Joint Pathology Center Veterinary Pathology Services

WEDNESDAY SLIDE CONFERENCE 2018-2019

C o n f e r e n c e 25 1 May 2019

CASE I: N261/13 (JPC 4037902). Laboratory results: Both Bordetella bronchiseptica and Mycoplasma felis were Signalment: 5 month old, female, Devon cultured from samples of lung. rex, Felis domesticus, feline. Microscopic Description: Multifocally, History: This young, fully vaccinated, there is extensive necrosis of contiguous pedigree Devon rex cat with no previous alveolar walls with filling of associated history of illness became acutely ill with airspace with cell debris, -rich pyrexia and severe dyspnea, the morning fluid/fibrin, and admixed intact and following its attendance at a cat show where degenerate inflammatory cells it had won both classes of competition for (predominantly neutrophils with fewer which it had been entered. Despite immediate and ). Myriad veterinary attention and antibiotic treatment, loosely scattered to clumped basophilic the died within 36 hrs and was coccobacilli (), and small numbers of submitted for necropsy.

Gross Pathology: The carcass is well preserved with adequate body fat reserves. Periocular and perinasal regions are encrusted with light brown discharge. Tracheal and bronchial lumens filled with frothy mucopurulent exudate. There is multifocal consolidation in all lung lobes: on sectioning coalescing pale firm foci are present. The kidneys are bilaterally congested at the cortico-medullary junction. The stomach contains mucus only. Feces of Lung, cat. Approximately 33% of the section is effaced by normal color and consistency in rectum. The areas of hyper\cellularity centered on small airways, and urinary bladder is empty. there are multifocal, largely subpleural areas of hemorrhage and . (HE, 4X)

1 erythrocytes also noted in these areas. in this , typically manifests Bronchioles frequently contain similar when pulmonary defenses are impaired by inflammatory debris and bacteria, and feline calicivirus or herpes virus , focally, necrosis of bronchiolar epithelia is or by stressful environmental conditions detected. Widespread hyperemia with small (while occasional structures possibly number of medium-caliber blood vessels suspicious of basophilic intra-nuclear variously exhibiting fibrinoid necrosis and inclusions were observed in bronchiolar leucocytoclastic vasculitis. Occasional gland epithelium in this case, these were thrombi noted (not visible in all sections). considered equivocal: immunohistochemistry was not Contributor’s Morphologic Diagnoses: performed).2,4,10 The clinical consequences Lung: necrotizing bronchopneumonia, acute, on infection can vary dramatically: from mild severe with myriad bacteria (identified as pyrexia, coughing and sneezing to severe Bordetella bronchiseptica on culture). pneumonia and death as seen in this case.

Contributor’s Comment: Bordetella Details of the pathogenesis of infection in the bronchiseptica is a gram-negative cat are inferred from studies in other species. coccobacillus commonly carried in the Bacterial virulence is regulated by the nasopharynx of healthy cats. In a survey of Bordetella virulence gene (bvg) operon, respiratory in multi-cat (≥5 cats) which orchestrates the expression of the households, B. bronchiseptica was detected adhesin filamentous haemagglutinin (FHA), by PCR in 5% of cats from households with pertactin, and the fimbriae that facilitate disease, and in 1.3% of cats without.6 adherence to the ciliated epithelium, as well Considered a somewhat uncommon as to macrophages and neutrophils within the

Lung, cat. Areas of inflammation are centered on necrotic airways, which are filled with numerous viable and degenerate neutrophils, cell debris, fibrin, and bacterial colonies. The exudate has ruptured through the wall and spilled into the surrounding alveoli. (HE, 163X)

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lungs.2,6 Following attachment the pathogen immunocompromised individuals, have been secretes the RTX adenylate cyclase toxin reported.8,11 (hemolysin) that results in impaired leucocyte phagocytosis and oxidative burst, Mycoplasma felis can colonize the upper and may induce their apoptosis. A respiratory tract of cats, but evidence lipooligosaccharide with endotoxin activity suggests it is not a significant primary and a soluble peptidoglycan-derived tracheal pathogen. Molecular detection techniques cytotoxin are also produced that induce have found Mycoplasma spp in the lower ciliostasis and the apoptosis of ciliated respiratory tract of 15.4% of cats with epithelial cells.2,6 respiratory disease, with M. felis, M. gateae and M. feliminutum the species identified. Bacterial culture (as in this case) and PCR are However, the pathogenic significance of their used to conform the diagnosis, although both presence remains unclear.7 It is likely that M. lack sensitivity.6,9 In regions where vaccines felis contributed to the severity of the against B. bronchiseptica are available, it is pneumonia in this case through opportunist recommended that cats should not be secondary involvement. routinely vaccinated, given the typically mild disease that occurs. However, vaccination is This case represented a sudden and highly encouraged in units such a shelters where traumatic loss for the owner concerned as populations are in continuous turnover, and they witnessed the rapid clinical deterioration thus at increased risk of infectious disease.6 and death of their prize-winning animal over Organisms are shed in oral and nasal a period of 36 hrs. We may speculate that secretions of infected cats and infected contact with other cats at the cat show are considered an infection risk for cats.5,6 provided a source of the infection, and how Rare , typically in possibly the stress of transport or attendance

Lung, cat. Within affected areas there are discontinuous alveolar septa, characteristic of septal necrosis (black arrows.) (HE, 313X)

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at this event could have precipitated disease. In any event, the rapidity of disease progression suggests the strain of B. bronchiseptica involved was particularly virulent.

