A quick and robust MHC typing method for free-ranging and captive primate species
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Gene products of the major histocompatibility complex (MHC) of human and non-human primates play a crucial role in adaptive immunity, and most of the relevant genes not only show a high degree of variability (polymorphism) but also copy number variation (CNV) is observed. Due to this diversity, MHC proteins influence the capability of individuals to cope with various pathogens. MHC and/or MHC-linked gene products such as odorant receptor genes are thought to influence mate choice and reproductive success. Therefore, MHC typing of wild and captive primate populations is considered to be useful in conservation biology, which is, however, often hampered by the need of invasive and time-consuming methods. All intact Mhc-DRB genes in primates appear to possess a complex and highly divergent microsatellite, DRB-STR. A panel of 154 pedigreed olive baboons (Papio anubis) was examined for their DRB content by DRB-STR analysis of genomic DNA. Using the same methodology on DNA of feces samples, DRB variability of a silvery gibbon population (Hylobates moloch) (N = 24), an endangered species, could successfully be studied. In both species, length determination of the DRB-STR resulted in the definition of unique genotyping patterns that appeared to be specific for a certain chromosome. Moreover, the different STR lengths were shown to segregate with the allelic variation of the respective gene. The results obtained expand data gained previously on DRB-STR typing in macaques, great apes, and humans and strengthen the conclusion that this protocol is applicable in molecular ecology, conservation biology, and colony management, especially of endangered primate species.
KeywordsPrimates MHC Microsatellites Conservation biology Pre-clinical research
The major histocompatibility complex (MHC) class I and II molecules are cell-surface glycoproteins, which play a central role in immune-related processes as in control or susceptibility to infectious diseases, in transplantation research, and in reproduction immunology by presenting peptides to CD8 and/or CD4 T lymphocytes. The MHC genes/loci encoding these molecules are structured in gene families, the hallmark of which is the allelic and copy number variation (CNV) of most of their genes. Balancing selection exerted mainly by pathogen pressure is believed to represent one of the major forces driving MHC polymorphism and diversity (Bernatchez and Landry 2003; Piertney and Oliver 2006; Spurgin and Richardson 2010). Characterization of polymorphic and polygenic MHC genes is therefore an excellent measurement of genetic health and as such of value in conservation biology, as e.g., management of captive breeding programs for endangered species (Cai et al. 2015; Hans et al. 2015; Oliver and Piertney 2012; Pechouskova et al. 2015). One of the most variable MHC genes in many vertebrate species is the DRB gene encoding the beta chain of the class II DR molecule. Therefore, DRB typing has often been used as an indication for MHC diversity in various species and for diverse purposes, as e.g., the importance of the MHC for mate choice (Setchell et al. 2013; Setchell et al. 2011), heterozygous advantage testing against heterogenous pathogen pressures (Oliver et al. 2009; Osborne et al. 2015), identification of selective pressures acting on MHC haplotypes (Huchard et al. 2008), pathogen-driven balancing selection (Nishita et al. 2015), selection countering drift to maintain MHC polymorphism (Oliver and Piertney 2012), and understanding genetic variability for the management of captive breeding programs (Cai et al. 2015).
Based on these published data, we examined the possibility to type for DRB by using the DRB-STR microsatellite in another Old World monkey species, the olive baboon. Very little was known so far about the DRB allelic variation and haplotype composition of the olive baboon (Papio anubis, Paan-DRB), although the olive baboon serves as model species in renal (Poirier et al. 2015), and xeno-transplantation research (Le Bas-Bernardet et al. 2015), and for immunotoxicity studies (Poirier et al. 2014).
An endangered lesser ape species, the silvery gibbon shares a common ancestor with humans ∼18–20 mill years ago (Perelman et al. 2011). The natural habitat of the silvery gibbons is the island of Java, where they occupy lowland and hill and lower mountain rainforests. A population survey of the silvery gibbon carried out between 1994 and 2002 found that 4000 to 4500 individuals were present not only in habitat fragments in the west of Java but also in central Java (Nijman 2004). The rate of which its numbers appear to be declining, mainly due to deforestation, the silvery gibbon has a high chance of going extinct. There are three wild populations located in Java National Parks, and the necessity to improve habitat protection has been recognized (Campbell et al. 2008). The global captive population of silvery gibbons totals 119 individuals, 71 of which are located in zoological institutions in Indonesia, where attempts to breed the species have been largely unsuccessful (Campbell et al. 2008; Cocks 2000). Outside of Indonesia, however, there are 48 individuals spread across several zoological institutions and 24 animals at Port Lympne and Howletts parks within the UK (Stanbury 2015). Port Lympne and Howletts zoos have selected the species for an overall conservation program that includes both a captive breeding and re-introduction program. The International Union for Conservation of Nature (IUCN) recommends that conservation should be carried out at three levels, the ecosystem, the species, and the genetic level (Frankham 2010). For the latter purpose, MHC typing appears to be appropriate, and we therefore wanted to establish the DRB-STR typing method for this endangered species on DNA isolated from feces for animal welfare reasons. So far, nothing was known about the polymorphism and variability of the DRB genes of the silvery gibbon (Hymo-DRB). Fortunately, primers that had been developed for the amplification of the human DRB-STR could be successfully used for the amplification of Hymo- as well as Paan-DRB. For the latter, primers used for the amplification of the DRB-STR in macaques could additionally be employed. In such a way, a large cohort of olive baboons, which had been partially pedigreed beforehand, has been defined for their DRB content. Although it has been widely reported that both DNA yield and quality derived from fecal samples is lower than that extracted from blood or tissue (Chaves et al. 2006; Marrero et al. 2009; Wasser et al. 1997), feces-extracted DNA of 20 of 21 silvery gibbons, housed at Port Lymphe and Howletts parks, allowed DRB-STR typing as well. DRB genotyping and haplotyping results of olive baboons and silvery gibbons will be presented. The data open opportunities for MHC analysis in captured and endangered primates in the context of animal welfare, and the impact on colony management and/or conservation biology will be discussed.
