Cytauxzoonosis in Indiana, USA: a case series of cats infected with Cytauxzoon felis (2018-2022).

CASE SERIES SUMMARY: This case series describes six cases involving seven cats naturally infected with Cytauxzoon felis in Indiana, USA. Medical records were retrospectively reviewed and all available information on signalment, history, clinical and diagnostic findings, treatment, outcome and pathology was reported. Cats infected with C felis were domestic shorthairs, were aged between 2 and 9 years and all but one of the cats were male. The seven infected cats originated from five counties in southwestern Indiana. Six of seven cats were found to have acute cytauxzoonosis based on clinical signs, gross pathologic lesions, observation of C felis in tissues and/or detection of C felis DNA. One cat was identified as a subclinical survivor cat with no known clinical history of cytauxzoonosis. RELEVANCE AND NOVEL INFORMATION: The reported cases are the first confirmed reports of acute and chronic cytauxzoonosis in cats from Indiana and document an expansion in the range of C felis. Veterinary practitioners in Indiana should consider infection with C felis as a differential diagnosis for cats that present with fever, inappetence, lethargy, depression, dehydration, dyspnea, hemolytic crisis, anorexia or icterus. Administration of approved acaricides to cats currently offers the best protection and control against C felis infection.

Targeted metabolomics characterizes metabolite occurrence and variability in stable freshwater mussel populations.

Freshwater mussels (order Unionida) play a key role in freshwater systems as ecosystem engineers and indicators of aquatic ecosystem health. The fauna is globally imperilled due to a diversity of suspected factors; however, causes for many population declines and mortality events remain unconfirmed due partly to limited health assessment tools. Mussel-monitoring activities often rely on population-level measurements, such as abundance and age structure, which reflect delayed responses to environmental conditions. Measures of organismal health would enable preemptive detection of declining condition before population-level effects manifest. Metabolomic analysis can identify shifts in biochemical pathways in response to stressors and changing environmental conditions; however, interpretation of the results requires information on inherent variability of metabolite concentrations in mussel populations. We targeted metabolites in the haemolymph of two common mussels, Lampsilis cardium and Lampsilis siliquoidea, from three Indiana streams (USA) using ultra-high-performance liquid chromatography combined with quadrupole time-of-flight mass spectroscopy. The influence of species, stream and sex on metabolite variability was examined with distance-based redundancy analysis. Metabolite variability was most influenced by species, followed by site and sex. Inter- and intraspecies metabolite variability among sexes was less distinct than differences among locations. We further categorized metabolites by occurrence and variability in mussel populations. Metabolites with high occurrence (Categories 1 and 2) included those indicative of energy status (catabolism versus anabolism; arginine, proline, carnitine, nicotinic acid, pantothenic acid), oxidative stress (proline, glutamine, glutamate) and protein metabolism (thymidine, cytidine, inosine). Metabolites with lower occurrence (Category 3) are constituents of assorted metabolic pathways and can be important biomarkers with additional temporal sampling to characterize their variability. These data provide a reference for future temporal (before/after) monitoring and for studies of stressor-metabolite linkages in freshwater mussels.

Histopathologic survey of free-living populations of 2 species of freshwater mussels in Indiana.

Freshwater mussels are one of the most endangered groups of animals in Indiana, with nearly half of the native species either extirpated or listed as "state endangered" or of "special concern." Nationally, numerous freshwater mussel species are considered threatened. Freshwater mussel diseases are not well understood and few published accounts of freshwater mussel diseases with detailed histological descriptions exist. Mass mortality events within mussel populations are increasingly recognized, often with undetermined etiology. Our objective was to determine baseline histopathology in free-living populations of freshwater mussels. One-hundred twenty individual mussels representing 2 species-plain pocketbook (Lampsilis cardium) and fatmucket (Lampsilis siliquoidea)-were collected from 3 different locations within the Wildcat Creek watershed in central Indiana during June and July 2019. A cross-section through the visceral mass was obtained and immersed in 10% neutral-buffered formalin, with routine processing and hematoxylin and eosin staining. Branchial acariasis occurred in 43/60 fatmuckets and 22/60 plain pocketbooks. Infection with a bucephalid trematode was recognized in 18/60 fatmuckets, while infection of the gonadal duct with an unidentified trematode species was identified in 4/60 fatmuckets and 18/59 plain pocketbooks. Additional changes associated with unidentified trematodes, bacteria, fungi or oomycetes, and ciliates were observed. Other miscellaneous changes included mineralization, neuronal lipofuscinosis, and gonadal atrophy/atresia. A range of histological changes were observed. These changes likely represented background lesions: incidental findings, spontaneous infectious or endosymbiotic conditions, or normal physiological changes that routinely occur in free-living wild populations. Awareness of baseline lesions should inform future diagnostic investigations of mussel mortality events.

