Famous Rat Pathology (Phenotypes) Discussion Plan General References

2017 Brayton RAT PATHOLOGY

Discussion Plan Famous Rat Pathology 1. Non Infectious, Non Neoplastic (Phenotypes) 2. Non Infectious, Neoplastic 3. Infectious Cory Brayton, D.V.M., D.A.C.L.A.M., D.A.C.V.P. Associate Professor, Molecular and Comparative Pathobiology Director, Phenotyping Core Johns Hopkins University, School of Medicine Baltimore, MD 21205 TEL: 410 502 3050 ‐‐ FAX: 443‐287‐2954 [email protected] http://www.hopkinsmedicine.org/mcp/PHENOCORE/index.html 1 3

General References: RATS RAT Resources online

1. ACLAM Blue books https://www.elsevier.com/books/book‐series/american‐college‐of‐ laboratory‐animal‐medicine 1. DORA Diseases of Research Animals http://dora.missouri.edu/ – The Rat in Biomedical Research. Fox et al 2006 2. RAT Genome Database ‐‐ with strains Phenotypes etc – Laboratory Animal Medicine. Fox et al 2015 http://rgd.mcw.edu/ 2. Barthold S, Griffey S, Percy D. 2016. Pathology of Laboratory Rodents and Rabbits, Wiley‐ Blackwell. 3. Noah’s Arkive (now at CLDavis) http://noahsarkive.cldavis.org/ 3. Treuting P and Dintzis S, Eds. 2012. Comparative Anatomy and Histology: A Mouse and Human Atlas. Elsevier (Academic Press): London. pp. 361‐381. 4. NTP NON neoplastic lesion atlas http://ntp.niehs.nih.gov/nnl/ new edition in press 2017 –with RATS 5. Pathbase: European mutant mouse pathology database Primary Journals http://www.pathbase.net/ 1. Comparative medicine / JAALAS (Search Comp Med, JAALAS) 2. Journal of Comparative Pathology (search J Comp Pathol) 6. RENI Tissue trimming guide + INHAND terminology and diagnostic criteria http://www.goreni.org/ 3. [Laboratory animals (Sage); Lab An (NY) (Nature.com)] 4. Toxicologic Pathology (search toxicol pathol) 7. VSPO JPC https://www.askjpc.org/vspo/ 5. Veterinary Pathology (search vet pathol)

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Famous Rat Phenotypes Rat Lesions (phenotypes)

NOT Neoplastic Neoplastic 1. Non Infectious, Non Neoplastic Nephropathy (D) Pituitary tumors (D) (mineralization, Parathyroid a. Nephropathy (adenomas>> carcinomas) hyperplasia) b. Arteritis ‘Cardiomyopathy’ (D) Mammary tumors (D) (Degeneration, fibrosis, inflammation) (fibroadenoma etc) c. ‘Cardiomyopathy’ Arteritis – polyarteritis Pheochromocytomas . etc Hematopoietic (I) Axonal degeneration Histiocytic sarcomas 2. Non Infectious, Neoplastic Fibromas, sarcomas Adrenal cortical, a. Skin Tumors (most commonly seen) Infectious/parasitic Thyroid follicular or C cell; Zymbal Gland, Urinary bladder (I) b. Hematopoietic (most? Famous) BOLD – possible‐likely COD CCOD c. Pituitary etc (most? Common killer) Not bold ‐ Incidental finding/phenotype to expect D Role of Diet . etc I Role of Infectious agents? E.g. MYCOPLASMA, 7 Trichosomoides crassicauda 7 8 3. Infectious

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1.a. Rat Nephropathy 1.a. Rat Nephropathy  Most common significant lesion or COD  Glomeruli in many studies, especially – LargeMesangium . Cellularity – Older studies/diets . Synechiae – Ad lib feeding – Small  Gross : . Sclerotic  Tubules – Bumpy/pitted kidneys – Dilated, protein – LARGER  then smaller – Degen, regen  AKA – Mineral +/‐  Interstitium – CPN Chronic progressive nephropathy – Inflammation – CRD Chronic Renal Disease – Fibrosis RodentPath 10 9 – ORN Old rat nephropathy 10

1.a. Rat Nephropathy Soft tissue mineralization  Glomeruli – LargeMesangium  Severe nephropathy. Enlarged parathyroid. . Cellularity . Synechiae – Small . Sclerotic  Tubules – Dilated, protein – Degen, regen – Mineral +/‐  Interstitium – Inflammation RodentPath 11 – Fibrosis 11 12

Soft tissue mineralization Soft tissue mineralization

  Severe nephropathy? Enlarged parathyroids? Severe nephropathy? Enlarged parathyroids? Stomach Muscle HEART, arteries LUNG septa

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1.b. Arteritis, periarteritis 1.b. Rat PAN (aka polyarteritis nodosa (PAN))  Gross:  Nodular thickening – Thick, nodular, multicolored arteries, mesenteric arcade,  Arteritis, periarteritis, pancreas, spermatic cord etc chronic,  Histology: – Inflammation neutrophilic  lymphohistiocytic, lymphohistiocytic, transmural – periarterial transmural – Mural‐periarterial thickening by inflammation, proliferative –with proliferation (fibroplasia;) hemorrhage‐fibrinoid, – Fibroplasia mineralization – /+Thrombi – laminated, recanalized etc – Fibrinoid – VEINS ARE SPARED – Thrombi – Mineral etc RodentPath 18 17 18

1.b. Rat periarteritis / polyarteritis Rat PAN

 Earliest in mesentery,  Earliest? In pancreas, spermatic – Pancreas cord – Mesentery  Most obvious in – Spermatic cord mesentery  Nodular thickening  Best gross in +/‐ hemorrhage, – Mesentery mineral  Veins  More common in – Spared SD, SHR,

hypertensive rats RodentPath 19 19 20

Rat PAN Rat PAN

  Thrombi Disrupted elastic lamina – Fibrinoid deposition  Proliferation  Disrupted elastic – Subintimal lamina – Medial  Proliferation – Adventitial – Subintimal  Inflammation – Medial – Adventitial  Rat crystals are  Inflammation blood (Hb) origin

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1.c. Rat ‘Cardiomyopathy’ ‘Cardiomyopathy’

 Myofiber  = ‘heart muscle abnormality’ degeneration  loss   Replacement IN pets & people fibrosis – Impaired function diagnosed by ECG, US  Mild chronic – Big – dilated ? Hypertrophic? Small fibrotic inflammation – HOCM COCM RCM  Cf human MI  +/‐ mineral

– usually with ORN RodentPath 23 23 24

‘Cardiomyopathy’ ‘Cardiomyopathy’

 IN TOX ‐ refers to spontaneous changes – Typically multifocal – Inflammation (Rats > mice)

– Myofiber degeneration /necrosis/ loss) 1. Inflammation (Rats > mice) 2. Myofiber degeneration /necrosis/ loss) – Fibroplasia +/‐ collagen 3. Fibroplasia +/- collagen  vs toxic myocardial degeneration – Usually extensive‐global, compound related  Jokinen & al 2012

25 26  Jokinen & al 2012. TP.

Etc: Alveolar 1. Etc NON neoplastic histiocytosis  Lungs – Alveolar histiocytosis – White spots = foam cell foci – Renne & Al  intra‐alveolar  Crystals macrophages  Alimentary ‐ Ulcers – gastric/duodenal containing foamy – Stressors, dehydration, water / diet restriction cytoplasm  Often subpleural in aged animals  Increased with hi fat diet – Fldodh Magnuson Magnuson 1974 27 28

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Rat ‐ WYD? 1. Etc Non Neoplastic

A. Alveolar histiocytosis WYD? B. Acidophilic macrophage A. Blood derived Pneumonia hemoglobin C. Pneumocystosis crystals D. Induced alveolar B. Charcot leyden proteinosis Crystals C. Oxalate crystals D. YM1 crystals

1. Etc Non Neoplastic Etc: Rat crystals

 Eosinophilic crystals, Rat lung; WYD? – Rectangular and linear; – Extracellular, alveoli and capillaries. A. Blood derived – No associated inflammation, nor hemoglobin resemblance to eosinophilic crystalline pneumonia crystals  Inset: Okajima stain for B. Charcot leyden hemoglobin. – Considered an artifact of freezing, Crystals intracardiac puncture, and barbiturate C. Oxalate euthanasia. – found only in euthanized rats and not in crystals rats found dead or killed in snap‐traps. D. YM1 crystals – likely crystalized hemoglobin

Rats ‐ WYD? Rats ‐ WYD?

 Which is Normal?  Gravid  Intrauterine hemorrhage, fetal death mummification Cause/s?  Vaginal septa 6. Normal vagina, rat.  E coli 7. Vagina, rat, vaginal septum with necro-hemorrhagic fluid accumulation. 8. Vagina, rat, thin vaginal septum. 9. Vagina, rat, thick vaginal septum. 10. Vagina, rat, mucus plug accumulation in the proximal part of the vagina.

