Proceedings of the 2017 National Toxicology Program Satellite Symposium.

The 2017 annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri," was held in Montreal, Quebec, Canada at the Society of Toxicologic Pathology's 36th 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 other topics covered during the symposium included renal papillary degeneration in perinatally exposed animals, an atriocaval mesothelioma, an unusual presentation of an alveolar-bronchiolar carcinoma, a paraganglioma of the organ of Zuckerkandl (also called an extra-adrenal pheochromocytoma), the use of human muscle samples to illustrate the challenges of manual scoring of fluorescent staining, intertubular spermatocytic seminomas, medical device pathology assessment and discussion of the approval process, collagen-induced arthritis, incisor denticles, ameloblast degeneration and poorly mineralized enamel matrix, connective tissue paragangliomas, microcystin-LR toxicity, perivascular mast cells in the forebrain thalamus unrelated to treatment, and 2 cases that provided a review of the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) bone nomenclature and recommended application of the terminology in routine nonclinical toxicity studies.

Proceedings of the 2016 National Toxicology Program Satellite Symposium.

The 2016 annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri" was held in San Diego, CA, at the Society of Toxicologic Pathology's (STP) 35th 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. Some lesions and topics covered during the symposium included malignant glioma and histiocytic sarcoma in the rodent brain; a new statistical method designed for histopathology data evaluation; uterine stromal/glandular polyp in a rat; malignant plasma cell tumor in a mouse brain; Schwann cell proliferative lesions in rat hearts; axillary schwannoma in a cat; necrosis and granulomatous inflammation in a rat brain; adenoma/carcinoma in a rat adrenal gland; hepatocyte maturation defect and liver/spleen hematopoietic defects in an embryonic mouse; distinguishing malignant glioma, malignant mixed glioma, and malignant oligodendroglioma in the rat; comparison of mammary gland whole mounts and histopathology from mice; and discussion of the International Harmonization of Nomenclature and Diagnostic Criteria collaborations.

Comparison of Renal Amyloid and Hyaline Glomerulopathy in B6C3F1 Mice: An NTP Retrospective Study.

Due to potential misdiagnosis of hyaline glomerulopathy (HG) for amyloidosis, a retrospective study of B6C3F1 mice from the National Toxicology Program (NTP) archives was undertaken to determine whether HG had occurred in prior NTP studies and, if so, whether these 2 glomerular lesions could be routinely discriminated. Kidney slides from 7 amyloid-positive control mice, 2 HG-positive control mice, 3 normal or negative control mice, and 41 potential HG mice (with renal-only deposits previously diagnosed as amyloid) were evaluated using hematoxylin and eosin (H&E), periodic acid Schiff (PAS), Congo red (CR), and Masson's trichrome (MT) stains. Utilizing these techniques, HG was reliably distinguished from amyloidosis. All 41 potential HG mice had glomerular deposits histochemically inconsistent with amyloid; the deposits were PAS positive and CR negative. Four of the 41 mice were selected for transmission electron microscopy of the glomerular deposits; ultrastructurally, the deposits in these animals were consistent with HG and not amyloid. Our findings indicate that HG is a spontaneous lesion in B6C3F1 mice of low occurrence, is commonly misdiagnosed as amyloidosis, and is more likely than amyloid to cause glomerular deposits in mice without evidence of deposits in other tissues. Also, HG can be distinguished from amyloid on H&E evaluation; however, the distinction is improved with use of PAS or CR staining and/or ultraviolet evaluation.

Proceedings of the 2014 National Toxicology Program Satellite Symposium.

The 2014 annual National Toxicology Program (NTP) Satellite Symposium, entitled "Pathology Potpourri" was held in Washington, D.C., in advance of the Society of Toxicologic Pathology's 33rd annual meeting. The goal of this annual NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers' presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for audience voting and discussion. Some lesions and topics covered during the symposium included a pulmonary mucinous adenocarcinoma in a male B6C3F1 mouse; plexiform vasculopathy in Wistar Han (Crl:WI[Han]) rats; staging of the estrous cycle in rats and mice; peri-islet fibrosis, hemorrhage, lobular atrophy and inflammation in male Sprague-Dawley (SD) rats; retinal dysplasia in Crl:WI[Han] rats and B6C3F1 mice; multicentric lymphoma with intravascular microemboli and tumor lysis syndrome, and 2 cases of myopathy and vascular anomaly in Tg.rasH2 mice; benign thymomas in Crl:WI[Han] rats; angiomatous lesions in the mesenteric lymph nodes of Crl:WI[Han] rats; an unusual foveal lesion in a cynomolgous monkey; and finally a series of nomenclatures challenges from the endocrine International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) Organ Working Group (OWG).