Monocytic Myeloid-Derived Suppressor Cells from Tumor Tissue Are a Differentiated Cell with Limited Fate Plasticity.

Owing to ease of access and high yield, most murine myeloid-derived suppressor cell (MDSC) knowledge comes from the study of spleen-derived MDSCs rather than those isolated from the tumor. Although several studies have identified subtle differences in suppressive function between these MDSCs, a recent report demonstrated that the whole peripheral myeloid compartment poorly reflects myeloid populations found at the tumor. We confirm and extend these observations by presenting data that indicate extensive differences exist between peripheral and tumor MDSCs, suggesting that it may be inappropriate to use spleen MDSCs as surrogates for studying tumor MDSCs. Using cytospins, we observed that tumor MDSCs have undergone a morphologic shift from immature myeloid cell forms commonly seen in bone marrow (BM) and spleen MDSCs and acquired mature myeloid cell characteristics. Spleen and BM monocyte-like MDSCs (M-MDSCs) readily responded to differentiation signals for multiple myeloid cell types whereas tumor M-MDSCs had remarkably reduced cellular plasticity. At the time of isolation, M-MDSCs from BM or spleen have little to no T cell suppressive activity whereas those from the tumor possess immediate and efficient T cell suppressive function. Finally, microarray analysis revealed that the transcriptomes of tumor and spleen M-MDSCs possessed >4500 differentially expressed transcripts. We conclude that tumor M-MDSCs are more differentiated and mature, and that they are morphologically, genetically, and functionally distinct from spleen and BM M-MDSCs. These observations have important implications for the design of anti-MDSC therapies and suggest that preclinical studies using nontumor MDSCs could lead to results not applicable to tumor MDSCs.

Investigation of a common canine factor VII deficiency variant in dogs with unexplained bleeding on autopsy.

The factor VII (FVII) protein is an integral component of the extrinsic coagulation pathway. Deleterious variants in the gene encoding this protein can result in factor VII deficiency (FVIID), a bleeding disorder characterized by abnormal (slowed) clotting with a wide range of severity, from asymptomatic to life-threatening. In canids, a single FVIID-associated variant, first described in Beagles, has been observed in 24 breeds and mixed-breed dogs. Because this variant is present in breeds of diverse backgrounds, we hypothesized that it could be a contributing factor to unexplained bleeding observed in some canine autopsy cases. DNA was extracted from paraffin-embedded tissue samples from 67 anticoagulant-negative autopsy cases with unexplained etiology for gross lesions of hemorrhage. Each dog was genotyped for the c.407G>A (F71) variant. Experimental controls included 3 known heterozygotes and 2 known homozygotes for the F71 variant, 2 normal dogs with known homozygous wild-type genotypes (F7WF7W), and 5 dogs with bleeding at autopsy that tested positive for anticoagulant rodenticide and were genotyped as F7WF7W. All 67 cases tested homozygous for the wild-type allele, indicating that the common FVIID variant was not responsible for the observed unexplained bleeding. Our work demonstrates the usefulness of retrospective studies utilizing veterinary diagnostic laboratory databases and tissue archives for genetic studies. In the case of FVIID, our results suggest that a singular molecular test for the F71 variant is not a high-yield addition to postmortem screening in these scenarios.

Atrazine intoxication in cattle, confirmed by gas chromatography-mass spectrometry.