S. Lezmi et al. Vet Pathol 2010;48:964-969 S. Lezmi et al. Vet Pathol 2010;48:964-969 Copyright © by American College of Veterinary Pathologists Copyright © by American College of Veterinary Pathologists 34 35

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RAT ‐ WYD RAT

 Acute – Vacuolation, pyknosis, hemorrhage

 Chronic – Lipid laden macrophages

36 De Jonghe et al. Toxicol Pathol 2015;43:1141-1148 Copyright © by Society of Toxicologic Pathology 37 De Jonghe et al. Toxicol Pathol 2015;43:1141-1148 Copyright © by Society of Toxicologic Pathology

Miscellaneous Rat lesions (phenotypes)  Spinal cord Degenerative Myelopathy – Symmetrical demyelination, axon swelling & loss, 1. Non Infectious, Non Neoplastic Gitter cells, gliosis; ventral & lateral tracts T4 –L4 2. Non Infectious, Neoplastic – Sciatic and tibial nerve degeneration; polyradiculoneuropathy a. Skin / Mammary – most obvious b. Hematopoietic – most famous  Eye Retinal degeneration (drop out) albino rats c. Pituitary – most common?  Eye corneal dystrophy (+/‐ mineralization) – More Tumors: endocrine, reproductive; urinary  Hepatodiaphragmatic nodules (mini hernias) tract, etc.  Pancreas exocrine atrophy 3. Infectious  Pancreas islet pigment, fibrosis (not amyloid)

38  Adrenal cortex cystic degeneration 39

2.a. ‘Skin’ Tumors 2.a. Skin Tumors

Mammary Fibroadenoma (F >> M)  Most common tumor identified clinically Mammary fibroadenoma  Gross: small‐big, soft‐firm ‐‐ Often resectable  More common in Female  Histo ‐ Mesenchymal –often predominates – 40‐70% incidence in – Glands –can be hard to find SD! – Associated with hi Fibromas ‐ Sarcomas (M > F?) prolactin,  Gross: small‐big, soft‐firm – But not necessarily  Histo – Mesenchymal pituitary tumors – Fibrosarcoma – collagen  Trichrome – Myxoid – myxomatous – bluish  Alcian blue RodentPath 41 – PNST‐like (peripheral nerve sheath tumor ) 40 41 c 1990

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HCD Rat Mammary Tumors 2.a. Skin Tumors (HCD = Historical Control Data)

F344 F344 F344 SD CIT SD WIS LE Mammary fibroadenoma ad lib DR Max %  More common in Female Fibroadenoma 36 2 7-29 42 19-31 4-25 22 Adenofibroma 24 – 40‐70% incidence in SD! Adenoma 4 1 21 – Associated with hi prolactin, Cystadenoma 10 – But not necessarily pituitary Adenocarcinoma 8 2 0-2 20 9-17 1-13 7 Ductular 14 tumors carcinoma  Trichrome stains collagen ref Thurman Thurman Sommer Attia Sommer Sommer Sommer 94 94 97 95 97 97 97 – Fibro ~ blue  ROLE OF STRAIN – (Adeno (glands) ~pink/red) RodentPath 42  ROLE OF DIET 42 43

Rat – Mammary Tumors 4069626 Case 4 RAT  18 month‐old female – Strain:  Influences on tumor development

Exposure to estrogen  Increased prolactin levels  Parity  Ovariectomy  Pituitary tumors  Body weight (Ad lib feeding)   What (2) are associated with increased  pituitary and mammary tumors – Estrogens & body weight 44 45

4069626 Case 4 RAT 2.a. Skin Tumors 18 month‐old female Long – Evans Rat Axillary Mass – Neoplasm, multilobular composed of disorganized epithelial proliferation and connective tissue. Fibroma – Lobules are surrounded and separated by moderate to abundant dense, collagen‐rich connective tissue stroma.  More likely? than – Neoplastic epithelium forms variably sized acini and tubules with mild marked intralumenal eosinophilic colloid like material (galactorrhea) fibroadenoma in – neoplastic cells are cuboidal to irregular, arranged inmonolayer. male – moderate to abundant cytoplasm which is often vacuolated; numerous and small (microvesicular vacuolation) to large single lipid vacuoles  which expand cytoplasm with peripheral displacement of the nucleus. Sarcomas are – Nuclei are round to slightly irregular, vesicular to finely granular invasive chromatin and have a small nucleolus. – slight anisocytosis and anisokaryosis. – Myxoid – Mitotic figures are not observed 0/10HPF – PNST – occasionally, acini contain condensed ovoid proteinaceous concretions with internal lamination (corpora amylacea). – Osteo‐ – Mast cells, hemorrhage, inflammation – Hemangio‐ – (Histiocytic 49 50 Sarcoma)

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2.a. Skin Tumors 2.b. Rat Hematopoietic Tumors Other ‘skin’ tumors  Histiocytic sarcoma in skin Lymphoma, leukemia, histiocytic sarcoma – Also in literature as Fibrous histiocytoma or  Arguably most Famous rat tumors – MFH Malignant Fibrous Histiocytoma everyone has heard of them  Papilloma – Squamous cell carcinomas  LGL ‘leukemia’ aka mononuclear leukemia – Uncommon unless induced (skin paint) – especially F344  Zymbal gland tumors  Other lymphomas – Sebaceous gland anteroventral to external canal  Histiocytic sarcoma ‐ anywhere in rat – Sebaceous – squamous feature  Keratocanthoma sporadic, crateriform – Lung Lymphomas? 52 – More common in Guinea pig ? 53

2.b. Rat Hematopoietic Tumors Brayton‐Williford– F344 rat LGL  LGL ‘leukemia’ aka mononuclear leukemia leukemia – Especially F344 – Splenomegaly (homogeneously enlarged on gross) . Arise in spleen marginal zone – Hepatomegaly ‐ later – Leukemia – circulating mononuclear cells with azurophilic granules . Pale rat with watery blood

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68040 68040

Diagnoses  Splenomegaly  Icterus  Hepatopathy (neoplasia)

 Icterus dt?

56 Photos by E Ihms 57 Photos by E Ihms

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68040 4032917 ‐ case 2 F344 Rat  Intravascular leukocytosis Leukemia Diagnosis  Special stains ? IHC?  Pigment nephropathy

Cause?  Hemolysis  Immune mediated?

Smear: Spherocytosis, polychromasia/anisocytosis, thrombocytopenia, neutrophilia, leukemia 58 Photos by E Ihms 59

2.b. Rat Hematopoietic ‘Mononuclear cell leukemia’ Tumors  ‘LGL leukemia’  Spleen  Heterogeneous  NonT, non B, – Nodules  NK activity  Usually Lysosyme,  Lymphoma Esterase negative  TOX PROBLEM  Histiocytic – ‘equivocals’ Sarcoma ?  Relevant human condition? – Rare aggressive NK‐LGL leukemia ?

62 63

2.b. Rat Hematopoietic 2.b. Rat Hematopoietic Tumors Tumors Histiocytic Lung Lymphoma? Sarcoma – Pleomorphic histiocytoid cells  Association with  Spleen M pulmonis….

RodentPath 64 RodentPath 65 64 65

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2.c. Pituitary Tumors 4069626  18 month‐old female Long –Evans Rat  MOST common tumors in some chronic studies  Axillary mass; – 30% ‐ 80%  pituitary mass – Important COD in some  often – Bloody – Cystic – Large – Prolactin secreting  galactorrhea

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4069626 4069626  18 month‐old female Long –Evans Rat

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4069626 Case 4 RAT Rat Pituitary Tumors 18 month‐old female Long –Evans Rat Pituitary mass  Usually pars distalis / intermedia / nervosa ? – Compressing the medulla is a 4‐6mm discrete, faintly circumscribed and densely cellular, nodular , neoplastic  Usually adenomas / carcinomas ? mass composed of a uniform population of polygonal cells  Usually secrete ? arranged into solid islands separated by fine fibrovascular stroma.  Most common in Males or females? – Neoplastic cells have moderate amount of eosinophilic to FE Male Strain age refs lightly amphophilic cytoplasm, generally indistinct % % cytoplasmic borders, and round to slightly irregular nuclei, Attia 1995 …usually w mammary neoplasia in SD old 62 47 vesicular to finely granular chromatin anda small nucleolus. females (estrogen exposure??) – There is moderate anisocytosis and anisokaryosis, with SD 2y 84 62 McMartin & al 1992 few giant karyomegalic cells at the edge of the mass. – rare‐few mitotic figures ~10/10HPF. Wistar 110w 25 14 Nataraju 2016 - estrogen induced – Numerous large blood‐filled spaces lined by neoplastic F344 2y 41-65 30-77 Thurman&al 1994 – by food restriction in M cells (pituicytes). At one edge of the mass there are Pollard, asa, Snyder LOBUND Hi? possible remnants of the pre‐existing adeno‐hypophysis. Gonadotroph> lactotroph: FSH LH > prl ?? 70 75

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2.b. Pituitary Tumors Rat Pituitary Changes

 Origin of most common pituitary tumor in  Usually pars distalis Human / Rat / Mouse  Pituitary adenomas – compressive ‐ May have . Pars distalis – Pseudocapsule, hemorrhage, cystic changes . Pars intermedia – Chromophobes, acidophilic, basophilic, mixed . Pars nervosa  Hyperplastic Foci = proliferation of cells of normal – Most common secretory product size, no evidence of pseudocapsule and no significant . Prolactin (+ GH in human) compression of adjacent pituitary tissue.  Nodules of hypertrophic cells = larger cells without – Likely clinical Signs evidence of encapsulation . Galactorrhea in males;  Pituitary carcinomas are rare and require evidence of . acromegalic features or growing nose in elderly invasion or distant metastasis for their diagnosis. humans