Prevalence of Sarcocystis neurona and Neospora spp. infection in horses from Brazil based on presence of serum antibodies to parasite surface antigen.

Sera from 961 horses from Brazil were tested for antibodies against the major surface antigens SnSAG4 and NhSAG1 to determine the seroprevalence of Sarcocystis neurona and Neospora hughesi, respectively. Antibodies against SnSAG4 were detected in 669 (69.6%) of the horses, while antibodies against NhSAG1 were detected in only 24 (2.5%) of the horses. These serologic results suggest that there is a high concentration of S. neurona in the environment of Brazil, which results in marked exposure of horses to this parasite. Additionally, the data further confirm that infection with Neospora spp. is relatively uncommon in horses.

Enzyme-linked immunosorbent assays for detection of equine antibodies specific to Sarcocystis neurona surface antigens.

Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.

Recombinant NhSAG1 ELISA: a sensitive and specific assay for detecting antibodies against Neospora hughesi in equine serum.

Neospora hughesi is a recently identified cause of equine protozoal myeloencephalitis. However, the significance of this parasite is poorly understood. An enzyme-linked immunosorbent assay (ELISA) with a recombinant form of the N. hughesi 29-kDa surface antigen (rNhSAG1) was developed for serodiagnosis of equine N. hughesi infections. Parallel ELISA analysis showed that animals immunized or infected with N. hughesi exhibited greater antibody reactivity with rNhSAG1 than with the Neospora caninum homolog, rNcSAG1. The rNhSAG1 ELISA showed 94.4% sensitivity and 95.0% specificity when compared with N. hughesi western blot results for 1,006 samples. The N. hughesi seroprevalence was 3.4% for the 1,917 samples tested by ELISA, which is less than earlier reports. Importantly, western blot analysis of ELISA-positive sera revealed only 18 true seropositive samples for an even lower seroprevalence of 0.9%. These results imply that Neospora spp. infections are uncommon in horses. The sensitivity and specificity exhibited by the rNhSAG1 ELISA suggest that it has a potential use for serodiagnosis of N. hughesi infection in equids. Furthermore, the high-throughput capability of the ELISA will allow for screening large sample sets, which should provide a better understanding of N. hughesi epidemiology.

Analysis of the Sarcocystis neurona microneme protein SnMIC10: protein characteristics and expression during intracellular development.

Sarcocystis neurona, an apicomplexan parasite, is the primary causative agent of equine protozoal myeloencephalitis. Like other members of the Apicomplexa, S. neurona zoites possess secretory organelles that contain proteins necessary for host cell invasion and intracellular survival. From a collection of S. neurona expressed sequence tags, we identified a sequence encoding a putative microneme protein based on similarity to Toxoplasma gondii MIC10 (TgMIC10). Pairwise sequence alignments of SnMIC10 to TgMIC10 and NcMIC10 from Neospora caninum revealed approximately 33% identity to both orthologues. The open reading frame of the S. neurona gene encodes a 255 amino acid protein with a predicted 39-residue signal peptide. Like TgMIC10 and NcMIC10, SnMIC10 is predicted to be hydrophilic, highly alpha-helical in structure, and devoid of identifiable adhesive domains. Antibodies raised against recombinant SnMIC10 recognised a protein band with an apparent molecular weight of 24 kDa in Western blots of S. neurona merozoites, consistent with the size predicted for SnMIC10. In vitro secretion assays demonstrated that this protein is secreted by extracellular merozoites in a temperature-dependent manner. Indirect immunofluorescence analysis of SnMIC10 showed a polar labelling pattern, which is consistent with the apical position of the micronemes, and immunoelectron microscopy provided definitive localisation of the protein to these secretory organelles. Further analysis of SnMIC10 in intracellular parasites revealed that expression of this protein is temporally regulated during endopolygeny, supporting the view that micronemes are only needed during host cell invasion. Collectively, the data indicate that SnMIC10 is a microneme protein that is part of the excreted/secreted antigen fraction of S. neurona. Identification and characterisation of additional S. neurona microneme antigens and comparisons to orthologues in other Apicomplexa could provide further insight into the functions that these proteins serve during invasion of host cells.