Ten of 40 cows died within 48 h of gaining access to a barn in which various chemicals were stored. Some of the surviving cows exhibited drooling, muscle tremors, and agitation. Postmortem examinations of 2 cows were performed in the field, and revealed nonspecific, moderate-to-severe pulmonary congestion. Liver and rumen contents, each from a different cow, were analyzed using a qualitative, multi-residue GC-MS method validated for the detection of pesticides and other chemical analytes. Using this method, extracts from the liver and rumen content samples were compared to atrazine (neat standard) and matrix-matched, control samples fortified with atrazine. GC-MS analysis detected atrazine at 215 m/z (NIST match >97%) with a retention time of ~13 min in liver and rumen content samples from our case. Detection of atrazine in the samples from the cows in this herd, combined with the clinical history, indicate that atrazine toxicity was the likely cause of clinical signs and death observed in this herd.

Chronic copper toxicosis in a crossbred heifer calf.

An 8-mo-old, crossbred, heifer calf was presented to the Heeke Animal Disease Diagnostic Laboratory with a history of ataxia and altered mentation. Grossly, the liver was diffusely yellow-orange, turgid, and exuded watery, thin blood on cut section. The cortex and medulla in both kidneys were diffusely and markedly dark brown to black. The urinary bladder was filled with dark red urine. Histologically, centrilobular hepatocellular degeneration was observed, but these sections lacked necrosis. In the kidney, numerous cortical tubules contained intraluminal bright eosinophilic fluid and red-orange granular casts that stained positive for hemoglobin with the Dunn-Thompson method. The gross and histologic lesions supported a high level of suspicion for copper toxicosis. Feed and water samples from the farm were submitted for mineral analysis. The copper concentration in the feed was 118 mg/kg, and the molybdenum concentration was 0.9 mg/kg. Chronic copper toxicosis is rarely reported in cattle. The gross lesions in our case are a departure from, although similar to, previously reported cases, including lack of histologic hepatocellular necrosis. Collectively, gross and histologic lesions were compatible with copper toxicosis in this calf, and copper concentrations in the feed samples suggest a feed-mixing error.

Overexpression of wbkF gene in Brucella abortus RB51WboA leads to increased O-polysaccharide expression and enhanced vaccine efficacy against B. abortus 2308, B. melitensis 16M, and B. suis 1330 in a murine brucellosis model.

Brucella abortus RB51 is an attenuated, stable, spontaneous rough mutant derived in the laboratory from the virulent strain B. abortus 2308. Previous studies discovered that the wboA gene, which encodes a glycosyltransferase required for synthesis of the O-polysaccharide, is disrupted in strain RB51 by an IS711 element. However, complementation of strain RB51 with a functional wboA gene (strain RB51WboA) does not confer it a smooth phenotype but results in low levels of cytoplasmic O-polysaccharide synthesis. In this study, we asked if increasing the potential availability of bactoprenol priming precursors in strain RB51WboA would increase the levels of O-polysaccharide synthesis and enhance the protective efficacy against virulent Brucella challenge. To achieve this, we overexpressed the wbkF gene, which encodes a putative undecaprenyl-glycosyltransferase involved in bactoprenol priming for O-polysaccharide polymerization, in strain RB51WboA to generate strain RB51WboAKF. In comparison to strain RB51WboA, strain RB51WboAKF expressed higher levels of O-polysaccharide, but was still attenuated and remained phenotypically rough. Mice immunized with strain RB51WboAKF developed increased levels of smooth LPS-specific serum antibodies, primarily of IgG2a and IgG3 isotype. Splenocytes from mice vaccinated with strain RB51WboAKF secreted higher levels of antigen-specific IFN-γ and TNF-α and contained more numbers of antigen-specific IFN-γ secreting CD4+ and CD8+ T lymphocytes when compared to those of the RB51 or RB51WboA vaccinated groups. Immunization with strain RB51WboAKF conferred enhanced protection against virulent B. abortus 2308, B. melitensis 16M and B. suis 1330 challenge when compared to the currently used vaccine strains. Our results suggest that strain RB51WboAKF has the potential to be a more efficacious vaccine than its parent strain in natural hosts.

Anaplasma ovis as the suspected cause of mortality in a neonatal elk calf.