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Pituitary T MORE rat tumors: endocrine

 Not just prolactin:  Adrenal: Multi‐hormone Producing – Cortical F> M ? Proliferative Lesions in – Medullary – pheochromocytoma M > F? Estrogen‐induced Rat . Up to 70% in Wistar rats Pituitary Prolactinoma – Some with Ganglioneuroma . Prolactin . TSH  Thyroid: . α SU = anti‐glycoprotein – Follicular tumors – thyroglobulin + /‐ hormone alpha‐subunit of . Follicular, cystic, papillary, solid Glycoprotein hormones LH – Interstitial C cell (M>F) –Calcitonin + FSH TSH HCG . more common than in mice esp in BN, WH? – negative for GH, LHβ, ACTH, and S‐100.  Islets – insulinoma ‐ BN rats Takekoshi & al. 2014. Acta Histochem Cytochem. 47(4): 155–164. – Vs hypertrophy in fat insulin resistant rats ? 78 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4164703/ 83

MORE rat tumors: endocrine Pheochromocytoma

 Adrenal  ~80% Wistar !! – Pheochromocytoma  ~30% SD & (medullary) F344 – Often . Bloody . Large  Often bloody . Invade Vessels  Invades  Another diagnosis? vasculature – ORN –chronic nephropathy

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Pheochromocytoma Ganglioneuroma

 ‘packeted’ ‘neuroendocrine’ pattern  Look like Ganglion cells  Usually w pheo

 Also in Trp53 TM mice

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MORE rat tumors: 68040 Another expected finding in F344 male >1yo  Reproductive: – Testes: Interstitial C cell (F344!!) > Sertoli, seminoma – Vagina /cervix / uterus – polyps, stromal tumors  Urinary tract – Kidney –renal carcinoma (LE rats, Eker rats); Nephroblastoma, Renal mesenchymal tumor – Ureter/Bladder ‐ BN rats – T crassicauda?  Mesothelioma – especially F344  Etc….

88 89 Photos by E Ihms

Creasy & al. 2012 RAT Interstitial Cell Tumors Rat testis. Leydig cell Tumors.  Aka Leydig cell tumors – usually benign 55. Adenoma  May be multiple or bilateral 56. Adenoma  – Expansile mass of pale foamy polygonal Hemorrhage, necrosis are common vacuolated cells  Hyperplasia (usually < 3 tubules diameter) – +/‐ small basophilic fusiform cells progresses to ICT (usually > 3 tubules – +/‐ hemorrhage, necrosis diameter) 57. Adenoma, retiform. 58. Adenoma, retiform.  Induced in rats by increasing luteinizing 59. Carcinoma hormone (LH) levels 60. Carcinoma  Mice –not common 90 Creasy et al. Toxicol Pathol 2012;40:40S‐121S Copyright © by Society of Toxicologic Pathology 91 – Induced in by hyperestrogenism

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Granulosa Cell tumor WYD

Synonym(s) – Tumor, sex cord stromal, benign, granulosa cell type.  Non‐cystic, solid proliferative lesion larger than a CL.  Cell morphology resembles normal granulosa cells.  >70% granulosa cells. (+ Stromal component)  Variable luteinization  Atypia can be Minimal.  Mitotic figures may be rare‐few.  Call‐Exner bodies uncommon.  Cystic, microfollicular, solid , trabecular but usually NOT subclassified by pattern.  Hemorrhage necrosis lipofuscin in larger tumors.

 Benign: Compressive  Malignant: Invasion/metastasis, size, hemorrhage, necrosis, atypia

95 Dixon&al female Repro INHAND 2014 JTP 97

AB RAT Uterus RAT Uterus

 Diagnosis/es?  Diagnosis/es? A. Endometrial A. Endometrial Carcinoma Carcinoma B. Endometrial B. Endometrial hyperplasia hyperplasia C. Leiomyoma C. Leiomyoma D. Polyp Glandular D. Stromal Sarcoma E. Polyp Stromal F. Stromal  Predisposed Sarcoma strains(s)?

Elmore et al Elmore et al 98Copyright © 2016 Society of Toxicologic Pathology https://www.ncbi.nlm.nih.gov/pubmed/27821709 99Copyright © 2016 Society of Toxicologic Pathology https://www.ncbi.nlm.nih.gov/pubmed/27821709

UTERUS Rat REPRO Rat

 Diagnosis/es?  Diagnosis/es? a) Endometrial a) Endometrial adenocarcinoma adenocarcinoma b) Endometrial b) Endometrial Stromal Sarcoma Stromal Sarcoma c) Granular Cell tumor c) Granular Cell tumor d) Histiocytic Sarcoma d) Histiocytic Sarcoma e) Schwannoma e) Schwannoma  Useful IHC? EM  Special stains? features? – PAS+ – S‐100  Useful IHC?

Dixon et– al. 2014presence of basement Dixon et al. 2014 100 101 – S‐100+ vimentin+ Copyright © membraneby Society of Toxicolo gicon Patholo EMgy https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4253081/ Copyright © by Society of Toxicologic Pathology https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4253081/

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Hereditary ‘Renal Cell Carcinoma’ RAT (RCC)  EKER rat (or LE )  Diagnosis/es? – Arose in inbred Wistar – Tsc 2 tuberous sclerosis2 tumor A. Renomegaly suppressor gene mutation B. Renal amyloidosis – autosomal dominant, homozygous C. Nephroblastoma lethal – Metastasis 0‐rare D. Renal cell carcinoma  Often multiple and bilateral renal E. Renal Mesenchymal adenomas tumor  Histo: simple cysts, papillary cystadenomas, solid eosinophilic adenomas, or solid basophilic adenomas

McDorman & Wolf Copyright © 2002 Society of Toxicologic Pathology 102 Gal et al 2012. https://openi.nlm.nih.gov/detailedresult.php?img=PMC3511877_1751-0147-54-50-2&req=4 103http://journals.sagepub.com/doi/pdf/10.1080/01926230290168542

Who is this Nephroblastoma famous rat? • Blastemal (primitive mesenchymal with abundant mitoses) cells around ducts • Basophilic ill‐defined primitive tubule or rosettes distinct from trapped tubules • Glomeruloid bodies

• NTP: predominantly Female , heritable in some SD lines? • Gross: Usually Spherical‐multilobular at 1 pole • Rapid growth, early death, may metastasize  What is its famous mutation? Hard et al Copyright © 2016 Society of Toxicologic Pathology 104 http://journals.sagepub.com/doi/full/10.1177/0192623316638960

Rat renal mesenchymal tumor Rat kidney tumors (RMT)

• Non circumscribed, heterogeneous Gross/behavior Histo etc comments primitive mesenchymal cells swirling Multiple bilateral Cysts/adenomas Renal Cell ‘Carcinoma’ around entrapped preexisting tubules, May be cystic Eker & LE rats – familial • All RMT contain many remnants of Usually at a pole Primitive blastema, epithelial ; Nephroblastoma Circumscribed but rapid primitive tubules, ‘glomeruloid preexisting, entrapped tubules and, growth bodies sometimes, hyperplastic pelvic Mesenchymal + Entrapped Renal mesenchymal urothelium Non circumscribed tubules , hyperplastic tumor • Invasion of renal pelvis, with urothelial urothelium Renal sarcoma, hyperplasia is a common feature hemangiosarcoma occasional/sporadic • More common than Nephroblastoma in lipomatous tumors NTP studies Hematopoietic tumors • May metastasize

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68683 ‐ 71 wo female Rat MORE rat tumors:  Diagnosis/es?  SD with Thy1:GFP transgene – Mesothelioma often involves/arises in Spermatic cord

 Another tumor ?

 Likely Strain ?

RodentPath 111 111 112

68683 ‐ 71 wo female Rat MORE rat tumors:  SD with Thy1:GFP transgene  expressing GFP under Thy1 promoter  axons constitutively express green fluorescent protein (GFP)  Diagnosis/es?