Contributing Institution: Room 012, Veterinary Sciences Centre, School of Veterinary Medicine, University Lung, cat. Large colonies of bacillii are present within College Dublin, Belfield, Dublin 2, Ireland airways and alveoli in close proximity. (HE, 400X) http://www.ucd.ie/vetmed/ role in fatal pneumonia as exemplified in this JPC Diagnosis: Lung: Bronchopneumonia, case. B. bronchiseptica has been identified in fibrinosuppurative and necrotizing, between 12 and 78% of dogs with lower multifocal to coalescing, severe, with respiratory tract infections, and 5-13% of numerous bacterial colonies. cats.9 At a major university veterinary school, a retrospective study identified B. JPC Comment: The contributor has bronchiseptica via immunohistochemistry provided an excellent review of Bordetella and/or bacterial culture in 8 of 36 of canine bronchiseptica in this case, as well as the and 14 of 31 feline cases of fatal pathogenesis of the disease across species bronchopneumonia. Upon close review, lines. Bordetella bronchiseptica is an bacteria could be visualized among the cilia important, if sporadic, pathogen of the in 4 of 36 canine cases and 5 of 31 feline respiratory system in a wide range of cases.9 Close review of columnar epithelium mammalian species. Previous WSC cases in this case also disclosed the presence of include Bordetella bronchopneumonia cilia-associated bacteria; it was seen only on infection in a chinchilla (WSC 2014-2015, intact cells, as necrotic or attenuated Conf 18, Case 4), an African green monkey epithelium has probably already lost its cilia. (WSC 2011-2012, Conf 22, Case 3), squirrel This may be an important finding in such monkeys (WSC 2007-2008, Conf 9, Case 3, cases, as the lesions associated with the pigs (WSC 1996-1997, Conf 20, Case 2), a bronchopneumonia in these cases, in the rabbit (WSC 1995-1996), among others. authors’ opinion, were not specific for any particular pathogen. There are three species of Bordetella of veterinary importance, including B. Bordetella bronchiseptica can be an bronchiseptica (whose dermonecrotic toxin opportunistic pathogen in humans as well, also causes atrophic rhinitis in pigs and particularly immunosuppressed individuals rabbits), B. hinzii which causes limited and cystic fibrosis patients. B. respiratory disease in turkey poults and rare bronchiseptica and B. pertussis, (the septicemia in humans, and B. pertussus a causative agent of whooping cough) exhibit potent pathogen in humans which may cause little genetic variation,1 with B. pertussis lower respiratory disease in lambs. being a more recent derived, human adapted bacterium derived as a consequence of gene Well known for its involvement in non-fatal deletions and loss of genetic regulatory tracheobronchitis (aka “kennel cough”), a functions. Both organisms possess the recent publication9 has detailed Bordetella’s Bordetella virulence gene as discussed

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of pneumonia in the lung across animal species. In this particular case, attendees were split on whether the appropriate morphologic diagnosis would be bronchopneumonia (based on the traditional pathogenesis of this aerogenous bacteria and the obvious necrosis within airways) or bronchointerstitial, based on the Lung, cat: Careful evaluation of the HE slide may disclose the presence of bacilli lining cilia widespread necrosis of columnar airway epithelium (arrows). Necrotic or attenuated epithelium likely lack cilia, so of alveolar septa, are not worthy of inspection. (HE, 600X) pulmonary vessels, and exudative above. In a large children’s CF center, 7 alveolitis in much of the section patients had multiple repeated isolates of B. bronchiseptica from their airways. All References: patients had documented exposure to pets or lived on a farm or an operating kennel, 1. Brady C, Ackerman P, Johnson M, resulting in an overall infection of as many McNamara J. Bordetella as 12% of children in the center at any time.1 bronchiseptica in a pediatric cystic fibrosis center. J Cystic Fibrosis, One of the issues associated with persistence 2014 13:43-48 of Bordetella infections in human patients 2. Caswell JL, Williams KJ: The leading to vaccine failures and re-emergence Respiratory System. In: Jubb, of disease has been recently elucidated – the Kennedy and Palmer’s Pathology of ability for this bacterium to produce Domestic , ed. Maxie MG, biofilms.3 This is yet another feature of the 5th ed., vol. 2, Philadelphia, PA: BvgAS gene, which regulates the expression Elsevier; 2007: 216-219. of genes encoding surface membrane, secreted, and regulatory from host 3. Cattelan N, Dubey P, Arnal L, cells, as well as filamentous hemagglutinin, Yantorno OM, Deora R. FEMS pertactin, fimbriae and various toxins which Pathog Dis 2016; doi contribute to the extracellular matrix 10.1093/femspd/ftv108. comprising the Bordetella biofilm.3 4. Coutts AJ, Dawson S, Binns S, Hart The moderator reviewed the general patterns CA, Gaskell CJ,Gaskell RM. Studies

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on natural transmission of Bordetella 10. Welsh RD. Bordetella bronchiseptica bronchiseptica in cats. Vet Microbiol infections in cats. J Am Anim Hosp 48:19–27, 1996. Assoc 32: 153–158, 1996.

5. Dawson S, Gaskell CJ, McCracken 11. Woolfrey BF, Moody JA. Human CM, Gaskell RM, Hart CA, Jones D. infections associated with Bordetella Bordetella bronchiseptica infection bronchiseptica. Clin Microbiol Rev in cats following contact with 4: 243–255, 1991. infected dogs. Vet Rec 146: 46–48, 2000.

