Chronic Inhalation Exposure to Antimony Trioxide Exacerbates the MAPK Signaling in Alveolar Bronchiolar Carcinomas in B6C3F1/N Mice.

Antimony trioxide (AT) is used as a flame retardant in fabrics and plastics. Occupational exposure in miners and smelters is mainly through inhalation and dermal contact. Chronic inhalation exposure to AT particulates in B6C3F1/N mice and Wistar Han rats resulted in increased incidences and tumor multiplicities of alveolar/bronchiolar carcinomas (ABCs). In this study, we demonstrated Kras (43%) and Egfr (46%) hotspot mutations in mouse lung tumors (n = 80) and only Egfr (50%) mutations in rat lung tumors (n = 26). Interestingly, there were no differences in the incidences of these mutations in ABCs from rats and mice at exposure concentrations that did and did not exceed the pulmonary overload threshold. There was increased expression of p44/42 mitogen-activated protein kinase (MAPK) (Erk1/2) protein in ABCs harboring mutations in Kras and/or Egfr, confirming the activation of MAPK signaling. Transcriptomic analysis indicated significant alterations in MAPK signaling such as ephrin receptor signaling and signaling by Rho-family GTPases in AT-exposed ABCs. In addition, there was significant overlap between transcriptomic data from mouse ABCs due to AT exposure and human pulmonary adenocarcinoma data. Collectively, these data suggest chronic AT exposure exacerbates MAPK signaling in ABCs and, thus, may be translationally relevant to human lung cancers.

Inter-pathologist agreement on diagnosis, classification and grading of canine glioma.

Histopathological evaluation of tumours is a subjective process, but studies of inter-pathologist agreement are uncommon in veterinary medicine. The Comparative Brain Tumour Consortium (CBTC) recently published diagnostic criteria for canine gliomas. Our objective was to assess the degree of inter-pathologist agreement on intracranial canine gliomas, utilising the CBTC diagnostic criteria in a cohort of eighty-five samples from dogs with an archival diagnosis of intracranial glioma. Five pathologists independently reviewed H&E and immunohistochemistry sections and provided a diagnosis and grade. Percentage agreement and kappa statistics were calculated to measure inter-pathologist agreement between pairs and amongst the entire group. A consensus diagnosis of glioma subtype and grade was achieved for 71/85 (84%) cases. For these cases, percentage agreement on combined diagnosis (subtype and grade), subtype only and grade only were 66%, 80% and 82%, respectively. Kappa statistics for the same were 0.466, 0.542 and 0.516, respectively. Kappa statistics for oligodendroglioma, astrocytoma and undefined glioma were 0.585, 0.566 and 0.280 and were 0.516 for both low-grade and high-grade tumours. Kappa statistics amongst pairs of pathologists for combined diagnosis varied from 0.352 to 0.839. 8 % of archival oligodendrogliomas and 61% of archival astrocytomas were reclassified as another entity after review. Inter-pathologist agreement utilising CBTC guidelines for canine glioma was moderate overall but varied from fair to almost perfect between pairs of pathologists. Agreement was similar for oligodendrogliomas and astrocytomas but lower for undefined gliomas. These results are similar to pathologist agreement in human glioma studies and with other tumour entities in veterinary medicine.

Immunohistochemical evaluation of immune cell infiltration in canine gliomas.

Evasion of the immune response is an integral part of the pathogenesis of glioma. In humans, important mechanisms of immune evasion include recruitment of regulatory T cells (Tregs) and polarization of macrophages toward an M2 phenotype. Canine glioma has a robust immune cell infiltrate that has not been extensively characterized. The purpose of this study was to determine the distribution of immune cells infiltrating spontaneous intracranial canine gliomas. Seventy-three formalin-fixed, paraffin-embedded tumor samples were evaluated using immunohistochemistry for CD3, forkhead box 3 (FOXP3), CD20, Iba1, calprotectin (Mac387), CD163, and indoleamine 2,3-dioxygenase (IDO). Immune cell infiltration was present in all tumors. Low-grade and high-grade gliomas significantly differed in the numbers of FoxP3+ cells, Mac387+ cells, and CD163+ cells (P = .006, .01, and .01, respectively). Considering all tumors, there was a significant increase in tumor area fraction of CD163 compared to Mac387 (P < .0001), and this ratio was greater in high-grade tumors than in low-grade tumors (P = .005). These data warrant further exploration into the roles of macrophage repolarization or Treg interference therapy in canine glioma.