Anaplasma ovis infection is known to occur in elk experimentally, but without clinical signs or significant clinicopathologic changes. An elk farm in southern Indiana experienced the death of 3 neonates. Gross findings suggested hemolytic anemia as the cause of death. Splenic impression smears revealed numerous intra-erythrocytic parasites compatible with Anaplasma spp. Products of a semi-nested PCR targeting the msp4 gene of A. ovis were sequenced and had 100% identity with published A. ovis sequences. Given the clinical presentation, vertical transmission of A. ovis was suspected. Pathologic and molecular findings confirmed that natural A. ovis infection occurred in an elk calf.

Clinical and Pathologic Characterization of an Outbreak of Highly Pathogenic Avian Influenza H7N8 in Commercial Turkeys in Southern Indiana.

Highly pathogenic avian influenza (HPAI) is a systemic lethal disease of poultry caused by several subtypes of influenza A virus and classified on the basis of serologic reactions to hemagglutinin and neuraminidase surface glycoproteins. In January 2016, a novel subtype of HPAI-H7N8-was diagnosed in a commercial turkey (Meleagris gallopavo) flock in southern Indiana. Clinical signs and history included increased mortality, dyspnea, head tremors, recumbency, and somnolent or unaware birds. Postmortem examination of six recently dead birds showed red-tinged mucous in the choana and trachea and marked pulmonary edema. Histologic lesions in the brain included severe, multifocal lymphohistiocytic meningoencephalitis with foci of malacia, neuronal necrosis, and neuronophagia. All anatomic locations of the brain were affected, although histologic changes in the cerebellum were considered mild. Other histologic lesions included pulmonary congestion and edema, splenic congestion and lymphoid depletion, fibrinoid necrosis of vessels within the spleen, and multifocal pancreatic acinar necrosis. Immunohistochemistry (IHC) was weakly positive for influenza A in the brain; IHC was negative in other tissues tested. The clinical and pathologic characteristics of this case matched previously published material concerning HPAI and add to instances of known or suspected mutation of a low pathogenic virus to a highly pathogenic virus.

Cholesterol Sulfonation Enzyme, SULT2B1b, Modulates AR and Cell Growth Properties in Prostate Cancer.

UNLABELLED: Cholesterol accumulates in prostate lesions and has been linked to prostate cancer incidence and progression. However, how accumulated cholesterol contributes to prostate cancer development and progression is not completely understood. Cholesterol sulfate (CS), the primary sulfonation product of cholesterol sulfotransferase (SULT2B1b), accumulates in human prostate adenocarcinoma and precancerous prostatic intraepithelial neoplasia (PIN) lesions compared with normal regions of the same tissue sample. Given the enhanced accumulation of CS in these lesions, it was hypothesized that SULT2B1b-mediated production of CS provides a growth advantage to these cells. To address this, prostate cancer cells with RNAi-mediated knockdown (KD) of SULT2B1b were used to assess the impact on cell growth and survival. SULT2B1b is expressed and functional in a variety of prostate cells, and the data demonstrate that SULT2B1b KD, in LNCaP and other androgen-responsive (VCaP and C4-2) cells, results in decreased cell growth/viability and induces cell death. SULT2B1b KD also decreases androgen receptor (AR) activity and expression at mRNA and protein levels. While AR overexpression has no impact on SULT2B1b KD-mediated cell death, the addition of exogenous androgen is able to partially rescue the growth inhibition induced by SULT2B1b KD in LNCaP cells. These results suggest that SULT2B1b positively regulates the AR either through alterations in ligand availability or by interaction with critical coregulators that influence AR activity. IMPLICATIONS: These findings provide evidence that SULT2B1b is a novel regulator of AR activity and cell growth in prostate cancer and should be further investigated for therapeutic potential. Mol Cancer Res; 14(9); 776-86. ©2016 AACR.

Characterization of autoimmune inflammation induced prostate stem cell expansion.