4067775 –Rat 113 116

Zymbal’s gland Zymbal’s gland neoplasia ?

 what is it ?  Types – multilobulated sebaceous holocrine gland – Sebaceous cell adenoma, located anterior‐ventral to the ear canal – Squamous cell papilloma, 3‐4 lobules each have intralobular ducts – Carcinoma of sebaceous and/or ductal epithelial origin (Squamous epithelium) that empty into the  Causes of Zymbal’s gland neoplasia ? excretory duct and then into ear canal. – Target organ of many carcinogens  Do mice have it? – incidence a high as 67% (versus 0% in controls) – E.g.2‐Acetylaminofluorine, Tris carbonium pamoate,  How common are spontaneous tumors ? urethan, heterocyclic amines, benzene, 3‐3 – Not very common but ~ 1% or less in aging dichlorobenzidine, 4‐aminostilbene, azoxymethane, methychorlanethrene, and cupferron. studies in various strains 120 121

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MORE rat tumors: 60200  Diagnosis/es?  Diagnosis/es? A. Cardiomyopathy A. Cardiomyopathy B. Myocardial B. Myocardial degeneration fibrosis degeneration fibrosis inflammation inflammation C. Endocardial C. Endocardial fibroelastosis fibroelastosis D. Endocardial D. Endocardial Schwannoma Schwannoma  IHC?  IHC? A. Vimentin, S100

60200 123 124

RAT Heart RAT CNS cardiac schwannomas elastic fibers and collagen WHICH is: (arrow), produced by the • Astrocytoma neoplastic Schwann cells. • Oligodendrogliom, Anitschkow cells – wavy • Medulloblastoma caterpillar like ovoid nucleus • Granular cell tumor and central condensed • Granular cell tumor chromatin + PAS Antoni A pattern, characterized by nuclear palisading

Bertrand et al. . Elmore et al Copyright © 2014 Society of Toxicologic Pathology http://journals.sagepub.com/doi/full/10.1177/0192623313518114 126Copyright © 2016 Society of Toxicologic Pathology https://www.ncbi.nlm.nih.gov/pubmed/27821709

RAT CNS QUIZ

A)Astrocytoma B)Malignant high-grade oligodendroglioma, C)Malignant low-grade- mixed glioma, D)Medulloblastoma E)Benign granular cell tumor F) Benign granular cell tumor + PAS

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FELASA 2014 recommendations Great Rat lesions (phenotypes) Rats ‐ Viruses 1. Non Infectious, Non Neoplastic  3 month testing 2. Non Infectious, Neoplastic 1. Parvovirus Kilham rat virus (RV)  Annual testing 3. Infectious 2. Parvovirus Rat minute virus (RMV) 1. MAD1 (FL) 3. Parvovirus Rat parvovirus (RPV) 2. MAD2 (K87) a. Viruses 4. Parvovirus Toolan’s H‐1 virus (H1) 3. Reovirus type 3 b. Bacteria 5. Pneumonia virus of Mice (PVM) 4. Sendai virus c. Eukaryota 6. Rat coronavirus (SDAV) 7. Rat Theilovirus (RTV) (see mice) a. Fungi 8. Hantaviruses b. Protozoa c. Metazoa a. Helminths 139 b. Arthropod 140 http://www.uni-heidelb.de/md/ibf/gesundzeugnis/hp-lab_anim-2014--178-92.pdf

Rat Serology: Results Pritchett‐Corning, K. R., J. Cosentino, et al. (2009). "Contemporary prevalence of infectious “Virus” prevalence – Rats agents in laboratory mice and rats." Lab Anim 43(2): 165‐173. 16 Prevalence % Agent (assay abbreviation) Method N NA Europe Total 14 Adenovirus (MAV 1 and 2) Serology 35,084 0.06 0.16 0.07 Hantaviruses (HANT) Serology 23,248 0.01 1.09 0.07 12 Lymphocytic choriomeningitis virus (LCMV) Serology 37,709 0.00 0.00 0.00 Parvovirus generic assay (NS-1) Serology 63,808 2.00 3.34 2.05 10 - H-1 (Toolan's) Serology 79,451 1.37 2.85 1.41 2002-2008 - Rat virus (RV, KRV) Serology 86,764 1.23 4.05 1.30 8 2009 - Rat minute virus (RMV) Serology 47,596 1.39 2.99 1.46 2010 - Rat parvovirus (RPV) Serology 85,901 1.19 1.86 1.21 6

Pneumonia virus of mice (PVM) Serology 79,954 0.06 1.24 0.10

Rat coronavirus (RCV/SDAV) Serology 82,733 0.28 0.61 0.29 4

Rat respiratory virus (RRV) Histopathology 3901 6.36 na 6.36 2 Rat theilovirus (RTV, GD-VII) Serology 35,920 1.43 1.32 1.43 Reovirus-3 (REO-3) Serology 72,886 0.01 0.00 0.01 0 Sendai virus (SEND) Serology 80,851 0.01 0.51 0.02 (P car) RRV Parv NS-1 RTV 141 142

Virus typea Sample type Family or genus No. of pools Most similar species Known Serum Anelloviridae 22 Unclassified anellovirus Serum Bocavirus 9 Porcine bocavirus NYC rats 201 Serum Mastadenovirus 1 Murine adenovirus A 3.a. Rat Viruses Serum Parvovirus 19 Kilham rat virus Fecal Bocavirus 16 Porcine bocavirus Fecal Calhevirus 10 Calhevirus 1  Boring ‐ usually subclinical seroconversion Fecal Cardiovirus 21 Boone cardiovirus / Rat theilovirus 1 Pig stool-associated circular ssDNA virus Fecal Circovirus 21 Chicken anemia virus Fecal Hunnivirus 19 Ovine hungarovirus Fecal Mamastrovirus 19 Rat astrovirus / Murine astrovirus 1. Parvoviruses Fecal Mastadenovirus 18 Murine adenovirus A . Seroconversion = only sign? Fecal Parvovirus 24 Kilham rat virus Fecal Rotavirus 3 IDIR agent . Historical /experimental disease Fecal Other 31 Porcine reproductive and respiratory syndrome Novel Serum Arterivirus 2 2. Rat Theilovirus virus Serum Hepacivirus 16 Rodent hepacivirus . Seroconversion = only sign? African horse sickness virus / Serum Orbivirus 3 Epizootic hemorrhagic disease virus Serum Pegivirus 15 Rodent pegivirus 3. SDAV  clinical Disease Serum Pestivirus 4 Bungowannah virus / Classical swine fever virus Fecal Hepeviridae-like 1 Hepatitis E virus / Betatetravirus  Cowpoxvirus – ZOONOTIC Fecal Kobuvirus 19 Mouse kobuvirus / Aichi virus Fecal Parechovirus 5 Ljungan virus / Sebokele virus . Pet rat reports Fecal Phlebovirus 1 Echarate virus Fecal Picobirnavirus 22 Human picobirnavirus / Otarine picobirnavirus . Not in standard surveillance or FELASA lists Fecal Picornaviridae 33 Feline picornavirus / Bat picornavirus 3 143 Fecal Rosavirus 20 Rosavirus M-7 144 Fl Si 13 H i /P i i

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Rat Parvoviruses Rat Parvoviruses (RV, H‐1, RMV, RPV‐1a) (RV, H‐1, RMV, RPV‐1a)  Parvoviridae  No disease recognized in natural infections by – nonenveloped SS DNA viruses. – Toolan’s H1 virus H1  Incidence: low. ? – Rat Parvovirus 1RPV  Transmission: Direct contact, fomites. – Rat Minute virus RMV  Clinical: Usually none; except maybe RV  Rare disease in natural infections by  Pathology: Usually none; except maybe RV – Kilham rat virus aka Rat virus (RV)  Immune effects ? Lymphotropic/lympholytic?  Diagnosis: serology MFI, IFA; fecal PCR (or mesenteric nodes)  Tumor effects? oncolytic ? – RPV 1a is a Model oncolytic agent… 145 146 – Transient? biliary hyperrplasia?

Rat Parvoviruses Adult RV disease Rat Virus (RV)  Clinical: usually none; may cause:  1983 report of – fertility, fetal resorption, small litters, runting. Outbreak in SPF naïve rats – Juvenile‐young adult male: lethal hemorrhage  Hemorrhage and necrosis in brain, testes, epididymides .  Pathology: usually none  (Infamous scrotal hemorrhage) – infects actively replicating cells  CPE cytolysis  Rare intranuclear inclusion – IN utero infection:  cerebellar hypoplasia bodies in endothelium, bile duct – In young males  hemorrhage, thrombi, infarction epithelium . Notably: Scrotal hemorrhage . Liver multi focal degeneration/ necrosis, hypertrophy

147 149

Pathology, organ distribution, and immune clinical‐grade parvovirus h1 (H1PV) response after single and repeated intravenous injection of rats with clinical‐grade parvovirus H1.  nonpathogenic except in  Parvovirus H1 (H1PV) ‐ natural host is rat. rat and hamster fetuses  Nonpathogenic except in rat and hamster fetuses and and newborns newborns.  natural host is rats  Strongly oncolytic in human cancer models  IV in adult rats  – Clinical‐grade H1PV was produced according to Good Manufacturing – No significant pathology! Practices. – Antivirus Ig G – Minimal effects (IgG & biliary hyperplasia) in rats  Due to the confirmation of the favorable safety profile of H1PV  Studied for oncolytic in a permissive animal model, a phase I/IIa clinical trial of H1PV brain tumor treatment ! in brain tumor patients could be initiated.  Geletneky & al. (2015).  Geletneky & al 2015 Comp Med.