6. Egberink H, Addie D, Belák S, CASE II: 2014 Case 2 (JPC 4050143). Boucraut-Baralon C, Frymus T, Gruffydd-Jones T, Hartmann K, Signalment: Juvenile (<30 months), Hosie MJ, Lloret A, Lutz H, Marsilio unreported gender, unreported breed, Bos F, Grazia Pennisi M, Radford AD, taurus, bovine. Thiry E, Truyen U, Horzinek MC. Bordetella Bronchiseptica Infection History: The bovine was presented for in Cats: ABCD guidelines on slaughter at a United States Department of prevention and management. J Fel Agriculture (USDA) federally inspected Med Surg 11:610-614, 2009. slaughter facility and passed antemortem inspection. The carcass was retained for 7. Reed N, Simpson K, Ayling R, further diagnostic testing after lesions Nicholas R, Gunn-Moore D. suspicious for bovine were Mycoplasma species in cats with observed in lymph nodes of the head, lower airway disease: improved thoracic, and abdominal cavities and lungs detection and species identification during postmortem inspection. using a polymerase chain reaction assay. J Fel Med Surg 14: 833–840, 2012.

8. Register KB, Sukumar N, Palavecino EL, Rubin BK, Deora R. Bordetella bronchiseptica in a paediatric cystic fibrosis patient: Possible transmission from a household cat. Zoonoses Public Hlth 59: 246–250, 2012.

9. Taha-Abdelaziz K, Bassel LL, Harness ML, Clark ME, Register KB, Caswell JL. Cilia-associated bacteria in fatal Brodetella bronchiseptica pneumonia of dogs and cats. J Vet Diagn Investig 2016; 28(4)369-376. , ox: The node is effaced by multiple, often coalescing granulomas. (HE, 5X)

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Gross Pathology: None within areas of necrosis and the cytoplasm of macrophages and giant cells. Laboratory results: PCR from formalin- fixed, paraffin-embedded tissues were Contributor’s Morphologic Diagnoses: positive for IS6110 ( Lymph node: Lymphadenitis, tuberculosis complex) and negative for IS900 necrogranulomatous, locally extensive, (M. avium paratuberculosis) and 16S rDNA chronic, severe, Bos taurus. (M. avium complex). Culture recovered M. bovis. Contributor’s Comment: causes tuberculosis in many , Microscopic Description: including cattle and is a zoonotic disease. Lymph node. Up to 80% of the parenchyma Cattle slaughtered for consumption in the (there is some slide variability) is effaced by United States in federally inspected abattoirs coalescing to locally extensive granulomas. undergo inspection by personnel from the Granulomas are composed of central areas of Food Safety Inspection Service (FSIS) of the caseous necrosis and variable amounts of USDA to insure they are safe and wholesome basophilic granular material (mineral) for entry into the market. In accordance with rimmed by large numbers of epithelioid the USDA’s Bovine Tuberculosis macrophages and multinucleated giant cells Eradication Program, FSIS personnel retain surrounded peripherally by lower numbers of carcasses when lesions resembling lymphocytes, plasma cells, fibroblasts, and tuberculosis are identified. Suspect fibrous connective tissue. Giant cells have up granulomas from these cattle are collected to 10 peripheral nuclei (Langhans type). and submitted to the National Veterinary There are rare, ~5µm long, acid-fast bacilli

Lymph node, ox: Granulomas have a thick, often central core of cellular debris, which is occasionally mineralized. The intervening remnant node is hyperplastic. (HE, 23X)

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Services Laboratories (NVSL) for program is focused on identifying M. bovis, histopathology and culture.8 primers used in the PCR are limited to those for M. tuberculosis complex (MTBC, of Microscopic lesions consistent with which M. bovis is a member), M. avium tuberculosis in cattle are often multicentric complex (MAC, common environmental and coalescing with central areas of caseous mycobacteria) and M. avium necrosis and mineral. Epithelioid paratuberculosis (MAP, the bacterium that macrophages with small to moderate causes Johne’s disease in cattle). A recent numbers of Langhans-type multinucleated report7 of mycobacteria cultured from giant cells surround the necrosis with smaller clinical samples submitted to the NVSL numbers of peripheral lymphocytes, plasma stated that the majority of mycobacteria cells, and occasional neutrophils. 2 The cultured from cattle were M. bovis (32%) typical tuberculous lesion caused by followed by M. avium complex (25.5%). The Mycobacterium bovis has rare to occasionally next most common species, M. fortuitum/M. moderate numbers of acid-fast bacteria fortuitum complex, comprised 10.1%.7 present within the cytoplasm of macrophages and giant cells as well as in areas of necrosis The microscopic features of the current case 2 were consistent with bovine tuberculosis and FFPE tissue was tested by PCR for Bovine cases that are histologically mycobacterial DNA using our primers for compatible with tuberculosis undergo MTBC, MAC, and MAP. PCR was positive additional testing at NVSL by means of PCR for the MTBC primer sets and negative for on formalin-fixed, paraffin-embedded MAC and MAP. False negative results for (FFPE) tissue to test for mycobacterial DNA. mycobacterial DNA can occur in some cases. Because the scope of the TB eradication The more common reasons for false negative

Lymph node, ox: The periphery of the node contains numerous epithelioid macrophages and aggregates of neutrophils, and more peripherally (HE, 238X)