BACKGROUND: The presence of inflammation in prostate cancer (PCa) and benign prostate hyperplasia (BPH) has been well described but the cellular mechanisms by which inflammation modulates the prostate are currently unclear. Prostate stem cells (PSC) not only maintain prostate homeostasis but also are considered to be the cell of origin of PCa and an important contributor to BPH. However, the impact of inflammation on PSC is not well understood. Therefore, we initiated studies to evaluate the effect of inflammation on PSC. METHOD: Ovalbumin specific CD8(+) T cells were intravenously delivered to intact and castrated prostate ovalbumin expressing transgenic-3 (POET-3) mice to induce inflammation. Lin (CD45/CD31)(-) Sca1(+) CD49f(+) cells (LSC) and progenitor cells within LSC were determined by flow cytometry. Sorted LSC were subjected to a prostate sphere forming assay to evaluate PSC clonal propagation, proliferation, immediate differentiation, and self-renewal ability. Density of individual spheres was measured by a cantilever-based resonator weighing system. Morphology and characterization of prostate spheres was determined by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). Finally, immediate PSC differentiation in sphere formation was determined by immunofluorescence for epithelial cytokeratin markers cytokeratin (CK) 5 and CK8. RESULT: Data presented here demonstrate a significant expansion of the proliferative (BrdU(+) ) LSC population, including CK5(+) , p63(+) , CK18(+) cells, as well as intermediate cells (CK5(+) /CK8(+) ) in inflamed prostates. Histological images reveal that PSC from inflamed prostates produce significantly larger spheres, indicating that the enhanced proliferation observed in LSC is sustained in vitro in the absence of inflammatory mediators. In addition, cultures from inflamed PSC yielded increased number of tubule-like spheres. These tube-like spheres grown from PSCs isolated from inflamed mice exhibited stratification of a CK8(+) luminal-like layer and a CK5(+) basal-like layer. Notably, the numbers of spheres formed by inflamed and non-inflamed PSC were equal, suggesting that even though proliferation is enhanced by inflammation, the homeostatic level of PSC is maintained. CONCLUSION: Induction of inflammation promotes PSC expansion and immediate differentiation through highly proliferative progenitor cells while the homeostasis of PSC is maintained.

Impact of prostate inflammation on lesion development in the POET3(+)Pten(+/-) mouse model of prostate carcinogenesis.

Evidence linking prostatitis and prostate cancer development is contradictory. To study this link, the POET3 mouse, an inducible model of prostatitis, was crossed with a Pten-loss model of prostate cancer (Pten(+/-)) containing the ROSA26 luciferase allele to monitor prostate size. Prostatitis was induced, and prostate bioluminescence was tracked over 12 months, with lesion development, inflammation, and cytokine expression analyzed at 4, 8, and 12 months and compared with mice without induction of prostatitis. Acute prostatitis led to more proliferative epithelium and enhanced bioluminescence. However, 4 months after initiation of prostatitis, mice with induced inflammation had lower grade pre-neoplastic lesions. A trend existed toward greater development of carcinoma 12 months after induction of inflammation, including one of two mice with carcinoma developing perineural invasion. Two of 18 mice at the later time points developed lesions with similarities to proliferative inflammatory atrophy, including one mouse with associated carcinoma. Pten(+/-) mice developed spontaneous inflammation, and prostatitis was similar among groups of mice at 8 and 12 months. Analyzed as one cohort, lesion number and grade were positively correlated with prostatitis. Specifically, amounts of CD11b(+)Gr1(+) cells were correlated with lesion development. These results support the hypothesis that myeloid-based inflammation is associated with lesion development in the murine prostate, and previous bouts of CD8-driven prostatitis may promote invasion in the Pten(+/-) model of cancer.

Characterization and treatment of transitional cell carcinoma of the abdominal wall in dogs: 24 cases (1985-2010).