150 151

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RAT THEILOVIRUS (RTV1) Rat coronaviruses SDAV RCV  Picornaviridae  Etiology: enveloped RNA virus – non‐enveloped, SSRNA virus (Cardiovirus) – Rat coronavirus (RCV or Parker’s rat coronavirus) – Sialodacryoadenitis virus (SDAV)  Incidence: ‘moderate’ (seropositives)  Incidence: ‘low’ (seropositives).  Transmission: Fecoral  Transmission: highly contagious by aerosol etc.  Clinical: usually none  Clinical & Pathology findings: can be none  Pathology: usually none – worst in immunodeficient w SDAV  Diagnosis: Serology MFI, IFA; fecal PCR  Diagnosis: Serology MFI, IFA; PCR in acute dz – SDAV from Harderian or submandibular gland. – RCV from lung

152 153

‘Rat coronaviruses’ Rat coronaviruses SDAV RCV  Gross: SDAV RCV – RCV Parker’s rat coronavirus causes respiratory – Large salivary glands + EDEMA infection – Large Cervical lymph nodes – SDAV infects upper respiratory tract, – Rhinitis, maybe pneumonia . Submandibular + parotid glands ‐ sialoaedintis . Harderian + exorbital glands – dacryoadenitis , – Occasional eye lesions (keratoconjunctivitis, corneal . Pulmonary disease is possible in young rats. opacities, megaloglobus, hypopyon, hyphema, etc.)  Clinical: Usually none, or classic SDA   Histology: . Neck swelling ‐ Sialoadenitis – Necrosis of gland & ductal epithelium of parotid and . Porphyrin oculonasal discharge ‐ DacryoAdenitis submaxillary and lacrimal glands . Also exophthalmos  keratitis &corneal ulcers. – Squamous metaplasia common during healing – Then fibrosis in chronic disease 154 155 – Sublingual gland usually spared

SDAV SDAV

Diagnosis /es? CLINICAL Disease!! in – Edema, neck competent animal – Sialodacryoadenitis – Hi morbidity Cause/s? (Outbreak) – sialodacryoadenitis virus (SDAV)  DORA – Swollen neck – Coronavirus (enveloped) . Salivary gland Affected tissues? edema – Respiratory tract, – Chromodacryorrhea – Harderian and exorbital lacrimal glands, . Consider – Submandibular and parotid salivary dehydration & glands; Harderian and lacrimal glands stressors RodentPath 156 – Not Sublingual glands 156 157 http://dora.missouri.edu/rats/coronaviruses‐rcv‐and‐sdav/

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Chronic infection Paramyxoviruses in Rats  ‘repair’ squamous metaplasia PVM Sendai virus – Especially in Harderian – pneumovirus – ~ Parainfluenza 1 gland ducts acini  Just seroconversion ?  Just seroconversion ?  Secondary ocular lesions due to lacrimal gland  Focal & perivascular  Wasting + pneumonia damage infiltrates ? in nude rats ? – Corneal ulcers   Serology & PCR  Serology & PCR – Keratitis  – Hyphema  – (exophthalmos, megaloglobus in some) 158 https://www.askjpc.org/vspo/show_page.php?id=747 159

DNA viruses X‐SCID rat when you see inclusions consider:  Parotid gland  Adenovirus –MAd1/2 – seroconversions  Prostate – Unconfirmed subclinical infections with  Diagnosis/es? intranuclear inclusion bodies (INIB) in enterocytes A. Cytokaryomegaly  Cytomegalovirus –RCMV – wild rats B. Intranuclear inclusion bodies – subclinical INIB in salivary & lacrimal glands C. Inflammation D. Hyperplasia?  Papillomavirus – wild rats subclinical  Cause/s?  Polyomavirus Rat –PyV–nude rat reports A. Cytomegalovirus? – Wasting pneumonia parotid sialoadenitis B. Polyomavirus? – INIB in epithelia kidneys parotid lungs

FELASA 2014 recommendations NYC Rats ‐ Firth & al 2014 Virus typea Sample type Family or genus #pools Most similar species Rats ‐ Viruses Known Serum Anelloviridae 22 Unclassified anellovirus Serum Bocavirus 9 Porcine bocavirus Serum Mastadenovirus 1 Murine adenovirus A Serum Parvovirus 19 Kilham rat virus  3 month testing Fecal Bocavirus 16 Porcine bocavirus Fecal Calhevirus 10 Calhevirus 1 1. Parvovirus Kilham rat virus (RV)  Annual testing Fecal Cardiovirus 21 Boone cardiovirus / Rat theilovirus 1 Pig stool-associated circular ssDNA virus Fecal Circovirus 21 2. Parvovirus Rat minute virus (RMV) 1. MAD1 (FL) Chicken anemia virus Fecal Hunnivirus 19 Ovine hungarovirus Fecal Mamastrovirus 19 Rat astrovirus / Murine astrovirus 3. Parvovirus Rat parvovirus (RPV) 2. MAD2 (K87) Fecal Mastadenovirus 18 Murine adenovirus A Fecal Parvovirus 24 Kilham rat virus 4. Parvovirus Toolan’s H‐1 virus (H1) 3. Reovirus type 3 Fecal Rotavirus 3 IDIR agent Fecal Other 31 5. Pneumonia virus of Mice (PVM) 4. Sendai virus Novel Serum Arterivirus 2 Porcine reproductive and respiratory syndrome virus Serum Hepacivirus 16 Rodent hepacivirus African horse sickness virus / 6. Rat coronavirus (SDAV) Serum Orbivirus 3 Epizootic hemorrhagic disease virus (see mice) Serum Pegivirus 15 Rodent pegivirus 7. Rat Theilovirus (RTV) Serum Pestivirus 4 Bungowannah virus / Classical swine fever virus Fecal Hepeviridae-like 1 Hepatitis E virus / Betatetravirus 8. Hantaviruses Fecal Kobuvirus 19 Mouse kobuvirus / Aichi virus Fecal Parechovirus 5 Ljungan virus / Sebokele virus Fecal Phlebovirus 1 Echarate virus Fecal Picobirnavirus 22 Human picobirnavirus / Otarine picobirnavirus Fecal Picornaviridae 33 Feline picornavirus / Bat picornavirus 3 Fecal Rosavirus 20 Rosavirus M-7 Fecal Sapovirus 13 Human sapovirus / Porcine sapovirus 162 http://www.uni-heidelb.de/md/ibf/gesundzeugnis/hp-lab_anim-2014--178-92.pdf 163

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3.a. Rat Viruses – Summary Table 2. Incidents/outbreaks identified by the review, by zoonotic agent and animal category.  Boring  usually subclinical seroconversion

 Historically significant – SDAV –not so common but still around

 Zoonotic – Hantavirus – wild rodents – subclinical seroconversion – LCMV – seropositive urban and seaport rats . Pet Hamsters & immunodeficient (mice)

Halsby KD, Walsh AL, Campbell C, Hewitt K, Morgan D (2014) Healthy Animals, Healthy People: Zoonosis Risk from Animal – Cowpox –pet rats Contact in Pet Shops, a Systematic Review of the Literature. PLoS ONE 9(2): e89309. doi:10.1371/journal.pone.0089309 http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0089309 – (monkeypox giant Gambian rat) 164 165

WYD?Figure 2. Skin. Cause?

6) Pharynx 7) Pharynx 8) Lung 9) Lung 10) Skin 11) Brain

 Name the inclusion

FELASA 2014 recommendations 3.b. Bacteria Rats – Bacteria & Fungi  Mostly opportunists today  3 month testing  Annual testing – Most research rats are immune sufficient 1. Clostridium piliforme 1. Filobacterium rodentium 2. Helicobacter spp. (CAR bacillus) 1. Gram positive: S aureus ~ 23% +  H bilis 2. Pneumocystis spp. 2. Helicobacters ~ 6% 3. Mycoplasma pulmonis 3. Salmonella spp. 4. Pasteurella pneumotropica 4. Streptobacillus moniliformis 3. Gram Negative: P pneumotropica > others 5. Strep b‐haemolytic (not group D) 6. Streptococcus pneumoniae 4. Mycoplasmosis – should be rare now …. . M pulmonis often with CARbacillus in Chronic Respiratory Disease – Pet and wild rats 172 http://www.uni-heidelberg.de/md/ibf/gesundzeugnis/hp-lab_anim-2014--178-92.pdf 173

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Rat Bacteriology: Results ‐ 2009 Pritchett‐Corning, Cosentino & Clifford 2009" Bacterial prevalence ‐ Rats

PREVALENCE % J facilities 30 Bacterium Method N NA Europe Total NH 07 Bordetella bronchiseptica Culture 8282 0.00 0.00 0.00 Cilia-associated respiratory bacillus Serology 26,057 0.27 4.63 0.48 25 Corynebacterium kutscheri Culture 8289 0.00 0.00 0.00 Gram+ Helicobacter genus (any sp.)* PCR 8852 6.59 7.14 6.63 - Helicobacter hepaticus PCR 8915 0.46 0.31 0.45 - Helicobacter bilis PCR 8915 0.42 0.31 0.42 20 Klebsiella oxytoca Culture 7315 0.37 0.00 0.37 Klebsiella pneumoniae Culture 7513 0.55 0.00 0.53 2002-2008 Culture 3433 0.06 nt 0.06 15 Mycoplasma pulmonis Serology 81,524 0.16 2.57 0.23 3? 2009 PCR 3734 0.29 nt 0.29 2010 Pasteurella multocida Culture 8223 0.00 0.00 0.00 10 Pasteurella pneumotropica Culture 8241 4.92 3.99 4.81 8 -Other Pasteurella spp. Culture 7346 0.45 0.00 0.44 Salmonella sp. Culture 8235 0.00 0.00 0.00 5 Staphylococcus aureus Culture 7365 23.50 30.63 23.61 ~40% Streptobacillus moniliformis Culture 797 0.00 0.00 0.00 Streptococcus pneumoniae Culture 8289 0.00 0.00 0.00 Streptococcus sp. – β-haemolytic, Group B Culture 7503 3.74 1.34 3.67 0 Streptococcus sp. – β-haemolytic, Group G Culture 8314 0.01 2.71 0.35 174 H hep H bil H any P pneum S aureus Clostridium174 piliforme ~2% 175

