Transcript Diagnosis of active fur mite infestation by quantitative PCR and

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Sequencing of Myocoptes musculinus
(Diagnosis of active fur mite infestation by quantitative PCR and RT-PCR)
ACLAM Forum
May 7, 2014
Alexander Sheh
Division of Comparative Medicine, MIT
Mites
• Common rodent fur mites
– Myocoptes musculinus
– Myobia musculi
– Radfordia affinis and ensifera
• Other mites
• Demodex musculi, arvicolae and flagellurus
• Psorergates simplex
• Mesostigmatic mites
– Laelaps echidnina (spiny rat mite)
– Ornithonyssus bacoti (tropical rat mite)
• Liponyssoides sanguineus (house mouse mite)
• Dermatophagoides farinae and pteronyssus (house dust
mites)
M. musculinus and M. musculi
• Most common fur mites in lab mice.
• Nonburrowing, feeding on skin
secretion and interstitial fluid
• Often present in mixed infections
– Myobia ~ head/shoulder pelage
– Myocoptes ~ inguinal, ventral abdomen
and dorsum
• Transmission is direct (not through
bedding) and requires hair shafts
http://www.idexxbioresearch.com
Effects of mite infestation
• General health complications
– None
– Ruffled fur to alopecia to erythema to pruritus to ulcerative dermatitis
• Strain dependent
– Decreased life span, body weight and reproductive indices
– Chronic infestations -> dermatitis may provoke secondary amyloidosis
– Self-trauma (secondary bacterial infections)
• Research concerns
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Immunological research
Behavioral research
Endocrine research
Toxicology research
Treatment
mosup.com
• Drugs (Ivermectin, Selemectin, Permethrins,
Chlorpyrifos, Dichlorvos, Moxidectin, and others)
– Injection, in water, in feed, spot on treatment, bedding,
and nestlet soak.
• Varied results with drug treatment based on species
and treatment method
• Rederivation may be needed to clean up a colony
• Prevention is preferred over treatment
Diagnostic methods
• Methods
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Direct exam of pelage
Cellophane tape testing
Hair plucks
Skin scraping
PCR
• Typically, detection of fur mites is a
visual process and poses difficulties
in terms of throughput, sampling
and personnel requirements.
http://www.idexxbioresearch.com
Diagnostics by PCR
• Charles River and IDEXX RADIL offer fur mite
PCR assays targeting rRNA genes.
• PCR increases throughput/sampling and
reduces hands on time with good specificity
and sensitivity.
RT-PCR: How do you know you
eradicated mites?
• Due to DNA’s stability, residual mite tissue on treated mice may
be a potential source of PCR false positives (Ricart Arbona et al. 2010).
• While DNA dominates the forensic sciences, understanding
RNA and its degradation may offer insights into cause of death,
the age of wounds/injuries and the post-mortem interval(Bauer 2007).
– 16S rRNA detection from Chlamydia pneumoniae was better
associated to active infection than detection of specific antigens(Meijer et
al., 2000).
• Can we use RNA degradation to complement DNA based
assays?
Developing a RT-PCR assay for mites
• Sequence rRNA and mitochondria
– Myocoptes musculinus
– Myobia musculi
– Radfordia affinis
• Develop and test specific primers
Available fur mite sequences on NCBI
• 935 bp sequence Myocoptes musculinus 18s
ribosomal RNA gene
• Closely related Myocoptes japonensis has
available 18S and 28S sequences
• Myobia musculi 18S, ITS, 5.8S, ITS, partial 28s
rRNA
(S.Compton (2011) and S. Feldman (2011)).
• No mitochondrial sequences available
Phylum Arthropoda, Class Arachnida
The common
murine fur mites diverge at superorder Acariformes.
Superorder
Parasitormes
Laelaps echidnina, Ornithonyssus bacoti, Liponyssoides sanguineus
The genera
Myobia and Radfordia are both in the Myobiidae family.
Order
Trombidiformes
Myobia musculi, Radfordia spp., Psorergates simplex, Demodex musculi and
The genus
Myocoptes
is within
themites)
superfamily Sarcoptoidea and the
other
(Tetranychus
urticae
– spider
familySarcoptiformes
Myocoptidae. Myocoptes musculinus and Dermatophagoides spp.
Order
diverge at musculinus,
suborder Psoroptidia.
Myocoptes
Dermatophagoides spp. and other (Sarcoptes scabiei scabies)
Adapted from www.tolweb.org
Processing Myocoptes samples
• Mites obtained from an experimentally infested
colony at MSKCC (Dr. Neil Lipman).
• Genomic DNA was extracted from fur plucks from
individual mice using the QIAamp® DNA Micro kit
(Qiagen) using carrier RNA. DNA was pooled.
Primer selection
• Three pairs of rRNA primers
based on Myocoptes japonensis
sequences.
• Mitochondrial primers based on
complete mitochondrion
sequences for Dermatophagoides
pteronyssus (EU884425) and
Dermatophagoides farinae
(NC_013184).
– More nonspecific bands!
rRNA PCR
MiSeq Sequencing
• Gel extracted PCR amplicons were pooled into
rRNA or mitochondria samples
• Amplicons were sonicated, size-selected and
ligated to sequencing adapters.
• Samples were ligated and amplified on flow
cell for sequencing.
Illumina
MiSeq Sequencing
• 150 bp paired end reads were sequenced and
assembled by Velvet.
• Contigs analyzed by Geneious
Illumina
Ribosomal RNA alignment
• Generated a 6.35Kb contig from assembly of rRNA sample reads
• Compared to Myobia musculi, Dermatophagoides spp., Myocoptes
japonensi and Myocoptes musculinus, with Mus musculus as negative
control (77%)
• Data from Charles River showed a 92% homology between Myobia
musculi and Myocoptes musculinus (Henderson and Perkins seminar)
D. farinae
92.9%
M. japonensis
98.9%
M. musculinus
99.9%
Increased taxonomic similarity
Myobia
84.6%
Ribosomal RNA alignment
Sequenced Myocoptes contig
28S % similarity
M. japonensis (partial 1334bp) 94.7%
D. farinae
89.4%
D. pteronyssus
89.9%
M. musculi (partial 444bp)
68.7&
Phylogenetic analysis of subclass Acari
18S rRNA sequences
• Sanity check – created a phylogenetic tree using 18S
sequences from 223 species from the subclass Acari
Trombidiformes,
incl. Myobia
Parasitiformes, Trombidiformes,
Sarcoptiformes
Sarcoptiformes
Mitochondrial genome alignment
• More difficulties with mitochondrial genome
• Have generated 5.2Kb out of ~14Kb
– Blue denotes new sequences matching reference
– Green and red denote reference genome
– Purple denotes PCR products
• 71.1-72.9% homology to D. farinae and
pteronyssus, respectively.
• Sequenced cox1-3, atp6, atp8, and cytB genes
Future directions
• Sequence rest of Myocoptes musculinus
mitochondria, and mitochondria and rRNA of
Myobia musculi and Radfordia affinis
• Design primers and test on PCR products and
T7-generated ssRNA under diverse
degradation conditions.
• Obtain frozen samples from Ivermectintreated and untreated mice
Acknowledgements
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ACLAM
James G. Fox
Mark Whary
DCM postdocs
– Laura Cacciopo, Courtnye Jackson, Courtney Ek
• Neil Lipman - MSKCC
• BioMicro Center
References
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http://www.idexxbioresearch.com/radil/userfiles/download_files/MitesFlyer_US_Rev062012.pdf
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