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PCR results include tissue being fixed in badgers, Meles meles), and New Zealand formalin for an extended period of time (>7 (brushtail possums, Trichosurus vulpecula).6 days) and and/or extremely low numbers of AFB present in the lesion or tissue section. Disease manifestation can vary, but M. bovis Formalin fixation causes irreversible cross- typically causes granulomatous linking between DNA and protein, which inflammation in the lungs and lymph nodes becomes worse as the tissue fixes over time.3 of the head (retropharyngeal), chest (tracheobronchial and mediastinal) and/or Culture, which is considered the gold abdomen (mesenteric), often reflecting the standard for definitive diagnosis of bovine route of transmission (inhalation or tuberculosis2 and can take up to 10 weeks to ingestion).2,7 Grossly, the classic tubercle is complete with slow-growing mycobacteria, encapsulated, pale yellow, and often has a recovered M. bovis. caseous core that may be variably mineralized.2 Clinically, the only sign of Mycobacterium bovis is a member of the M. infection may be chronic weight loss tuberculosis complex, which includes M. (), weakness, loss of appetite, tuberculosis, M. africanum, M. canettii, M. fluctuating fever, cough, exercise pinnipedii, M. caprae, M. microti,5 and the intolerance, and lymphadenomegaly.9 Most newly described M. mungi.1 M. bovis can animals that are infected with M. bovis do not cause disease in cattle as well as humans and develop clinical disease.2 other domesticated and wild mammals. Currently, M. bovis is endemic in various Contributing Institution: populations of wildlife and are a source for National Centers for Animal Health, Ames, re-infection of domesticated animals in IA regions of the United States (white-tailed http://www.ars.usda.gov/main/site_main.ht , Odocoileus virginianus), Spain (wild m?modecode=36-25-30-00 boar, Sus scrofa), United (Eurasian http://www.aphis.usda.gov/nvsl

Lymph node, ox: Numerous Langhans giant cells are present at the periphery of the granulomas. (HE, 238X)

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JPC Diagnosis: Lymph node: Lymphadenitis, granulomatous, multifocal to coalescing, severe, with diffuse moderate follicular and paracortical hyperplasia.

JPC Comment: The contributor does an excellent job reviewing the thorough protocol used to investigate tissue suspected of - Mycobacterium bovis infection at the National Veterinary Services Laboratory, an important link in food safety in the United States.

Lymph node, ox. Three acid-fast bacilli, approximately 5- M. bovis is a member of the growing M. 7µm, within the cytoplasm of a multinucleated giant cell. tuberculosis complex as described above. A Modified Ziehl-Neelsen., 1000X) recent publication4 noted the divergent pathogeneses in non-bovine hosts in several In western Europe, wild boars serve as countries of the world which have been wildlife reservoirs of Mycobacterium bovis. incriminated in maintaining infection in local This species, well known as highly livestock. susceptible to M. bovis, has been used as a sentinel species to screen for M. bovis in In the North America, the white-tailed deer, Hawaii and New Zealand. Disseminated and to a lesser extent, elk harbor the infection infection is the rule in this species, with and potentiate the infection though contact lesions in multiple anatomic regions, with cattle in their pasturing area. Infected including cranial lymph nodes and lymph nodes in deer often have soft centers, mandibular lymph nodes, lungs, liver, and resembling abscesses; however neutrophils, spleen.4 as seen in cattle, are not a feature of the lesion. 4 Approximately 30-35% of infections In New Zealand, the brushtail possum exhibit spread to the thoracic viscera, and (Trichosurus vulpecula) is considered a often the pleural surfaces, where they form source of infection for cattle. Highly pearlescent nodules reminiscent of susceptible, they manifest the disease by mesothelioma. infection of subcutaneous lymph nodes, forming draining fistulous tracts. Terminally In England and Ireland, the Eurasian badger ill possums attract cattle by wandering (Meles meles) has been indicted as a major erratically across pastures, and curious cattle factor in outbreaks of cattle with bovine may sniff and lick them, thereby receiving tuberculosis. In the badger, respiratory exposure to the bacteria. In these regions, infection is most common with 50% cases infected wild ferrets may also be infected, but showing pulmonary infection, and 35% of their role in spreading the infection to cattle cases involving lymph nodse. Elongate is controversial – a reduction in possum radial lesions may be seen in the kidneys, and population in these areas results in a miliary lesions may be seen in the liver and proportional decrease in infection of this spleen. Many infected badgers show no carnivore, suggesting that the ferret is a visible gross lesions.4 spillover host at best.4

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need to know. Vet Microbiol 112: The participants agreed that an alternate JPC 181-190, 2006 diagnosis of multiple lymph node 8 Thacker T, Robbe-Austerman S, Harris B, granulomas would be acceptable as well. As Palmer MV, Waters WR: Isolation of this was the last conference of the 2018-2019 mycobacteria from clinical samples year, the box containing the age-old collected in the United States from discussion of “granulomatous versus 2004 to 2011. BMC Vet Res 9:100, granuloma” was quickly slammed shut. 2013 9 Thoen CO: Overview of tuberculosis and References: other mycobacterial infections. In: The Merck Veterinary Manual Online, eds. Aiello 1 Alexander KA, Laver PN, Michel AL, SE, Moses MA. 2012 Williams M, van Helden PD, Warren RM, Gey van Pittius NC: Novel Mycobacterium tuberculosis complex CASE III: S-2017-20 (JPC 4100993). pathogen, M. mungi. Emerg Infect Dis 16: 1296-1299, 2010 Signalment: 6-year and 5-month old female 2 Caswell JL, Williams KJ: Respiratory neutered crossbreed (Canis lupus system. In: Jubb, Kennedy, and familiaris). Palmer's Pathology of Domestic Animals, ed. Maxie MG, pp. 523- History: A 6-year 5 months-old female 653. Saunders Elsevier, St. Louis, neutered Springer Spaniel Cross presented 2007 for investigation of pyrexia of unknown 3 Fang SG, Wan QH, Fujihara N: Formalin origin of 5 months duration. The owner also removal from archival tissue by noted lethargy and mild gastrointestinal signs critical point drying. Biotechniques during this time (inappetence and diarrhoea). 33: 604-611, 2002 Previously the patient had responded well to 4. Fitzgerald SD and Kaneene JB. Wildlife oral prednisolone and potentiated reservoirs of bovine tuberculosis amoxicillin, however symptoms recurred worldwide: hosts, pathology, surveillance, and control. Vet Pathol 2013; 50(3):488-499. 5 Olsen I, Barletta RG, Thoen CO: Mycobacterium. In: Pathogenesis of Bacterial Infections in Animals, pp. 113-132. Wiley-Blackwell, 2010 6 Palmer MV, Thacker TC, Waters WR, Gortázar C, Corner LAL: Mycobacterium bovis: A model pathogen at the interface of livestock, wildlife, and humans. Vet Med Int 2012: 17, 2012 7 Palmer MV, Waters WR: Advances in bovine tuberculosis diagnosis and Omentum, dog. The omentum is markedly expanded, and pathogenesis: What policy makers largely effaced by multifocal to coalescing pyogranulomas. (HE, 4X)