Staphylococci in rats Staphylococci in rats

 Gram‐positive cocci  Common isolates – S aureus, S xylosus from normal skin or  Incidence: VERY common – Dermatitis   Transmission: contact, fomites, wounds  Clinical & Pathology Findings: Usually none  Furunculosis in nude – Opportunistic infections rats  Diagnosis: Culture, PCR, histopathology

176 177 http://dora.missouri.edu/rats/

Helicobacters in rats Helicobacters in rats Identified by PCR  Gram‐negative, microaerophilic spiral motile Uncertain significance – H. bilis, ganmani, H. muridarum, H. rodentium , – Hepatobiliary dz ? H. trogontum H. typhlonius Incidence: The – Zoonotic concern? incidence of infection is moderate.  Incidence: pretty common H spp, H ganmani >  Transmission: fecal oral – H hepaticus or bilis  Clinical & Pathology Findings: Usually none H bilis in nu/nu rats  Diagnosis: Fecal PCR – Proliferative and ulcerative typhlitis, colitis, proctitis

Haines, D.C., et al., Inflammatory large bowel disease in immunodeficient rats RodentPath 179 naturally and experimentally infected with Helicobacter bilis. Vet Pathol, 1998. 178 179 35(3): p. 202‐208.

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Rat Helicobacters Pasteurella pneumotropica  Gram‐negative, short pleomorphic rod H bilis in nu/nu rats – Poor (gram or silver) staining on histo Proliferative and ulcerative  Incidence: common in mice & rats typhlitis, colitis, proctitis  Transmission: aerosol, fecal‐oral, contact 1. Colon  Clinical: Often none, opportunist 2. Cecum – Conjunctivitis, respiratory, reproductive tract infections  Pathology: often none – Inflammation necrosis, inconspicuous bacteria – Abscesses especially in immunodeficient Haines, D.C., et al., Inflammatory large bowel disease in immunodeficient rats RodentPath 180 naturally and experimentally infected with Helicobacter bilis. Vet Pathol, 1998. 180 35(3): p. 202‐208. 181 Diagnosis: PCR most sensitive, specific

Naturally Occurring Disseminated Group B Streptococci Streptococcus Infections in Postnatal Rats  On the list: B hemolytic + S Pneumoniae – Beta‐hemolytic species  Group B Streptococcus (Streptococcus agalactiae, GBS) . cause complete rupture of red blood cells – (Beta hemolytic) . E.g. S pyogenes, S pneumoniae, S agalactiae, S equi  10 of 26 (38.5%) 21‐ to 24‐d‐old rat pups died or were . Low grade Opportunists in immune sufficient euthanized due to a moribund state . Undesirable in immune deficient – tg on Munich–Wistar–Frömter background. – Streptococcus pneumoniae  4 had intralesional coccoid bacteria in various organs . Secondary pathogen –human risk ? To or from rats? without accompanying inflammation. . Fibrinosuppurative poly serositis pleuropneumonia  GBS isolated from liver of 2, skin abscesses in 3 littermates.  GBS is a potentially clinically significant spontaneous – Enterococci E durans, E faecalis (formerly group D) infection research rats…. . Gamma hemolytic (incomplete hemolysis)  Shuster & al 2013 COMP MED . Historical ‘outbreaks’ in weanling rats: diarrhea death . Gram positive cocci adhered to villus surfaces 182 183

3.b. Bacteria – summary 3.b. Bacteria – summary

1. Gram positive: S aureus ~ 23%* ! (+S xylosus)  HISTORICAL –should not see in lab rats today… – Dermatitis, Abscesses, opportunist infections – Mycoplasma pulmonis + CARbacillus (CRD) 2. Helicobacters ~6% . BUT ON THE LIST & in pets etc near you! – Enterohepatic – lower GI Tract – Cl Piliforme – megaloileitis + liver necrosis 3. Gram Negative: P pneumotropica – Salmonella enterica typhimurium – liver necrosis – C kutscheri – pseudotuberculosis (abscessed Lymph – Opportunists, inflammatory lesions, abscesses nodes +/‐ pneumonia)  + HISTORICAL rare/unlikely in lab rats… – Klebsiella pneumoniae  + Zoonotic rare/unlikely in lab rats…

184 * % positive of submitted specimens – CRL 185

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Murine Respiratory Murine Respiratory Mycoplasmosis Mycoplasmosis M pulmonis & CARbacillus

 MRM, Chronic Respiratory Disease of rats  M pulmonis – mollicutes – Tiny, lack a cell wall  Pneumonia  Perivascular  Cilia‐associated respiratory (CAR) bacillus  Bronchiectasis peribronchiolar – Filobacterium rodentium  Neutrophilic  chronic lymphocytic cuffing – Gram‐negative filamentous rod, motil  M pulmonis & – Similar to Flavobacterium, Flexibacter CARbacillus are  + upper respiratory lymphocyte mitogens  Incidence: Rare now; historical significance disease

186 http://www.radil.missouri.edu/info/dora/RATPAGE/Bac.htm 187

Brayton– Rat CARbacillus – Wild Urban Rats

 Lymphoplasmacytic cuffs, lung  Associated with what agents ?

Wild Urban Rats Wild Urban Rats Norway (Rattus norvegicus) and black rats (Rattus rattus)  Diagnosis/es 5. Nose, rhinitis  BALT hyperplasia – Lumen purulent exudate  Peribronchiolar and – Submucosa Lymphocytes and perivascular lymphocytic plasma cells cuffs may not actually be inflammatory lesions but  Cause/s rather inducible BALT iBALT, 6. Nose, rhinitis  May be response to – Basophilic bacilli between cilia (tangled M. pulmonis, a recognized cilia) B‐ and T‐cell mitogen in rats, – Inset: Steiner’s silver stain. Black  or to antigens of CARBacillus filamentous bacteria among the cilia. which was identified more – CARBacillus commonly

Rothenburger et al. Vet Pathol 2015;52:1210-1219 Rothenburger et al. Vet Pathol 2015;52:1210-1219 190 Copyright © by American College of Veterinary Pathologists 191 Copyright © by American College of Veterinary Pathologists

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FELASA 2014 recommendations Other mycoplasmas RATS – Other Agents

 Mycoplasma haemomuris  Bacteria etc  Unlikely in – (Hemobartonella muris) 1. Bordetella bronchiseptica contemporary 2. Corynebacterium kutscheri colonies – Extracellular RBC parasite  infectious anemia or splenomegaly in rodents. 3. Encephalitozoon cuniculi 4. Klebsiella oxytoca – Dx by PCR, blood smear 5. Klebsiella pneumoniae – transmitted by rat louse Polyplax sp  Found in healthy 6. Other Pasteurellaceae animals & 7. Pseudomonas aeruginosa Too common to  Mycoplasmas in tissue culture 8. Staphylococcus aureus practically exclude – Common ‐ Most are NOT transmissible to rodents

193 194 http://www.uni-heidelberg.de/md/ibf/gesundzeugnis/hp-lab_anim-2014--178-92.pdf

Rat WYD? Diagnosis/es? a) Pneumonia  Lung: Pneumonia, b) Bronchopneumonia embolic, necrosuppurative, subacute, multifocal to c) Interstitial coalescing, marked, with mild pleuritis, and d) Embolic numerous bacilli, etiology consistent with Corynebacterium Cause/s? kutscheri, Long‐Evans rat, a) M pulmonis rodent.  Cause: Corynebacterium b) CARbacillus kutscheri c) Streptococcus pneumoniae  Condition Synonyms: Murine d) Corynebacterium kutscheri pseudotuberculosis e) Murine Pseudotuberculosis

195 http://www.askjpc.org/vspo/show_page.php?id=543 197 http://www.askjpc.org/vspo/show_page.php?id=543

3.b. Bacteria – Zoonotic Human/rodent helicobacters… ?

H bilis? hepaticus? ganmani ?  Helicobacter pylori, Leptospirosis –rat urine contaminated water  Helicobacter bilis, Salmonella enterica typhimurium  Helicobacter hepaticus, RBF = rat bite fever due to  Helicobacter ganmani . Spirillum minus – spirillary RBF, sodoku, Haverhill fever Streptobacillus moniliformis – Streptobacillary RBF  trend of a higher presence of Helicobacter Yersinia pestis ? –black plague Via rat fleas ? spp. in patients with biliary tract cancers compared with normal controls or those with benign biliary diseases… – Shen & al 2014 200 201

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Fatal Rat‐Bite Fever in a Child San Diego County, California, 2013

 CDC.gov – Healthy 10yo boy  rigors, fevers, vomiting, headaches, leg pains, death – PCR Pos Streptobacillus moniliformis (Lung, liver, epiglottis) – owned 2 pet rats; 1 PCR Pos oropharyngeal swab Streptobacillus moniliformis  ‘Nearly all domestic and wild rats carry S. moniliformis. Elliot 2007  17% of NYC R norvegicus… Firth& al 2014  Or may be not …. 202 205  Blaming gerbils from Asia now….