Material and methods
Olive baboons, originally obtained from the Centre National de la Recherche Scientifique Centre de Primatologie (Rousset, France), were housed at the large animal facility of the INSERM unit 1064 (Nantes, France). Blood sampling was performed under anesthesia in accordance with the institutional ethical guidelines. The 154 animals analyzed in this study were partly pedigreed and descended from 34 sires and 105 dames. In most cases, the sires were known but often two males could possibly have sired the offspring.
Twenty-four silvery gibbon individuals reside at Port Lympne and Howletts, UK, zoological institutions, of which the pedigree was partly known. Fecal samples were allocated to specific individuals either by direct observation, and two fecal samples per individual were collected immediately or by hand feeding foodstuffs containing maize that could then be identified on subsequent days. Samples were immediately frozen upon collection.
DNA extraction from EDTA blood of olive baboons
DNA was extracted from fresh EDTA or citrate blood samples using a conventional phenol/chloroform method. Briefly, white blood cells (WBC) were harvested after the lysis of red blood cells and removing the plasma by spinning down the WBCs. WBC pellets were then lysed using a 0.2% SDS and 100 μg/ml proteinase K solution and incubated overnight at 55 °C. An equal volume of phenol was then added to the lysate, and the incubation tubes were intensively mixed and then centrifuged. The upper aqueous layer was harvested and an equal volume of phenol and then of chloroform were added. Tubes were again intensively mixed, centrifuged, and the upper aqueous layer was harvested. Finally, an equal volume of chloroform was added, tubes were mixed, centrifuged, and the upper aqueous layer was harvested again. DNA were precipitated at −20 °C with two volumes of 96% ethanol. The DNA pellets were washed using 70% ethanol and then air-dried before reconstitution in sterile water. DNA concentration were adjusted at 50 ng/μl and stored at 4 °C.
DNA extraction from fecal samples of the silvery gibbon
DNA was extracted from the frozen fecal samples using the QIAamp DNA stool Mini Kit (Qiagen) following the Stool Larger Volumes protocol. This commercially available kit was chosen as it included a step that involved binding secondary compounds found in plant matter that is present in herbivorous diets, which is applicable to the silvery gibbon species. Plant secondary compounds negatively impact the PCR process by interfering with the Taq polymerase enzymatic reaction (Marrero et al. 2009). For each DNA extraction, an amount of 400 mg of frozen stool was used ensuring that both internal and external surfaces of the feces were present as a precautionary measure as sloughed epithelial cells may not be homogenously distributed throughout the sample (Piggott MaT 2003).
Amplification of the relevant DNA segment in olive baboon and silvery gibbon was performed as described for humans and rhesus macaques using the same primer sets (Doxiadis et al. 2007). Briefly, the relevant DNA was amplified with a labeled forward primer located at the end of exon 2, 5′HLA-DRB-STR_VIC and a reverse primer in intron 2, namely 3′HLA-DRB-STR or the unlabeled primer 5′Mamu-DRB-STR together with the labeled macaque specific primer 3′Mamu-DRB-STR_VIC. The labeled primers were synthesized by Applied Biosystems (Foster City, USA) and the unlabeled primers by Invitrogen (Paisley, Scotland). The PCR reaction was performed in a 25-μl reaction volume containing 1 unit of Taq polymerase (Invitrogen, Paisley, Scotland) with 0.1 μM of the forward primer, 0.1 μM of the reverse primer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 1× PCR buffer II (Invitrogen, Paisley, Scotland), and 100 ng DNA.
The cycling parameters were a 5-min 94 °C initial denaturation step, followed by 5 cycles of 1 min at 94 °C, 45 s at 58 °C, and 45 s at 72 °C. Then the program was followed by 25 cycles with 45 s at 94 °C, 30 s at 58 °C, and 45 s at 72 °C. A final extension step was performed at 72 °C for 30 min. The amplified DNA was prepared for genotyping according to the manufacturer’s guidelines using the GeneScan™ 350Rox™ Size Standard and analyzed on an ABI 3130 genetic analyzer (Applied Biosystems). STR analysis was performed with the Genemapper software 5 program (Applied Biosystems) and all samples were at least analyzed twice. For Paan-DRB genotyping both primer pairs, HLA-DRB-STR and Mamu-DRB-STR were used. STRs defined by the HLA primers are named STR-H, and those detected by the macaque primers are called STR-M. Allele bins are defined beforehand.