11 after the treatment was stopped. On physical examination, marked pyrexia of 40.3C was noted, with tachycardia of 160 beats per minute and normal synchronous pulses. A normal respiratory rate of 32 breaths per minute with normal respiratory effort was noted. Auscultation of the thorax was unremarkable. Mucous membranes were pink and moist, with CRT <2s. Mild Omentum, dog. Pyogranulomas are centered on large discomfort was noted on deep palpation of colonies of filamentous bacilli which range up to a mm in the cranial abdomen, which was subjectively diameter. (HE, 63X) mildly bloated. Rectal examination was unremarkable and peripheral lymph nodes distribution, are large numbers of were within normal limits. Abdominal macrophages, neutrophils and lesser numbers ultrasound showed free fluid within the of lymphocytes, which are often forming abdomen. At exploratory laparotomy the moderately sized to large groups. Small to abdomen was diffusely filled with creamy moderate numbers of plasma cells are present pink fluid and there were multiple masses in multifocally, often at the periphery of these the omentum and mesentery of the jejunum, groups and multifocally within the which were sampled for histopathology. connective tissue and adipose. Large numbers of macrophages and neutrophils Gross Pathology: In a multifocal to often surround large colonies of filamentous coalescing pattern, the greater omentum was Gram-positive bacteria, measuring expanded by irregular, tan to brown to dark approximately 1x3-7µm. Bacteria are red and moderately firm mass-like lesions admixed with large (approximately 500µm with cream to tan, multifocally red cut diameter) accumulations of finely granular surfaces. The adjacent omentum was tan to basophilic to eosinophilic material which brown, and soft. radiates outwards from a central focus (Splendore-Hoeppli material; “sulphur Laboratory results: Haematology: WBC granules”). These colonies often show a count 18.2x109/L (ref 6-17), otherwise rosette-like arrangement and are visible unremarkable. Biochemistry: C-reactive subgrossly. Small amounts of eosinophilic protein 26.3mg/L (ref 0-8.2) and material, cellular debris, haemorrhage and hypoalbuminaemia 17g/L (ref 25-41). ALT small numbers of haemosiderin-laden was mildly decreased and AST was mildly macrophages (haemosiderophages) increased. Abdominal fluid sample: Cytology multifocally surround the colonies. Further to of aspirated abdominal fluid showed the periphery of these foci are haphazardly neutrophilic macrophagic inflammation with arranged, hypertrophied fibroblasts phagocytosed bacteria and was consistent embedded in fibrous connective tissue which with a septic exudate. Culture of omental often progresses to thick bands of mature masses: spp. was cultured. fibrous connective tissue, containing scattered to moderate numbers of Microscopic Description: neutrophils, lymphocytes, plasma cells, and Expanding and effacing the collagenous fewer macrophages. connective tissue and adipose tissue of the omentum, in a multifocal to coalescing

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Contributor’s Comment: The histopathological findings in this case were consistent with abdominal , a diagnosis that was supported by the microbiology results. The patient had an abdominal drain placed at the time of exploratory laparotomy and was subsequently hospitalized for 6 days. Omentum, dog. There is mild granulomatous Antibiotic treatment with amoxicillin- inflammation focused on areas of remnant adipose tissue. clavulanic acid commenced following receipt (HE, 150X) of the histopathology and culture results, and treatment resulted in good clinical The omentum is lined by flattened to improvement and a positive outcome. multifocally plump mesothelial cells and exhibits multifocal ulceration. Underlying Actinomycosis is caused by filamentous, ulcerated areas there are frequent plump gram-positive bacteria from the family reactive fibroblasts, aligned in parallel to , Actinomyces, and each other and perpendicular to adjacent, to a lesser extent to the related genus newly formed blood vessels (angiogenesis) . These bacteria are and to the ulcerated surface (granulation normally present in the mucous membranes, tissue formation). At the edges of the section especially in the oropharynx, and in the the omentum exhibits a multifocal folded genital and gastrointestinal tracts, having appearance and contains numerous, however the potential to cause opportunistic frequently congested, blood vessels and is infection when inoculated into tissues in expanded by variable numbers of association with other bacteria when there is macrophages, lymphocytes, neutrophils, mechanical disruption of normal mucosal plasma cells, and fibroblasts. barriers (such as deeply penetrating wounds or migration of foreign material).7,14 Contributor’s Morphologic Diagnoses: Greater omentum: Peritonitis, Canine actinomycosis is most common in pyogranulomatous, lymphocytic, and young adult to middle-aged large breed dogs plasmacytic, chronic, locally extensive, (median age of 5 years old) that have outdoor severe; with: access, especially retriever and hunting a. Numerous accumulations of breeds. The development of actinomycosis in Splendore-Hoeppli material outdoor dogs is frequently related to with intralesional exposure to plant penetrating material, such microcolonies of Gram- as grass awns.7,14 After ingestion or positive filamentous rod inhalation, plant awns become contaminated bacteria, morphology with Actinomyces spp. and other bacteria consistent with Actinomyces from the oropharynx, and then migrate to spp.; various sites.6,7,14 b. Granulation tissue formation, chronic, multifocal, marked; Actinomyces spp. can also be inoculated into and tissues by bite injury. Other co-infecting c. Fibrosis, chronic, multifocal, aerobic and anaerobic bacteria from the oral marked. cavity or intestinal tract impair normal host