IRGM1 links mitochondrial quality control to autoimmunity.

Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7 and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial DNA-dependent induction of type I interferon. Here, we show that mice lacking the GTPase IRGM1 (IRGM homolog) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired the execution of mitophagy with cell-specific consequences. In fibroblasts, mitochondrial DNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent type I interferon, whereas in macrophages, lysosomal Toll-like receptor 7 was activated. In vivo, Irgm1-/- tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology was abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.

Constitutive expression of progesterone receptor isoforms promotes the development of hormone-dependent ovarian neoplasms.

Differences in the relative abundances of the progesterone receptor (PGR) isoforms PGRA and PGRB are often observed in women with reproductive tract cancers. To assess the importance of the PGR isoform ratio in the maintenance of the reproductive tract, we generated mice that overexpress PGRA or PGRB in all PGR-positive tissues. Whereas few PGRA-overexpressing mice developed reproductive tract tumors, all PGRB-overexpressing mice developed ovarian neoplasms that were derived from ovarian luteal cells. Transcriptomic analyses of the ovarian tumors from PGRB-overexpressing mice revealed enhanced AKT signaling and a gene expression signature similar to those of human ovarian and endometrial cancers. Treating PGRB-overexpressing mice with the PGR antagonist RU486 stalled tumor growth and decreased the expression of cell cycle-associated genes, indicating that tumor growth and cell proliferation were hormone dependent in addition to being isoform dependent. Analysis of the PGRB cistrome identified binding events at genes encoding proteins that are critical regulators of mitotic phase entry. This work suggests a mechanism whereby an increase in the abundance of PGRB relative to that of PGRA drives neoplasia in vivo by stimulating cell cycling.

Previously Diagnosed Reticulum Cell Hyperplasia in Decalcified Rat Bone Marrow Stain Positive for Ionized Calcium Binding Adapter Molecule 1 (Iba1): A Monocytic/Macrophage Cell Marker.

Reticulum cell hyperplasia (RCH) was a term used for many years by the National Toxicology Program (NTP) to describe a certain non-neoplastic bone marrow lesion of rats. Retrospective microscopic evaluation of RCH lesions and immunohistochemistry analyses were performed to reassess and further characterize these lesions. The NTP database was searched to identify femoral bone marrow specimens diagnosed with RCH from 1981 to 2014 (n = 254). The diagnosis last occurred in 2003, after which the term "cellular infiltration" was used. Eighty-three RCH slides, spanning 22 years, representing 34 different chemicals, were selected for microscopic review, and a subset (23) was chosen for ionized calcium binding adapter molecule 1 (Iba1) immunohistochemical staining; initial investigations revealed Iba1 worked as a macrophage marker on decalcified tissue. The following diagnoses were made upon reevaluation: 36 were consistent with cellularity increased, macrophage, 22 with histiocytic sarcoma, 8 with increased myeloid cells, 4 with autolysis, and 13 were normal appearance. All 23 RCH lesions stained positive for Iba1. Fifty-eight of 83 bone marrows previously diagnosed with RCH are consistent morphologically and immunohistochemically with cells of histiocytic origin. These results will help with interpretation of historical data and demonstrates that Iba1 can be used in decalcified bone marrow sections.

Proceedings of the 2019 National Toxicology Program Satellite Symposium.

The 2019 annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri," was held in Raleigh, North Carolina, at the Society of Toxicologic Pathology's 38th annual meeting. The goal of this symposium was to present and discuss challenging diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers' talks along with select images that were used by the audience for voting and discussion. Various lesions and topics covered during the symposium included aging mouse lesions from various strains, as well as the following lesions from various rat strains: rete testis sperm granuloma/fibrosis, ovarian cystadenocarcinoma, retro-orbital schwannoma, periductal cholangiofibrosis of the liver and pancreas, pars distalis hypertrophy, chronic progressive nephropathy, and renal tubule regeneration. Other cases included polyovular follicles in young beagle dogs and a fungal blood smear contaminant. One series of cases challenged the audience to consider how immunohistochemistry may improve the diagnosis of some tumors. Interesting retinal lesions from a rhesus macaque emphasized the difficulty in determining the etiology of any particular retinal lesion due to the retina's similar response to vascular injury. Finally, a series of lesions from the International Harmonization of Nomenclature and Diagnostic Criteria Non-Rodent Fish Working Group were presented.

