Itp.lucidcentral.org 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 – – – – 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 – – – – – 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 • • • • ACLAM James G. Fox Mark Whary DCM postdocs – Laura Cacciopo, Courtnye Jackson, Courtney Ek • Neil Lipman - MSKCC • BioMicro Center References • • • • • • • • • • • • • • • • • • • • • • • Pathology of Laboratory Rodents and Rabbits. Percy and Barthold, eds. 2007, 3rd ed. The Mouse in Biomedical Research, 2nd Edition. Volume II, Diseases. 2007. Fox et al., eds.. Flynn’s Parasites of Laboratory Animals. Second Edition. 2007. Baker, ed. Ricart Arbona et al. Treatment and eradication of murine fur mites: I. Toxicologic evaluation of ivermectin-compounded feed. JAALAS 2010;49(5):564-70 Ricart 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), 2008. 272-276. Clifford and Watson. Old enemies, still with us after all these years. ILAR Journal, 49(3), 2008. 291-302. Watson. New building, old parasite: Mesostigmatid mites-an ever-present threat to barrier rodent facilities. ILAR Journal, 49(3), 2008. 303-309. http://tolweb.org/tree/ Hill et al. Contemp Top Lab Anim Sci. 1999 Nov;38(6):13-18. Demodex musculi in the Skin of Transgenic Mice. Dermauw et al. 2009 BMC GENOMICS. 10. The complete mitochondrial genome of the house dust mite Dermatophagoides pteronyssinus (Trouessart): a novel gene arrangement among arthropods http://www.criver.com/about-us/eureka/wp-content/uploads/Fur-mite-PCR-AALAS-KSH-talk.pdf http://www.idexxbioresearch.com/radil/userfiles/download_files/MitesFlyer_US_Rev062012.pdf Weiss 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. 574-578(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. 773-781(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. 69-73(5) Bauer. RNA in forensic science. Forensic Science International: Genetics Volume 1, Issue 1, March 2007, Pages 69–74 Meijer 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.