13 defenses and reduce oxygen tension, which allows Actinomyces spp. to endure.7,14 Abdominal actinomycosis, as seen in this case, may develop when ingested foreign bodies penetrate the gastrointestinal tract, which leads to the formation of intra- abdominal mass lesions and ascites or it can also occur due to direct extension from subcutaneous tissues or hematogenous Omentum, dog. At the edge of the section, there are spread of the organism to abdominal organs mesothelial lined papillary projections of fibrous such as the liver.9,14 connective tissue, and bacterial colonies (“granules”) within the extracellular space. (HE, 150X)

Actinomyces spp. that have fimbriae can bind to specific cell surface receptors on other meningoencephalitis,5,14 and ocular bacteria, especially streptococci, and this infections such as keratitis and bacterial co-aggregation prevents the endophthalmitis,3 may also occur. capacity of neutrophils to phagocytize the organisms.13,14 Dense colonies of In cattle, actinomycosis, caused by Actinomyces spp. form, and these ´Sulfur’ , often affects the bones of granules can be macroscopically visible in the jaw, with development of granulomatous exudates in actinomycosis as small yellow and fibrosing osteomyelitis (‘lumpy jaw’). free granules.15 The colonies are Less commonly it can also affect the progressively encircled by concentric musculature of the tongue, leading to the accumulations of neutrophils, macrophages, development of chronic fibrosing nodular and plasma cells, with the ensuing myositis.16 Actinomyces spp. may also cause development of pyogranulomatous hepatic abscesses in different species.4 In inflammation14 and are often bordered by swine, Actinomyces suismastidis has been star-like or club-shaped-like amorphous associated with the development of eosinophilic material (Splendore-Hoeppli pyogranulomatous mastitis10 and material). Actinomyces hyovaginalis has been associated with necrotic pulmonary lesions in The connective tissue is destroyed by the same species.2 In horses, Actinomyces proteolytic enzymes from the associated spp. can cause abscesses, which are most bacteria, macrophages and degranulated commonly located in the submandibular and neutrophils, which allows the inflammatory retropharyngeal regions8 and Actinomyces process to extend through normal tissue denticolens has been associated with the planes. Less frequently Actinomyces spp. development of submandibular spreads hematogenously to distant sites. In lymphadenitis.1 some cases, the inflammatory reaction is accompanied by mass formation and Contributing Institution: extensive fibrosis, as in this case. The most Department of Veterinary Medicine, common clinical forms of actinomycosis in University of Cambridge, Madingley Road, dogs and cats involve the cervicofacial Cambridge CB3 0ES, UK. region, thorax, abdomen, and subcutaneous http://www.vet.cam.ac.uk/ tissue, but central nervous system infections including meningitis and JPC Diagnosis: Omentum: Peritonitis and

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steatitis, chronic-active and pyogranulomatous, multifocal to coalescing, severe, with large colonies of filamentous bacilli and marked mesothelial hyperplasia

JPC Comment: The contributor has provided an excellent review of actinomycotic infection in the dog and a wider range of species. Actinomyces is a genus of gram-positive bacteria, with new species being identified on a regular basis in species as diverse as mammals and mollusks. infections are polymicrobial in . Members of the genus Actinomyces are well Unlike dogs, the basis for thoracic infection documented in health and disease in humans is usually aspiration of oropharyngeal as well. A number of species of Actinomyces, secretions, and results in pulmonary including A. odontolyticus, A. oris, and A. abscessation. Extrapulmonary extension into naeslundii, are common commensals in the the thorax (common in small animals) is oral cavity, and are components of the relatively uncommon in humans, with sepsis biofilm on teeth at all ages. A number of being a more common result. Abdominal commensal actinomycetes colonize the infections are often the result of abdominal gastrointestinal tract, where they are surgery or invasive infection such as proposed to help in the breakdown of appendicitis; pelvic infections have been complex sugars as well as producing associated with prolonged use of intrauterine antibiotics to maintain balance of the devices for contraception. Less common bacterial flora.12 manifestations are cutaneous and musculoskeletal infections (generally caused The pathology of actinomycosis in humans by traumatic implantation, and cerebral and was first identified in 1892 by Kruse, when disseminated actinomycosis (usually the bacterium was known as Streptomyces resulting from sepsis). An excellent review israelii (now A. israelii).11 Twenty-five of human infection, to include specific forms species of pathogenic actinomycetes have of infection and associated actinomycotic now been identified in humans. These species species was published by Kononen et al. in of Actinomyces are endogenous inhabitants 2015.11 of mucosal membranes which are introduced The moderator discussed the nature of into deeper tissues by trauma, surgery, or the Splendore-Hoeppli material and various introduction of foreign bodies. In humans, theories concerning its makeup, and possible actinomycosis is classified as mechanisms of infection the dog and cat, as orocervicofacial, thoracic, and abdominal. well as actinomycotic infections in other Omentum, dog: Filamentous bacilli stain positively with species. a tissue gram stain. (Brown and Brenn, 400X)

More than half of all cases of human actinomycosis are orocervicofacial, an unsurprising fact considering the ubiquity of References: multiple species in the oral cavity, and many