Efficient CD4Cre-Mediated Conditional KRas Expression in Alveolar Macrophages and Alveolar Epithelial Cells Causes Fatal Hyperproliferative Pneumonitis.

The CD4Cre transgenic model has been widely used for T cell-specific gene manipulation. We report unexpected highly efficient Cre-mediated recombination in alveolar macrophages (AMFs), bronchial epithelial cells (BECs), and alveolar epithelial cells (AECs) in this strain of mice. Different from CD4 T cells, AMFs, AECs, and BECs do not express detectable Cre protein, suggesting that Cre protein is either very transiently expressed in these cells or only expressed in their precursors. Mice carrying a conditional constitutively active KRas (caKRas) allele and the CD4Cre transgene contain not only hyperactivated T cells but also develop severe AMF accumulation, AEC and BEC hyperplasia, and adenomas in the lung, leading to early lethality correlated with caKRas expression in these cells. We propose that caKRas-CD4Cre mice represent, to our knowledge, a novel model of proliferative pneumonitis involving macrophages and epithelial cells and that the CD4Cre model may offer unique usefulness for studying gene functions simultaneously in multilineages in the lung. Our observations, additionally, suggest that caution in data interpretation is warranted when using the CD4Cre transgenic model for T cell-specific gene manipulation, particularly when lung pathophysiological status is being examined.

ERBB2 Regulates MED24 during Cancer Progression in Mice with Pten and Smad4 Deletion in the Pulmonary Epithelium.

ERBB2 is an oncogenic driver with frequent gene mutations and amplification in human lung tumors and is an attractive target for lung cancer therapy. However, target therapies can be improved by understanding the in vivo mechanisms regulated by ERBB2 during lung tumor development. Here, we generated genetic mouse models to show that Erbb2 loss inhibited lung tumor development induced by deletion of Pten and Smad4. Transcriptome analysis showed that Erbb2 loss suppressed the significant changes of most of the induced genes by ablation of Pten and Smad4. Overlapping with ERBB2-associated human lung cancer genes further identified those ERBB2 downstream players potentially conserved in human and mouse lung tumors. Furthermore, MED24 was identified as a crucial oncogenic target of ERBB2 in lung tumor development. Taken together, ERBB2 is required for the dysregulation of cancer-related genes, such as MED24, during lung tumor development.

Do GISTs Occur in Rats and Mice? Immunohistochemical Characterization of Gastrointestinal Tumors Diagnosed as Smooth Muscle Tumors in The National Toxicology Program.

The majority of the tumors in the gastrointestinal (GI) tract of rats and mice, with spindle cell morphology, are diagnosed as smooth muscle tumors (SMTs). Similarly, several decades ago human GI tumors with spindle cell morphology were also diagnosed as SMTs. However, later investigations identified most of these tumors in humans as gastrointestinal stromal tumors (GISTs). The GISTs are considered to arise from the interstitial cells of Cajal located throughout the GI tract. Positive immunohistochemical staining with CKIT antibody is a well-accepted diagnostic marker for GISTs in humans. Since there is a considerable overlap between the histomorphology of SMTs and GISTs, it is not possible to distinguish them on hematoxylin and eosin stained sections. As a result, GISTs are not routinely diagnosed in toxicological studies. The current study was designed to evaluate the tumors diagnosed as leiomyoma or leiomyosarcoma in the National Toxicology Program's 2-year bioassays using CKIT, smooth muscle actin, and desmin immunohistochemistry. The results demonstrate that most of the mouse SMTs diagnosed as leiomyoma or leiomyosarcoma are likely GISTs, whereas in rats the tumors are likely SMTs and not GISTs.

JNK1/2 represses Lkb1-deficiency-induced lung squamous cell carcinoma progression.

