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1 Division of Comparative Medicine, MIT Itp.lucidcentral.orgSequencing of Myocoptes musculinus (Diagnosis of active fur mite infestation by quantitative PCR and RT-PCR)ACLAM ForumMay 7, 2014Alexander ShehDivision of Comparative Medicine, MIT
2 Mites Common rodent fur mites Other mites Myocoptes musculinus Myobia musculiRadfordia affinis and ensiferaOther mitesDemodex musculi, arvicolae and flagellurusPsorergates simplexMesostigmatic mitesLaelaps echidnina (spiny rat mite)Ornithonyssus bacoti (tropical rat mite)Liponyssoides sanguineus (house mouse mite)Dermatophagoides farinae and pteronyssus (house dust mites)
3 M. musculinus and M. musculi Most common fur mites in lab mice.Nonburrowing, feeding on skin secretion and interstitial fluidOften present in mixed infectionsMyobia ~ head/shoulder pelageMyocoptes ~ inguinal, ventral abdomen and dorsumTransmission is direct (not through bedding) and requires hair shafts
4 Effects of mite infestation General health complicationsNoneRuffled fur to alopecia to erythema to pruritus to ulcerative dermatitisStrain dependentDecreased life span, body weight and reproductive indicesChronic infestations -> dermatitis may provoke secondary amyloidosisSelf-trauma (secondary bacterial infections)Research concernsImmunological researchBehavioral researchEndocrine researchToxicology research
5 Treatmentmosup.comDrugs (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 methodRederivation may be needed to clean up a colonyPrevention is preferred over treatment
6 Diagnostic methods Methods Direct exam of pelageCellophane tape testingHair plucksSkin scrapingPCRTypically, detection of fur mites is a visual process and poses difficulties in terms of throughput, sampling and personnel requirements.
7 Diagnostics by PCRCharles 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.
8 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?
9 Developing a RT-PCR assay for mites Sequence rRNA and mitochondriaMyocoptes musculinusMyobia musculiRadfordia affinisDevelop and test specific primers
10 Available fur mite sequences on NCBI 935 bp sequence Myocoptes musculinus 18s ribosomal RNA geneClosely related Myocoptes japonensis has available 18S and 28S sequencesMyobia musculi 18S, ITS, 5.8S, ITS, partial 28s rRNA (S.Compton (2011) and S. Feldman (2011)).No mitochondrial sequences available
11 Phylum Arthropoda, Class Arachnida 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.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)Adapted from
12 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.
13 Primer selectionThree 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
14 MiSeq SequencingGel extracted PCR amplicons were pooled into rRNA or mitochondria samplesAmplicons were sonicated, size-selected and ligated to sequencing adapters.Samples were ligated and amplified on flow cell for sequencing.Illumina
15 MiSeq Sequencing150 bp paired end reads were sequenced and assembled by Velvet.Contigs analyzed by GeneiousIllumina
16 Ribosomal RNA alignment Generated a 6.35Kb contig from assembly of rRNA sample readsCompared 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%D. farinae92.9%Increased taxonomic similarityM. japonensis98.9%M. musculinus99.9%
18 Phylogenetic analysis of subclass Acari 18S rRNA sequences Sanity check – created a phylogenetic tree using 18S sequences from 223 species from the subclass AcariTrombidiformes,incl. MyobiaParasitiformes, Trombidiformes,SarcoptiformesMyocoptes musculinusSarcoptiformes
19 Mitochondrial genome alignment More difficulties with mitochondrial genomeHave generated 5.2Kb out of ~14KbBlue denotes new sequences matching referenceGreen and red denote reference genomePurple denotes PCR products% homology to D. farinae and pteronyssus, respectively.Sequenced cox1-3, atp6, atp8, and cytB genes
20 Future directionsSequence rest of Myocoptes musculinus mitochondria, and mitochondria and rRNA of Myobia musculi and Radfordia affinisDesign primers and test on PCR products and T7-generated ssRNA under diverse degradation conditions.Obtain frozen samples from Ivermectin-treated and untreated mice
21 Acknowledgements ACLAM James G. Fox Mark Whary DCM postdocs Laura Cacciopo, Courtnye Jackson, Courtney EkNeil Lipman - MSKCCBioMicro Center
22 ReferencesPathology of Laboratory Rodents and Rabbits. Percy and Barthold, eds. 2007, 3rd ed.The Mouse in Biomedical Research, 2nd Edition. Volume II, Diseases Fox et al., eds..Flynn’s Parasites of Laboratory Animals. Second Edition Baker, ed.Ricart Arbona et al. Treatment and eradication of murine fur mites: I. Toxicologic evaluation of ivermectin-compounded feed. JAALAS 2010;49(5):564-70Ricart Arbona et al. Treatment and Eradication of Murine Fur Mites: III. Treatment of a Large Mouse Colony with Ivermectin-Compounded Feed. JAALAS 2010;49(5):633-7.Ricart Arbona et al. Treatment and Eradication of Murine Fur Mites: II. Diagnostic Considerations. JAALAS 2010;49(5):583-7.Lindstrom et al. Soiled Bedding Sentinels for the Detection of Fur Mites in Mice. JAALAS 50(1)p54-60.Mook et al. Use of selamectin and moxidectin in the treatment of mouse fur mites. JAALAS 2008 May;47(3):20-4.Burdett et al. Evaluation of five treatment regimens and five diagnostic methods for murine mites. JAALAS 1997 Mar;36(2):73-6.Carty, A. Opportunistic infections of mice and rats: Jacoby and Lindsey revisited. ILAR Journal, 49(3),Clifford and Watson. Old enemies, still with us after all these years. ILAR Journal, 49(3),Watson. New building, old parasite: Mesostigmatid mites-an ever-present threat to barrier rodent facilities. ILAR Journal, 49(3),Hill et al. Contemp Top Lab Anim Sci. 1999 Nov;38(6): Demodex musculi in the Skin of Transgenic Mice.Dermauw et al BMC GENOMICS. 10. The complete mitochondrial genome of the house dust mite Dermatophagoides pteronyssinus (Trouessart): a novel gene arrangement among arthropodsWeiss et al. Comparison of a Fur Mite PCR Assay and the Tape Test for Initial and Posttreatment Diagnosis during a Natural Infection JAALAS Volume 51, Number 5, September 2012 , pp (5)Rice et al. Evaluation of Diagnostic Methods for Myocoptes musculinusAccording to Age and Treatment Status of Mice (Mus musculus). JAALAS Volume 52, Number 6, November 2013 , pp (9)Karlsson, EM et al. Combined Evaluation of Commonly Used Techniques, Including PCR, for Diagnosis of Mouse Fur Mites. JAALAS, Volume 53, Number 1, January 2014 , pp (5)Bauer. RNA in forensic science. Forensic Science International: Genetics Volume 1, Issue 1, March 2007, Pages 69–74Meijer et al. Chlamydia pneumoniae in vitro and in vivo; a critical evaluation of in situ detection methods J. Clin. Pathol., 53 (2000), pp. 904–910.Tolker-Nielsen et al. Effects of stress treatments on the detection of Salmonella typhimuriumby in situ hybridization Int. J. Food Microbiol., 33 (1997), pp. 251–258.