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1. Albini S, Korczak BM, Abril C, et al. 11. Kononen E, Wade WG. Actinomyces Mandibular lymphadenopathy caused by and related organisms in human infections. Actinomyces denticolens mimicking Clin Microbiol Rev doi: strangles in three horses. Vet Rec. 10.1128/CMR.00100-14. 2008;162(5):158-9. 12. Li J, Li Y, Zhou Y, Wang C, Wu B, Wan 2. Aalbæk B, Christensen H, Bisgaard M, et J. Actinomyces and alimentary tract diseases al. Actinomyces hyovaginalis Associated a review of it. Biomed Res Int 2018; with disseminated necrotic lung lesions in https://doi.org/10.1155/2018/3820215 slaughter pactinomyces veterinary 13. Ochiai K, Kurita-Ochiai T, Kamino Y, et igs. J Comp Pathol. 2003;129 (1):70–77. al. Effect of co-aggregation on the 3. Barnes LD, Grahn BH. Actinomyces pathogenicity of oral bacteria. J Med endophthalmitis and pneumonia in a dog. Microbiol. 1993;39(3):183-90. Can Vet J. 2007;48(11):1155-8. 14. Sykes J. Bacterial diseases - 4. Brown DL, Van Wettere AJ, Cullen JM – Actinomycosis In: Sykes J, ed. Canine and Hepatobiliary System and Exocrine Pancreas Feline Infectious Diseases. St Louis, MO: In: Zachary FJ, ed. Pathologic Basis of Elsevier Saunders, 2014:399-408. Veterinary Disease: Elsevier Saunders, 15. Uzal FA, Plattner BL, Hostetter JM. 2017:442. Alimentary system. In: Maxie MG, ed. 5. Couto SS, Dickinson PJ, Jang S, et al. Pathology of Domestic Animals, Volume 2. Pyogranulomatous meningoencephalitis due 6th ed. St Louis, MO: Elsevier, 2015:253. to Actinomyces sp. in a dog. Vet Pathol. 16. Valentine B – Skeletal Muscle In: 2000;37(6):650-2. Zachary FJ, ed. Pathologic Basis of 6. Edwards DF, Nyland TG, Weigel JP. Veterinary Disease. 6th ed. St Louis, MO: Thoracic, abdominal, and vertebral Elsevier Saunders, 2017:926, 942. actinomycosis. Diagnosis and long-term therapy in three dogs. J Vet Intern Med. 1988;2(4):184-91. CASE IV: 12H5674 (JPC 4032439). 7. Edwards DF. Actinomycosis and . In: Greene CE, ed. Infectious Signalment: A six year old, spayed female Diseases of the Dog and Cat. 1st ed. Pembroke Welsh Corgi dog (Canis lupus Philadelphia: WB Saunders, 1990:585–590. familiaris). 8. Fielding CL, Magdesian KG, Morgan RA, Ruby RE, Sprayberry KA. Actinomyces History: The dog presented with tachypnea, species as a cause of abscesses in nine horses. increased respiratory effort, cardiomegaly, Vet Rec. 2008;162(1):18-20. panhypoproteinemia, mild anemia. 9. Gavhane, D.S., Moregaonkar, S.D., Mhase, A.K., Sawale, G.K. and Kadam, Gross Pathology: The dog had areas of D.P.A Case of Chronic Abdominal ecchymoses around the peripheral veins and Actinomycosis with Severe Hepatic clotted blood in the right nasal cavity and Involvement in a Dog. Israel J Vet Med. ethmoid conchae. Feces were black and tarry. 2016; 71(1):58. Lungs were diffusely firm and dark red. 10. Hoyles L, Falsen E, Holmström G, et al. There were pinpoint multifocal white circular Actinomyces suimastitidis sp. nov., isolated structures diffuse across the lung surface. from pig mastitis. Int J Syst Evol Microbiol. Other findings included: subendocarial 2001;51(Pt 4):1323-6.

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Lung, hemorrhage, congestion, and edema, multifocal, moderate

Contributor’s Comment: The arterial lesions coupled with the clinical and laboratory results are suggestive of plexiform pulmonary arteriopathy.2,6 The dog lacked heartworms and lacked evidence of a left to right cardiac shunt and thus the changes are consistent with idiopathic pulmonary arterial hypertension (IPAH).6 In a limited number of dogs (six) with pulmonary arteriopathy and hypertension 67% were female, as in this Lung, dog. At low magnification, there are areas of case. The pulmonary vascular changes likely hypercellularity around small arterioles. (HE, 7X) led to or were caused by pulmonary hypertension resulting in right heart failure petechiae and petechiae cranial to the trigone suggested by radiographic and ultrasound of the bladder. findings that included right ventricular dilation and hepatic centrilobular congestion. Laboratory results: The dog had a slight Hemorrhage and congestion are present in increase in neutrophils (11.81; range 3.0-11.4 the lung and are likely due to pulmonary x 10-3/ul), monocytes (2.35; range 0.15-1.35 hypertension possibly exacerbated by the x 10-3/ul), reduced hematocrit (36.9; range thrombocytopenia. The hypoproteinemia 37.0-55%), reduced platelets (43; range 200- and azotemia could not be fully explained. 500 x 10-3/ul), increased ALT (223; range 24- Right heart failure can lead to renal 90 IU/L), BUN (>180; range 10-30 mg/dl), dysfunction but typically not glomerular decreased calcium (8.6; range 9.7-11.3 leakage. mg/dl), increased phosphorus (10.7; range 3.2-6.0 mg/dl), increased glucose (202; range Some cases of IPAH have a mild 68-115 mg/dl), decreased sodium (122; range inflammatory component in the arterioles 141-151 mEq/L), decreased total protein which was evident in this case as well.6 It (4.6; range 5.2-7.1 gm/dl). could not be determined if the inflammation v variable numbers of lymphocytes was secondary or incidental to the (plexiform pulmonary arteriopathy). Multifocal alveoli are variably-filled with seroproteinaceous fluid and/or hemorrhage along with increased numbers of alveolar macrophages. There is moderate multifocal to diffuse vascular congestion.