Mechanisms of lung squamous cell carcinoma (LSCC) development are poorly understood. Here, we report that JNK1/2 activities attenuate Lkb1-deficiency-driven LSCC initiation and progression through repressing ΔNp63 signaling. In vivo Lkb1 ablation alone is sufficient to induce LSCC development by reducing MKK7 levels and JNK1/2 activities, independent of the AMPKα and mTOR pathways. JNK1/2 activities is positively regulated by MKK7 during LSCC development. Pharmaceutically elevated JNK1/2 activities abates Lkb1 dependent LSCC formation while compound mutations of Jnk1/2 and Lkb1 further accelerate LSCC progression. JNK1/2 is inactivated in a substantial proportion of human LSCC and JNK1/2 activities positively correlates with survival rates of lung, cervical and head and neck squamous cell carcinoma patients. These findings not only determine a suppressive role of the stress response regulators JNK1/2 on LSCC development by acting downstream of the key LSCC suppresser Lkb1, but also demonstrate activating JNK1/2 activities as a therapeutic approach against LSCC.

Male infertility in mice lacking the store-operated Ca(2+) channel Orai1.

Store-operated calcium entry (SOCE) is an important Ca(2+) influx pathway in somatic cells. In addition to maintaining endoplasmic reticulum (ER) Ca(2+) stores, Ca(2+) entry through store-operated channels regulates essential signaling pathways in numerous cell types. Patients with mutations in the store-operated channel subunit ORAI1 exhibit defects in store-operated Ca(2+) influx, along with severe immunodeficiency, congenital myopathy and ectodermal dysplasia. However, little is known about the functional role of ORAI1 in germ cells and reproductive function in mice, or in men, since men with loss-of-function or null mutations in ORAI1 rarely survive to reproductive age. In this study, we investigated the role of ORAI1 in male reproductive function. We reveal that Orai1(-/-) male mice are sterile and have severe defects in spermatogenesis, with prominent deficiencies in mid- to late-stage elongating spermatid development. These studies establish an essential in vivo role for store-operated ORAI1 channels in male reproductive function and identify these channels as potential non-steroidal regulators of male fertility.

Role of the store-operated calcium entry protein, STIM1, in neutrophil chemotaxis and infiltration into a murine model of psoriasis-inflamed skin.

Stromal interaction molecule 1 (STIM1) is a Ca(2+) sensor protein that initiates store-operated calcium entry (SOCE). STIM1 is known to be involved in the chemoattractant signaling pathway for FPR1 in cell lines, but its role in in vivo functioning of neutrophils is unclear. Plaque-type psoriasis is a chronic inflammatory skin disorder associated with chemoattractants driving neutrophils into the epidermis. We investigated the involvement of STIM1 in neutrophil chemotaxis in vitro, as well as during chronic psoriatic inflammation. To this end, we used conditional knockout (KO) mice lacking STIM1 in cells of myeloid lineage (STIM1(fl/fl) LysM-cre). We demonstrate that STIM1 is required for chemotaxis because of multiple chemoattractants in mouse neutrophils in vitro. Using an imiquimod-induced psoriasis-like skin model, we show that KO mice had less neutrophil infiltration in the epidermis than controls, whereas neither chemoattractant production in the epidermis nor macrophage migration was decreased. KO mice displayed a more rapid reversal of the outward signs of psoriasis (plaques). Thus, KO of STIM1 impairs neutrophil contribution to psoriatic inflammation. Our data provide new insights to our understanding of how STIM1 orchestrates the cellular behavior underlying chemotaxis and illustrate the important role of SOCE in a disease-related pathologic model.

Histopathological and Immunohistochemical Characterization of Methyl Eugenol-induced Nonneoplastic and Neoplastic Neuroendocrine Cell Lesions in Glandular Stomach of Rats.

Methyl eugenol induces neuroendocrine (NE) cell hyperplasia and tumors in F344/N rat stomach. Detailed histopathological and immunohistochemical (IHC) characterization of these tumors has not been previously reported. The objective of this study was to fill that data gap. Archived slides and paraffin blocks were retrieved from the National Toxicology Program Archives. NE hyperplasias and tumors were stained with chromogranin A, synaptophysin, amylase, gastrin, H(+)/K(+) adenosine triphosphatase (ATPase), pepsinogen, somatostatin, and cytokeratin 18 (CK18) antibodies. Many of the rats had gastric mucosal atrophy, due to loss of chief and parietal cells. The hyperplasias and tumors were confined to fundic stomach, and females were more affected than the males. Hyperplasia of NE cells was not observed in the pyloric region. Approximately one-third of the females with malignant NE tumors had areas of pancreatic acinar differentiation. The rate of metastasis was 21%, with liver being the most common site of metastasis. Immunohistochemically, the hyperplasias and tumors stained consistently with chromogranin A and synaptophysin. Neoplastic cells were also positive for amylase and CK18 and negative for gastrin, somatostatin, H(+)/K(+) ATPase, and pepsinogen. Metastatic neoplasms histologically similar to the primary neoplasm stained positively for chromogranin A and synaptophysin. Based on the histopathological and IHC features, the neoplasms appear to arise from enterochromaffin-like cells.

