzheimer, J., Thalwitzer, S., Hofer, H., Sommer, S. (2012).
Immunogenetic Variation and Differential Pathogen Exposure in Free-Ranging
Cheetahs across Namibian Farmlands. PLoS
ONE. Volume 7 (11), 1-8.
Cheetah Conservation
Fund. (2012). Cheetah Fact Sheet. Retrieved from: http://www.cheetah.org/?nd=cheetah_facts
Davidson College. Single-Strand Polymorphism Conformation
(SSCP). 2003. Retrieved from: http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Parker/method.html
Drake, G.J.C., Kennedy,
J.L., Auty, H.K., Ryvar, R., Ollier, E.R., Kitchener, A.C., Freeman, A.R.,
Radford, A.D. (2004). The use of reference strand-mediated conformational
analysis for the study of cheetah (Acinonyx
jubatus) feline leucocyte antigen class II DRB polymorphisms. Molecular Ecology. 13. 221-229.
Hedrick, P.W. (1996). Bottleneck(s)
or Metapopulation in Cheetahs. Conservation Biology, Volume 10 (3),
897-899.
Miller, P.S., Hedrick,
P.W. (1991). MHC Polymorphism and the Design of Captive Breeding Programs:
Simple Solutions Are Not the Answer. Conservation
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O’Brien, S.J., Johnson,
W.E. (2005). Big Cat Genomics. Annual
Review Genomics Hum. Genet. Volume 6. 407-429.
O’Brien, S.J. Roelke,
M.E., Marker, L., Newman, A. Winkler, C.A., Meltzer, D., Colly, L., Evermann,
J.F., Bush, M., Wildt, D.E. (1985). Genetic basis for species vulnerability in
the cheetah. Science. Vol. 227 (4693). 1428-1434.
Parham, P. (1999).
Virtual reality in the MHC. Immunological
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Wielebnowski, N.,
(1996). Reassessing the Relationship Between Juvenile Mortality and Genetic
Monomorphism in Captive Cheetahs. Zoo
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