Rat Eukaryota: Results 3.c. Eukaryotes Pritchett‐Corning, K. R., J. Cosentino, et al. (2009). "Contemporary prevalence of infectious agents in laboratory mice and rats." Lab Anim 43(2): 165‐173. 1. Fungi & Protists PREVALENCE % – P carinii (RRV common) Agent Method N NA Europe Total – T mentagrophytes – Ringworm ‐ unlikely Encephalitozoon cuniculi Serology 22,486 0.08 1.55 0.16 Lice Direct 8202 0.00 nr 0.00 – E cuniculi – microsporidia ‐ unlikely Mites Direct 9241 0.00 0.00 0.00 Oxyurids* 2. Protozoa Aspiculuris tetraptera Direct 9233 0.00 – Entamoebae & flagellates > Giardia & Spironucleus Syphacia muris Direct 9233 1.10 3.58 1.10 Syphacia obvelata Direct 9233 0.10 3. Helminths – Protozoa Chilomastix sp. Wet mount 7741 1.65 nr 1.65 – Pinworms – S muris > S obvelata etc Entamoeba sp. Wet mount 7741 3.18 nr 3.18 – Bladder worm – T crassicauda ‐ unlikely Giardia sp. Wet mount 9810 0.00 0.00 0.00 Hexamastix sp. Wet mount 7741 4.60 nr 4.60 – Cestodes (Hymenolepis, Rodentolepis, Cysticercus) Monocercomonoides sp. Wet mount 7741 0.09 nr 0.09 Retortamonas sp. Wet mount 7741 0.17 nr 0.17 4. Arthropods Spironucleus sp. Wet mount 7741 0.19 nr 0.19 – Mites –Fur mites & mesostigmatid > Fleas & lice Trichomonads Wet mount 7741 1.58 nr 1.58

206 207

Current nomenclature for Fungi important rodent Pneumocystis species

Host species Binomial or trinomial namea Comment  Pneumocystis (formerly protozoan) Human Pneumocystis jiroveci – P carinii & P wakefieldii in rats Mouse Pneumocystis murina Rat (1) Pneumocystis carinii major species in lab rats – P carinii = RRV (2) Pneumocystis wakefieldi minor species in lab rats  (3) Pneumocystis carinii f. sp. ratti secondi detected in wild rats Trichophyton mentagrophytes (4) Pneumocystis carinii f. sp. ratti tertii detected in wild rats – Ringworm –no recent reports (5) Pneumocystis carinii f. sp. ratti quarti detected in wild rats

 Microsporidian (also formerly in protozoa) • Weisbroth (2006) Lab Animal – Encephalitozoon cuniculi ‐ UNLIKELY – Unless rats or mice from pet store

208

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RADIL scientists prove there is Pneumocystis carinii no RRV; instead it’s P. carinii  Typical pneumocystosis in immunodeficient  Formerly known as RRV, Rat Respiratory  …fulfilled Koch’s postulates by proving that P. Virus in immunocompetent mice carinii is an etiologic agent of the idiopathic – Not a virus pneumonia historically associated with “rat respiratory virus” (“RRV”) infection.  It’s a fungus  AALAS 2010 – Used to be classified as protozoan

 Pathology in competent rats is very different from findings in immunodeficient animals

that can not clear the agent. 213 210 213

RRV Fig. 1. Percentage of 8 rats sampled each week that had gross lung lesions consistent with interstitial pneumonia.

Fig. 2. Lung; rat 7 weeks after exposure.

Copyright © by American College of Veterinary Pathologists Albers T M et al. Vet Pathol 2009;46:992-999 Copyright © by American College of Veterinary Pathologists Albers T M et al. Vet Pathol 2009;46:992-999 214 215

Fig. 4. Lung; rat 7 weeks after exposure. Pneumocystis

Only 4 spp ?:  In rats – P carinii – P wakefieldiae  In mice – P murina  In humans – P jirovecii (ICBN)

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Pneumocystis carinii Formerly known as Rat Respiratory Virus Brown Norway Pneumonia  PCR neg for Pc  Common (~6%)  BN used to Model – Consider this with seronegative lung disease allergic/reactive airway disease – Relationship to BN rat ‘allergic’? Pneumonia ? ‘asthma’  Diagnosis by pathology  Consider – Now ELISA + PCR stimulants/irritants  Gross – patchy lung lesions  resolve in bedding, – Be concerned about Sendai, Mycoplasma environment  Histology – perivascular – interstitial  These control BAL pneumonia ‐ mostly mononuclear were similar to OVA treated.. 219 – Resolves completely 220

FELASA –Rat – annual testing Protozoa /Protists Parasites /protists (protozoa)

Ectoparasites  Mostly commensals & pests Protozoa 1. Fleas – Small intestine – potentially pathogenic 1. Entamoeba 2. Mesostigmatid mites . Giardia Most to least common: 2. Giardia 3. Polyplax . Spironucleus . Coccidia 1. Hexamastix sp. 3. Spironucleus 4. Radfordia 2. Entamoeba sp. – Large Intestine 3. Chilomastix sp. 4. Tritrichomonas muris Helminths . Entamoebae 4. Trichomonads 1. Aspiculuris 5. Spironucleus sp. . Trichomonads 6. Retortamonas sp. 2. Syphacia . Chilomastix etc 7. Monocercomonoides sp. 3. Hymenolepis 8. Giardia sp.

222 4. Rodentolepis 223

Helminths Pinworms  Nematodes  Syphacia muris >>>> S obvelata – Pinworms, oxyurids, S muris, S obvelata – Tape test, eggs flat on 1 side – IN WILD RATS – S muris eggs are smaller 72‐82 x 25‐36 µm. . Bladder threadworm – T crassicauda . Rat Lungworm –A cantonensis – S obvelata eggs 118‐151 x 33‐55 µm more curve . Hepatic capillariasis ‐ Calodium hepaticum .Larvae + adults in cecum; Adults in colon . Gongylonema neoplasticum Forestomach Esoph . Heterakis spumosa LI  HISTO (intralumenal adults) . Mastophorus muris stomach – Platymyarian musculature . Strongyloides ratti Small Int – +/‐ lateral alae  Cestodes – Rhabditiform esophagus (corpus, isthmus, bulb) – Cysticerci – intermediate hosts – Hymenolepid (Rodentolepis, Hymenolepis)  Eggs on perineum, Thick shelled, embryonated

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Syphacia muris Arthropods

Meade & Watson JAALAS 2014  Fur mites  Neither cecal exam nor tape tests alone  – Radfordia ensifera (look like Myocoptes) reliably predicted pinworm infestation, – Incidence: low.  Peak egg shedding 1400 hrs/2pm – Distribution: intrascapular ‐dorsal cervical – Hiest sensitivity time for tape testing  Egg shedding periodicity, – Clinical Signs: usually none – decreasing to 0 at 2‐ to 3‐wk intervals. – Diagnosis: fur pluck etc, PCR more reliable – Host Sex had no consistent effect on shedding.  Mesostigmatid mites – blood sucking  Eggs aged up to 7 mo remained viable, – Gaseous chlorine dioxide ClO2 was an effective – Ornithonyssus bacoti ovicidal agent, kill rate of 99.7%. – Laelaps echidninus  [PCR is increasingly efficient ]  Lice 226 228 – Polyplax spinulosa is the sucking louse of rats

3.c. Eukaryotes – Summary 3.c. Eukaryotes – Summary  Historical/unexpected in laboratory rats  Expected – Fungi & Protists – Unlikely 1. Fungi . T mentagrophytes –Ringworm . P carinii (RRV common) . E cuniculi – microsporidiosis 2. Protozoa – the usual – Helminths ‐ Unlikely but GREAT LESIONS . Entamoebae & flagellates > Giardia & Spironucleus A cantonensis –Rat lungworm 3. Helminths – the usual C hepaticum –Hepatic capillariasis . Pinworms – S muris > S obvelata etc . T crassicauda – Bladder threadworm 4. Arthropods – the usual . Cysticercus fasciolaris ‐ where’s the cat ? . Fur mites – Radfordia & mesostigmatid Rodentolepis nana, – R microstoma , Hymenolepis diminuta  Unexpected: Historical &/or in Wild rodents – Arthropods – Unlikely but important disease (?) Zoonotic . Fleas – xenopsylla  black plague ? . Lice –polyplax Mycoplasma haemomuris 229 230

Who is it ? Des escargots pour le dîner?