Transactivating function (AF) 2-mediated AF-1 activity of estrogen receptor α is crucial to maintain male reproductive tract function.

Estrogen receptor alpha (ERα) is a ligand-dependent transcription factor containing two transcriptional activation function (AF) domains. AF-1 is in the N terminus of the receptor protein, and AF-2 activity is dependent on helix 12 of the C-terminal ligand-binding domain. We recently showed that two point mutations converting leucines 543 and 544 to alanines in helix 12 (AF2ER) minimized estrogen-dependent AF-2 transcriptional activation. A characteristic feature of AF2ER is that the estrogen antagonists ICI182780 and tamoxifen (TAM) act as agonists through intact AF-1, but not through mutated AF-2. Here we report the reproductive phenotype of male AF2ER knock-in (AF2ERKI) mice and demonstrate the involvement of ERα in male fertility. The AF2ERKI male homozygotes are infertile because of seminiferous tubular dysmorphogenesis in the testis, similar to ERα KO males. Sperm counts and motility did not differ at age 6 wk in AF2ERKI and WT mice, but a significant testis defect was observed in adult AF2ERKI male mice. The expression of efferent ductal genes involved in fluid reabsorption was significantly lower in AF2ERKI males. TAM treatment for 3 wk beginning at age 21 d activated AF-2-mutated ERα (AF2ER) and restored expression of efferent ductule genes. At the same time, the TAM treatment reversed AF2ERKI male infertility compared with the vehicle-treated group. These results indicate that the ERα AF-2 mutation results in male infertility, suggesting that the AF-1 is regulated in an AF-2-dependent manner in the male reproductive tract. Activation of ERα AF-1 is capable of rescuing AF2ERKI male infertility.

RAP80 is critical in maintaining genomic stability and suppressing tumor development.

The ubiquitin interaction motif-containing protein RAP80 was recently found to play a key role in DNA damage response (DDR) signaling by facilitating the translocation of several DDR mediators, including BRCA1, to ionizing irradiation (IR)-induced foci. In this study, we examine the effect of the loss of RAP80 on genomic stability and the susceptibility to cancer development in RAP80 null (RAP80(-/-)) mice. RAP80(-/-) mice are viable and did not exhibit any apparent developmental defects. Mouse embryonic fibroblasts (MEF) derived from RAP80(-/-) mice underwent premature senescence compared with wild-type (WT) MEFs, were more sensitive to IR, and exhibited a higher level of spontaneous and IR-induced genomic instability. RAP80(-/-) thymocytes were more sensitive to IR-induced cell death than WT thymocytes. RAP80(-/-) mice were more susceptible to spontaneous lymphoma development and the development of 7,12-dimethylbenz(a)anthracene-induced mammary gland tumors. Moreover, the loss of RAP80 accelerated tumor formation in both p53(-/-) and p53(+/-) mice. Our data indicate that RAP80-deficiency promotes genomic instability and causes an increase in cancer risk consistent with the concept that RAP80 exhibits a tumor suppressor function.

Role of estrogen receptor signaling required for endometriosis-like lesion establishment in a mouse model.

Endometriosis results from ectopic invasion of endometrial tissue within the peritoneal cavity. Aberrant levels of the estrogen receptor (ER), ERα and ERß, and higher incidence of autoimmune disorders are observed in women with endometriosis. An immunocompetent mouse model of endometriosis was used in which minced uterine tissue from a donor was dispersed into the peritoneal cavity of a recipient. Wild-type (WT), ERα-knockout (αERKO), and ßERKO mice were donors or recipients to investigate the roles of ERα, ERß, and estradiol-mediated signaling on endometriosis-like disease. Mice were treated with vehicle or estradiol, and resulting location, number, and size of endometriosis-like lesions were assessed. In comparison with WT lesions in WT hosts, αERKO lesions in WT hosts were smaller and fewer in number. The effect of ER status and estradiol treatment on nuclear receptor status, proliferation, organization, and inflammation within lesions were examined. αERKO lesions in WT hosts did not form distal to the incision site, respond to estradiol, or proliferate but did have increased inflammation. WT lesions in αERKO hosts did respond to estradiol, proliferate, and show decreased inflammation with treatment, but surprisingly, progesterone receptor expression and localization remained unchanged. Only minor differences were observed between WT lesions in ßERKO hosts and ßERKO lesions in WT hosts, demonstrating the estradiol-mediated signaling responses are predominately through ERα. In sum, these results suggest ER in both endometriosis-like lesions and their environment influence lesion characteristics, and understanding these interactions may play a critical role in elucidating this enigmatic disease.