Contributor’s Morphologic Diagnoses: Lung, arteriolitis and arteritis, proliferative, lymphocytic and fibrosing with plexiform Lung, dog. There is intimal hyperplasia and expansion of change, chronic moderate to severe the tunica medial by smooth muscle hyperplasia and increased amounts of collagen and extracellular matrix. Surrounding affected areioles, there is a proliferation of thin-walled arteriolar branches. (HE, 282X)

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proliferative change or if there is some cases fibrinoid necrosis (all present in this antigen (self or infectious agent) that section), the plexiform lesion is a unique triggered the lymphocytic infiltration. finding. This lesion is the result of a localized sac-like dilation of a branch of a muscular Contributing Institution: artery, in which fibrin accumulates from Department of Veterinary Pathology necrosis in the wall of the parent artery.1 College of Veterinary Medicine Over time, this fibrin is organized into small Iowa State University vessels populated by cells from the parent Ames, Iowa 50010-1250 artery as well. This pathogenesis also explains why the proliferating vessels are not JPC Diagnosis: Lung, small arterioles: circumferential but often present Plexiform (plexogenic) arteritis with marked eccentrically on only one side of affected intimal and medial hyperplasia and fibrosis, arterioles.1 recanalization, and multifocal fibrinoid necrosis and exudative alveolitis. These lesions have been well studied by

JPC Comment: Plexiform (plexogenic) arterial lesions are striking histologic lesions which are associated with pulmonary hypertension in man and animals. In general terms, plexiform lesions is a form of “dilatation” lesion developing in late stages of pulmonary hypertension, following a precipitous decrease in pulmonary blood flow and resultant irreversible pulmonary Lung, dog. Affected vessels occasionally contain extruded brightly eosinophilic protein and cellular debris within hypertension. While many small arterioles their walls (fibrinoid necrosis.) (HE, 400X) will show evidence of prolonged hypertension, to include intimal hyperplasia, sequential biopsies taken during the closure smooth muscle hyperplasia and disarray, and of congenital shunts in humans, and many of medial and adventitial fibrosis and in some the associated lesions are seen in this section. The first lesion is increased muscularity of the wall of small arterioles (medial hypertrophy). This is often followed over time by an intimal cellular proliferation, in which smooth muscle cells penetrate the internal elastic lamina and their proliferation at this point may occlude the lumen. They are often accompanied by a proliferation of both collagen and elastin fibers within the intima as well.4 At this point, dilatation lesions will develop proximally and concurrent fibrinoid necrosis in the wall of Lung, dog: Plexiform lesion demonstrate marked the artery begins the final development of the 4 asymmetric intimal hyperplasia and medical and plexiform lesion as described above. adventitial fibrosis, as well a proliferation of smaller arterioles (also fibrotic) at the periphery. (Masson’s, 400X) In humans, plexogenic arteriopathy may arise

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References:

1. Heath D, Smith P. Plexiform lesions with giant cells. Thorax 1982; 37:394-395. 2. Kolm US, Amberger CN, Boujon CE, Lombard CW. Plexogenic pulmonary arteriopathy in a

Lung dog – There is often polymerized fibrin, increased Pembroke Welsh Corgi. J Small numbers of alveolar macrophages, polymerized fibrin, Anim Prac 45(9):461-466, 2004 and patchy type II pneumocyte hyperplasia within 3. Patel M, Predescu D, Bardita C, proximity of plexiform lesions. (HE 282X) Chen J, Jegnathan N, Pritchard M, DiBartolo S, Machado, R, as a result of congenital cardiac shunting, but Predescu S. Modulation of may also be seen with acquired cardiac intersectin-1s lung expression shunts, hepatic cirrhosis with portal induces obliterative remodeling hypertension, schistosomiasis, and oral and severe plexiform arteriopathy ingestion of drugs for anorexia.4 in the murine pulmonary vascular bed. Am J Pathol 2017; The first description of pulmonary plexiform 187(3):529-542 arteriopathy (PPA), also in a Welsh Corgi, 4. Wagenvoort CA. Plexiform was published in 2004, and its etiology was arteriopathy. Thorax 1994: attributed to an untreated patent ductus 49:S39-245. arteriosus (the animal was 21 months at its 5. Wideman RF, Mason JG, Anthony death.)2 PPA was also identified in 4 of 6 NB, Cross D. Plexogenic animals in another study of dogs with arteriopathy in broiler lungs: idiopathic pulmonary hypertension. Evaluation of line, age and sex Plexiform lesions were identified primarily at influences. Poultry Sci 2015; branching points of terminal to alveolar duct 94:628-638. arteries.6 6. Zabka TS, Campbell FE, Wilson DE. Pulmonary arteriopathy and Plexiform lesions have also been identified in idiopathic pulmonary arterial a line of rapidly growing broiler chickens hypertension in six dogs. Vet which had a high incidence of idiopathic Pathol 43(4): 510-522, 2006 pulmonary arterial hypertension; however, as opposed to other species, plexiform lesions could be identified immediately post-hatch and did not require a period of pulmonary arterial hypertension.5 A line of genetically engineered mutant mice that develop plexiform arteriopathy has been developed as a result of decreased expression of intersectin-1s.3

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