 A gift of globalization…  Transmission to humans is by eating intermediate hosts, a tiny translucent slug … imperceptible on a leaf of lettuce ... slime from an infected slug is a transmission risk.  Eating raw or undercooked freshwater prawns, crabs, and frogs is also a risk factor….  Angiostrongylus  But OK to eat rats… (Rattostrongylus) cantonensis 231 232

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Rat Angiostrongyus cantonensis

Diagnosis/es? – Pulmonary artery nemotodiasis  Rat lungworm Cause/s?  Wild rats etc – Angiostrongylus cantonensis  Snail intermediate Definitive host? host  dog, human – Rats , R rattus etc eosinophilic Intermediate hosts? meningitis – Var snails Zoonotic risk?  Embryonated and – Rat feces? non embryonated – Snail slime in your salad?  H WALDEN UF CVM strongyle eggs in  Walden et al 2017 233 234 rat lungs Askjpc.org

RAT Calodium hepaticum

 Diagnosis/es? – pyogranulomatous hepatitis  Hepatic capillariasis with intralesional nematode  (Capillaria hepatica) eggs (with bipolar plugs) and  Wild rats etc rodents, peripheral fibrosis AND rabbits – Hepatic capillariasis (eggs) – Urban rats  Cause  Capillarid eggs with – Calodium hepaticum bipolar plugs  Zoonotic?

McGarry et al. 2015  Simoes & al 2014 235 http://onlinelibrary.wiley.com/doi/10.1111/zph.12116/full 236

RAT Trichosomoides crassicauda  Diagnosis/es? – Urinary bladder, verminous  Urinary bladder urocystitis… – Proliferative cystitis (usually modest inflammation) – cystitis proliferative  adult nematodes in lumen (modest inflammation)  150‐200 um diameter, smooth cuticle;  Cause  polymyarian, coelomyarian musculature; – Trichosomoides crassicauda  pseudocoelom,  Associated findings  bacillary bands, – Calculi; bladder tumors  intestine lined by numerous uninucleate cells,  Zoonotic?  thick‐walled uterus often contains eggs McGarry et al. 2015 – Golden brown 40x70um, bi‐operculate and larvated. http://onlinelibrary.wiley.com/doi/10.1111/zph.12116/epdf – Eggs also in urothelium 237 238

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Trichosomoides crassicauda Hymenolepid cestodes  Rodentolepis nana = dwarf tapeworm  Bladder threadworm – R microstoma in bile ducts – usually mice ?  Incidence: rare (in Laboratory rats).  Hymenolepis diminuta = rat tapeworm Incidence: rare.  Distribution: Urinary bladder, ureter renal pelvis.  Transmission: ingested intermediate hosts  Clinical Signs: Usually None – fleas, flour beetles, cockroaches  Pathology: Proliferative cystitis, calculi, worms – Direct cycle: Rodentolepis only. Ingested eggs.  Diagnosis: eggs in urine, fecal PCR; Path  Distribution: Small intestine –larva in mucosa. – Female: ~10 mm!  Clinical: Usually none – Male ~1mm in female repro tract  Diagnosis: Fecal flotation; Path – eggs: ~50x70µ, brown, w bipolar plugs 240 241  ZOONOTIC POTENTIAL ?

Hymenolepid cestodes Rodent tapeworms (& humans)

 Diagnostic morphology:  Rodentolepis (Hymenolepis) nana  Rodentolepis nana – common tapeworm in children – Adults ‐ 25‐40 mm + armed rostellum – SAME ? Species WAS common in rats, mice … – Strobilocerci (cysticerci) in lamina propria – Still found in pet stores & wild rodents – Eggs: 40‐60u, oval 2 membranes; 3 pairs polar . Roble & al 2012, McGarry et al. 2015 filaments – Human isolates do NOT infect mice, rats  Hymenolepis diminuta . Macnish & al 2002 – Adults: 10‐60x3‐4 wide without hooks . Tough to do the human infection expt … – Eggs: 54‐85 µm, yellow, spherical  ZOONOTIC POTENTIAL ? 242 243

RAT HELMINTHS Cysticerci (Strobilocerci) GI tract HISTO Fecal prevalence prevalence +/ n= 42  Calodium hepaticum –9.5%– Cysticercus fasciolaris /Taenia taeniaeformis Rodentolepis nana (small intestine) 14.3% – 4 – (Cysticercus taeniaformis, Strobilocercus fasciolaris) Heterakis spumosa (large intestine) 76.2% 7.1% 20 – Mice rats = INTERMEDIATE host Trichosomoides crassicauda *(bladder) – 31% – Mastophorus muris (stomach) 2.4% 11.9% 1* – Cat/carnivore = DEFINITIVE host *Eggs of ingested T. crassicauda also in stomach wall (2 samples)  Incidence: should not occur in lab rats

 Rodent‐specific, non‐zoonotic helminths were  Transmission: ingestion of cat/carnivore feces – spiruroid Mastophorus muris (16.0%) in the stomach,  – trichuroid Trichosomoides crassicauda in the urinary bladder (31.0%); Distribution: liver. – ascarid Heterakis spumosa was the commonest helminth in large intestine (76.2%).  Clinical Signs: usually none  McGarry et al. 2015  http://onlinelibrary.wiley.com/doi/10.1111/zph.12116/full  Diagnosis: Path: Cysticerci in liver

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Taenia Larval forms WYD? Figure 1. Liver; wild rat. in Intermediate Hosts  Coinfection with   Calodium Cysticercus intermediate host hepatica and – cystic structure with a single scolice – Taenia saginata, Taenia solium, Taenia  Cysticercus hydatigena, Taenia pisiformis, Taenia crassiceps, fasciolaris Taenia hydatigena and Taenia ovis  ….Hepatic  Strobilocercus Parasites in – (everted) scolice + body (strobila) + bladder Korean Wild Rats – Taenia taeniaeformis uses a strobilocercus form (Rattus norvegicus)  Coenurus and Their – numerous scolices (inverted) per cyst Association With – Taenia multiceps, Taenia serialis and Taenia Pulmonary 246 ArteriolarJ.-Y. Yi et al. Vet Pathol Medial 2009;47:292-297 247 Copyright © by American College of Veterinary Pathologists

‘Strobilocercus’ fasciolaris 3.c. Eukaryotes

 Sarcoma?   Zoonotic – Pneumocystis ? Species specific – Giardia? (G duodenalis ?) – Hymenolepis / rodentolepis tapeworms ? – Syphacia ? – Caodium hepaticum – Angiostrongylus cantonensis –Beware of snails – Fleas Xenopsylla cheopis  Yersinia pestis ? . Beware of gerbils….

Kumar et 2006 248 http://www.sciencedirect.com/science/article/pii/S0304401706003426 249

Rat testing RATS

 FELASA Recommendations  Not so much rat breeding in research facilities  http://www.uni‐  More all in, all out studies heidelberg.de/md/ibf/gesundzeugnis/hp‐  More clean rats from commercial vendors lab_anim‐2014‐‐178‐92.pdf  Try pet stores to see good infectious disease.  Immune deficient, gnotobiotics & other needs may warrant different testing.  A few more ‘rat’ lesions….

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Cotton rats –WYD?

 THANKS!  Pregnant females  Danke!  Hemochorial placentation  Trophoblast emboli  Bon Appetit!  La Perle, & al (2014).

Key Largo Woodrats Cotton rat (Neotoma floridana smalli)

 Diagnosis/es? Diagnosis/es? Causes?

S. P. Terrell et al. Vet Pathol 2011;49:710-716 S. Lezmi et al. Vet Pathol 2010;48:964-969 Copyright © by American College of Veterinary Pathologists 257 Copyright © by American College of Veterinary Pathologists 258

Neotoma sp Psammomys obesus

 WYD?  2/5 also had signs of diabetes  What are these  Patchy glomerular ‘rats’ used to immunoreactivity for IgG study ? in 2 cases, but Not IgA and IgM  CPN.  ad libitum high‐protein

diet. S. P. Terrell et al. Vet Pathol 2011;49:710-716 Copyright © by American College of Veterinary Pathologists T. J. Steinbach, and J. D. Kane Vet Pathol 2012;50:709-714 259 260 Copyright © by American College of Veterinary Pathologists

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Heterocephalus Heterocephalus Glaber Glaber

 WYD?  WYD? 1. Conspecific trauma 6. Testis 2. Calcinosis cutis 7. ICH 3. Calcinosis 8. Liver circumscripta 9. Hemosiderosis 4. Calcinosis 10. Adrenal cortex circumscripta hyperplasia 5. CPN 11. Heart ‐ myofiber 6. CPN variation

M. A. Delaney et al. Vet Pathol 2013;50:607-621 12. Normal lungsM. A. Delaney et al. Vet Pathol 2013;50:607-621 261 Copyright © by American College of Veterinary Pathologists 262 Copyright © by American College of Veterinary Pathologists

Figures 1–6. Damaraland mole rat (Cryptomys Heterocephalus glaber . Vimentin + damarensis) . CK NEG • Diagnosis/es? . CD3 NEG . S100 + . Melan A+  Diagnosis/es? – Amelanotic Melanoma  Cause/s? – Transponder?  Significance ? – FIRST report of

Sura et 2011neoplasia in this Copyright © by American College of Veterinary Pathologists http://journals.sagepub.com/doi/pdf/10.1177/0300985810377184species M. A.263 Delaney et al. Vet Pathol 2015;53:493-503 264

HYPOGEOMYS WYD ANTIMENA

 MALAGASY GIANT  Likely causes? JUMPING RAT Contributors?  http://www.bioone.org  Likely related lesions? /doi/full/10.1638/2012‐ 0116R1.1  Cause/Species ?  Developmental stage?

RodentPath 266 265 266

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WYD? WYD

 Possible causes or contributors?

RodentPath 268 267 268

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