Chondrosarcoma in a dromedary camel (Camelus dromedarius).

A 4-year 10-month-old, intact female dromedary camel had progressive left carpal joint swelling and lameness for 7 months. Radiographs showed multifocal lytic lesions in the carpal and proximal metacarpal bones. Surgical biopsy of the synovial capsule and carpal bones suggested neoplasia, and the camel was subsequently euthanized. At necropsy, a white to pale pink, firm, multilobulated, soft tissue mass was located on the palmar aspect of the left carpal joint. Two smaller masses were present on the dorsal aspect of the carpal joint. The masses infiltrated all the carpal bones and the proximal region of the metacarpal bone. The joint capsule was diffusely thickened. The articular surfaces of the carpal bones and the metacarpal bone were multifocally eroded. The lungs contained multiple, firm, raised, gray, randomly distributed nodules. The neoplastic cells stained positive for vimentin and S-100. Chondrosarcoma arising from around the carpal joint with infiltration of carpal and metacarpal bones, and pulmonary metastasis, was diagnosed based on the histopathological and immunohistochemical evaluation.

Swine models of cystic fibrosis reveal male reproductive tract phenotype at birth.

Nearly all male cystic fibrosis (CF) patients exhibit tissue abnormalities in the reproductive tract, a condition that renders them azoospermic and infertile. Two swine CF models have been reported recently that include respiratory and digestive manifestations that are comparable to human CF. The goal of this study was to determine the phenotypic changes that may be present in the vas deferens of these swine CF models. Tracts from CFTR(-/-) and CFTR(ΔF508/ΔF508) neonates revealed partial or total vas deferens and/or epididymis atresia at birth, while wild-type littermates were normal. Histopathological analysis revealed a range of tissue abnormalities and disruptions in tubular organization. Vas deferens epithelial cells were isolated and electrophysiological results support that CFTR(-/-) monolayers can exhibit Na(+) reabsorption but reveal no anion secretion following exposure to cAMP-generating compounds, suggesting that CFTR-dependent Cl(-) and/or HCO(3)(-) transport is completely impaired. SLC26A3 and SLC26A6 immunoreactivities were detected in all experimental groups, indicating that these two chloride-bicarbonate exchangers were present, but were either unable to function or their activity is electroneutral. In addition, no signs of increased mucus synthesis and/or secretion were present in the male excurrent ducts of these CF models. Results demonstrate a causal link between CFTR mutations and duct abnormalities that are manifested at birth.

Fatal canine adenoviral pneumonia in two litters of Bulldogs.

Five Bulldog pups, 4 weeks of age or younger, were presented over a 2-day period for postmortem examination and diagnostic evaluation. The pups originated from 2 different litters but had been cared for at a common facility since their birth. All 5 pups died after exhibiting symptoms consisting of lethargy, dyspnea, nasal discharge, anorexia, vomiting, diarrhea, and abdominal pain. Necropsy examination revealed locally extensive to diffusely red, firm, consolidated lungs in all pups. Histopathologically, the lungs were variably effaced by multifocal areas of necrosis. The alveolar lumens contained fibrin, edema fluid, macrophages, and neutrophils. Many of the bronchioles contained cellular debris and neutrophils admixed with sloughed bronchiolar epithelium, which often contained large intranuclear amphophilic inclusion bodies that peripherally displaced chromatin. Fluorescent antibody testing was positive for Canine adenovirus. An adenovirus isolated via cell culture was positive on direct fluorescent antibody test and was identified as Canine adenovirus serotype 2 via polymerase chain reaction. Electron microscopy revealed typical viral inclusions within bronchiolar epithelial cells. Hemolytic Escherichia coli was also isolated from the lungs in 3 of the 5 pups. The current case demonstrates a natural and rare fatal infection with a viral agent that is typically associated with immunosuppression in both animals and humans.