Sequencing of Myocoptes musculinus(Diagnosis of active fur mite infestation by quantitative PCR and RT-PCR)

ACLAM ForumMay 7, 2014

Alexander ShehDivision of Comparative Medicine, MIT

Itp.lucidcentral.org

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 shaftshttp://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– Immunological research– Behavioral research– Endocrine research– Toxicology research

Treatment

• 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

mosup.com

Diagnostic methods

• Methods– 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

Superorder ParasitormesLaelaps echidnina, Ornithonyssus bacoti, Liponyssoides sanguineusOrder TrombidiformesMyobia musculi, Radfordia spp., Psorergates simplex, Demodex musculi and other (Tetranychus urticae – spider mites) Order SarcoptiformesMyocoptes musculinus, Dermatophagoides spp. and other (Sarcoptes scabiei - scabies)

The common murine fur mites diverge at superorder Acariformes.

The genera Myobia and Radfordia are both in the Myobiidae family.

The genus Myocoptes is within the superfamily Sarcoptoidea and the family Myocoptidae. Myocoptes musculinus and Dermatophagoides spp. diverge at suborder Psoroptidia.

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

Illumina

• 150 bp paired end reads were sequenced and assembled by Velvet.

• Contigs analyzed by Geneious

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)

Myobia84.6%

M. japonensis98.9%

D. farinae92.9%

M. musculinus99.9%

Increased taxonomic sim

ilarity

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

Sarcoptiformes

Parasitiformes, Trombidiformes, Sarcoptiformes

Myocoptes musculinus

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 Ivermectin-treated and untreated mice

Acknowledgements

• ACLAM• James G. Fox• Mark Whary• DCM postdocs– Laura Cacciopo, Courtnye Jackson, Courtney Ek

• Neil Lipman - MSKCC• BioMicro Center

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