PCR, cloning, and sequencing
Forty-four different Paan-DRB alleles and 15 different Hymo-DRB alleles were sequenced from exon 2 to intron 2, including the microsatellite. Therefore, we used the same primers and PCR reaction as described for humans and rhesus macaques (Doxiadis et al. 2007), and at least 48 clones per animal have been picked and sequenced. All the gained Paan-DRB and Hymo-DRB sequences were unreported and have been deposited in the EMBL database. In addition the sequences are officially designated by the IPD/MHC database (de Groot et al. 2012; Ellis et al. 2006; Robinson et al. 2003).
Multiple sequence alignments of exon 2 of silvery gibbon DRB sequences together with some DRB sequences from humans and chimpanzees were performed using MacVector™ version 12.7.5 (Oxford Molecular Group) and phylogenetic analyses were then performed using MEGA version 4.0.2. Pairwise distances were calculated using maximum likelihood and Kimura-2 parameter for creating a phylogram. Confidence estimates of grouping were calculated according to the bootstrap method generated from 1000 replicates and the tree was rooted with Caja-DRB*W16:01.
DNA sequences are deposited to the EMBL gene bank with accessions numbers JQ666205–JQ666210, JQ666212–JQ666215, JQ666217–JQ666230, JQ666232–JQ666251, KJ701253–KJ701266, and LN867601).
In two non-human primate species, for which no MHC class II data have been available so far, the analysis of the Mhc-DRB allele content has been effectively performed by the presented robust, time-saving, and inexpensive microsatellite typing technique. In such a way, the DRB repertoire of an olive baboon population could be defined. The results obtained are of importance for biomedical research as in transplantation (Le Bas-Bernardet et al. 2015; Poirier et al. 2015), for which these animals are a model species. Due to the high CNV of the DRB genes in baboons and the possibility to define DRB haplotypes, the DRB content in these animals can be followed in addition for colony management reasons as parentage testing and calculation of the inbreeding coefficient.
As has been shown previously for a wild Assamese macaque population (Muller et al. 2014), DRB-STR typing was also successful when DNA isolated from non-invasively collected fecal material was used. This is especially important in the case of wild or free ranging endangered species such as the silvery gibbon. The quality of the isolated DNA was high, most probably due to the immediate freezing of the sample. As a consequence, cloning and sequencing of DRB exon 2 including the microsatellite was feasible, and in such a way the DRB-STR length could be linked to a certain DRB allele. Therefore, we were able to get an insight into the DRB repertoire of the silvery gibbon, which shows an important reduction in the number of lineages present and in CNV in comparison to chimpanzees, humans, and especially to the olive baboon analyzed. Due to the existence of several, mostly three, DRB genes with adjacent microsatellites per haplotype, DRB haplotypes could be defined for the silvery gibbon, too. Although the genetic repertoire as defined for Hymo-DRB appears to be reduced, we were able to confirm the pedigree of two related founder families as recorded in the studbook. In addition, results with this one microsatellite indicated that the father of one of the animals was incorrectly assigned.
Despite their conservation importance, distinct characteristics and high species diversity gibbons are mostly neglected by population genetic studies (Kim et al. 2011). However, several reports describe the high rates of chromosomal rearrangements that can be observed not only in the large number of rearrangements separating gibbons from other apes and humans (Carbone et al. 2006; Muller et al. 2014) but also the numerous rearrangements that separate different gibbon species from each other as well as rearrangements that are polymorphic within a species (Carbone et al. 2009; Van Tuinen et al. 1999). The possible mechanism for this genomic plasticity may be a gibbon-specific retrotransposon, LAVA (Carbone et al. 2014). The observed “patchwork” pattern of most of the DRB haplotypes of the silvery gibbon, which are built up of three DRB genes of only a few lineages, may also be the result of a near-instantaneous radiation (Carbone et al. 2014) and rearrangements in the DRB region of this species.
The complex microsatellite in DRB intron 2 is an evolutionarily old entity, which has been observed not only in apes and OWM but also in Platyrrhini species as in marmosets and owl monkeys (Trtkova et al. 1995), and it is observed even in sheep (Ballingall et al. 2008). In the two Aotus species it is highly polymorphic and has been used for DRB typing, too (Lopez et al. 2014). Thus, this typing method is applicable for nearly all primates. Since DRB-STR typing is highly informative and can additionally be easily performed by feces collection, this methodology can be used to observe differences in genetic fitness between wild and free-ranging animals. Additionally, this method is applicable for monitoring, e.g., reproduction, parentage typing, mate choice, parasite susceptibility, and conservation. Furthermore, in the case of breeding of non-human primates, the DRB microsatellite is valuable for parentage typing, avoidance of inbreeding and genetic reduction, and thus for colony management in general.
The authors wish to thank Francisca van Hassel for preparing the figures. The work was supported in part by NIH/NIAID project HHSN272201100013C.
Compliance with ethical standards
All applicable international, national, and/or institutional guidelines for the care and use of animals are followed.
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