OBJECTIVE: To determine uroplakin III expression, potential etiologic factors, biological behavior, and treatment response of transitional cell carcinoma (TCC) in the abdominal wall (ABWTCC) in dogs. DESIGN: Retrospective case series. ANIMALS: 24 dogs with TCC of the urinary tract that also had histopathologic confirmation of ABWTCC. PROCEDURES: Medical records, histologic slides, radiographs, and ultrasonographic images of dogs with ABWTCC between July 1, 1985, and December 31, 2010, were reviewed. In available tissue specimens, immunohistochemistry was used to detect uroplakin III expression in the ABWTCC and in the primary tumor. RESULTS: The ABWTCC lesions ranged from < 2 to > 20 cm in diameter. Uroplakin III was expressed in 19 of 20 primary tumors and 17 of 17 ABWTCCs. Transitional cell carcinoma in the abdominal wall developed significantly more often in dogs that had undergone cystotomy (18/177 [10.2%]) than in those that had not (6/367 [1.6%]). In 1 dog that had not undergone cystotomy, TCC had invaded through the urinary bladder wall and spread down the median ligament to the abdominal wall. None of 18 dogs that received anticancer drugs had remission of the ABWTCC once clinically detected; median survival time after ABWTCC detection was 57 days (range, 0 to 324 days). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that ABWTCC is uncommon, but once TCC becomes established and clinically detectable in the abdominal wall, it carries a poor prognosis. It is crucial to minimize risk of TCC seeding at surgery. Percutaneous sampling of TCC should be avoided. Uroplakin III is commonly expressed in ABWTCC.

Myocardial fibrosis associated with previous ingestion of yew (Taxus sp.) in a Holstein heifer: evidence for chronic yew toxicity in cattle.

Twenty-six 5-month-old Holstein calves were accidentally exposed to discarded branches of yew bushes (Taxus sp.). Several calves were found dead approximately 24 hr after exposure; however, a few calves died several days after exposure. One calf died 18 days after the initial exposure to Taxus sp. and was examined on the farm via necropsy. Gross lesions included ascites, and dilated and flaccid myocardial ventricles. Sections of formalin-fixed heart were submitted to the Indiana Animal Disease Diagnostic Laboratory for histopathologic examination; fresh rumen contents were submitted for toxicologic testing. Histologically, large areas of myocardium were replaced by fibrous connective tissue, suggesting previous myocardial necrosis. Taxus alkaloids were identified in the rumen contents using gas chromatography-mass spectrometry. Based on the clinical history, the gross and histologic lesions, the identification of Taxus alkaloids in the rumen contents, and lack of exposure to other known cardiotoxic agents, yew toxicity was considered the cause of death in this calf. Ingestion of taxines is known to cause acute and subacute toxicity in human beings and animals; however, a chronic clinical course and severe histologic lesions have not been previously associated with yew toxicity. Although only 1 calf was examined, this case suggests that yew toxicity can result in a prolonged clinical course in cattle and can cause histologic myocardial lesions.

Development of a gas chromatography-mass spectrometry technique to diagnose white snakeroot (Ageratina altissima) poisoning in a cow.

An 8-year-old, crossbred beef cow was referred to the Indiana Animal Disease Diagnostic Laboratory at Purdue University for a complete necropsy in October 2009. The cow was the sixth to die in a 7-day period. Affected cows were reportedly stumbling and became weak, excitable, and recumbent. Histologically, myonecrosis was severe in the skeletal muscles and mild in the heart and tongue. According to the submitter, exposure to a poisonous plant was suspected, and a plant specimen received from this case was identified as white snakeroot (Ageratina altissima). Using the white snakeroot specimen, a gas chromatography-mass spectrometry analytical method for the detection of tremetone and dehydrotremetone (2 components of white snakeroot) was developed. Both tremetone and dehydrotremetone were detected in the plant specimen. Dehydrotremetone was recovered from the liver, while neither component was recovered in the rumen content. In the past, because of the lack of standard reference material, the diagnosis of white snakeroot poisoning was based mainly on history of exposure and the presence of the plant in the rumen. The analytical method described herein can be used to document exposure to tremetone or dehydrotremetone in cases of suspected white snakeroot poisoning when coupled with the appropriate clinical signs and lesions.

Imaging diagnosis-magnetic resonance imaging features of metastatic cerebral lymphoma in a dog.

We describe histopathologically confirmed intracranial metastasis of cutaneous lymphoma. In magnetic resonance (MR) images there was a heterogeneous, contrast-enhancing, extraaxial mass in the right parietal and piriform lobes at the level of the optic chiasm. Our MR imaging findings are consistent with reports in humans in that lymphoma masses have indistinct borders that are iso- to hyperintense relative to adjacent gray matter on T2-weighted images. Our report varies from findings in humans in that the mass was extraaxial, whereas masses reported in humans are intraaxial. Contrast enhancement can be heterogeneous, as in our report, or homogeneous.