Role of Pre-Farrow Natural Planned Exposure of Gilts in Shaping the Passive Antibody Response to Rotavirus A in Piglets.

Natural planned exposure (NPE) remains one of the most common methods in swine herds to boost lactogenic immunity against rotaviruses. However, the efficacy of NPE protocols in generating lactogenic immunity has not been investigated before. A longitudinal study was conducted to investigate the dynamics of genotype-specific antibody responses to different doses (3, 2 and 1) of Rotavirus A (RVA) NPE (genotypes G4, G5, P[7] and P[23]) in gilts and the transfer of lactogenic immunity to their piglets. Group 1 gilts received three doses of NPE at 5, 4 and 3 weeks pre-farrow (WPF), group 2 received two doses at 5 and 3 WPF, group 3 received one dose at 5 WPF, and group 4 received no NPE (control group). VP7 (G4 and G5) and truncated VP4* (P[7] and P[23]) antigens of RVA were expressed in mammalian and bacterial expression systems, respectively, and used to optimize indirect ELISAs to determine antibody levels against RVA in gilts and piglets. In day-0 colostrum samples, group 1 had significantly higher IgG titers compared to the control group for all four antigens, and either significantly or numerically higher IgG titers than groups 2 and 3. Group 1 also had significantly higher colostrum IgA levels than the control group for all antigens (except G4), and either significantly or numerically higher IgA levels compared to groups 2 and 3. In piglet serum, group 1 piglets had higher IgG titers for all four antigens at day 0 than the other groups. Importantly, RVA NPE stimulated antibodies in all groups regardless of the treatment doses and prevented G4, G5, P[7] and P[23] RVA fecal shedding prior to weaning in piglets in the absence of viral challenge. The G11 and P[34] RVA genotypes detected from pre-weaning piglets differed at multiple amino acid positions with parent NPE strains. In conclusion, the results of this study suggest that the group 1 NPE regimen (three doses of NPE) resulted in the highest anti-RVA antibody (IgG and IgA) levels in the colostrum/milk, and the highest IgG levels in piglet serum.

Determination of optimal storage time and temperature for the detection of red blood cell and platelet surface-associated immunoglobulin by flow cytometry in healthy horses.

Differentiating immune-mediated causes from other causes of anemia and thrombocytopenia can be challenging. Flow cytometry can detect surface-associated immunoglobulin (sIg) on red blood cells (RBC) and platelets (PLT) in dogs and horses. Sample storage parameters for ideal assay performance has not been evaluated in horses. The study objective is to identify optimal storage time and temperature of equine whole blood for the detection of RBC-sIg and PLT-sIg via flow cytometry. Both assays were performed on samples at time 0, 4, 24, 48, and 72 h post collection. RBC-sIg samples were stored at 4 °C and PLT-sIg samples were stored at 4 °C and room temperature. RBC-sIg percentages were stable up to 72 h storage. Platelet surface-associated IgG percent positive platelets increased above baseline at all timepoints and percent positive platelets were inconsistent across timepoints for IgM and IgA. PLT-sIg testing should ideally be performed within 4 h of collection. In instances where this is not feasible, samples should be stored at 4 °C and analyzed no later than 24 h after collection. Whereas cutoff values for RBC-sIg remained similar across timepoints, results for PLT-sIg should be compared to time-specific cutoff or reference intervals established by the laboratory running the test.

Multiple myeloma presenting as blepharitis in a horse.

Myeloma-related disorders, including multiple myeloma, extramedullary plasmacytoma, and solid osseous plasmacytoma, are rare in horses. Clinical complaints for myeloma-related disorders are nonspecific, and when present, M-protein location is more variable on protein electrophoresis in horses relative to dogs and cats. Here, we describe a case of a 15-year-old Thoroughbred mare who presented with recurrent blepharitis. Marked hyperglobulinemia was an incidental finding on routine hematologic and biochemical testing. Bone marrow aspiration consisted of >30% plasma cells, and serum protein electrophoresis demonstrated a monoclonal gammopathy in the alpha 2 fraction leading to a diagnosis of multiple myeloma. Immunofixation and radial immunodiffusion confirmed the presence of an IgG M-protein. Based on a restricted peak in the alpha 2 location, the specific M-protein is suspected to be IgG(T), an IgG isotype unique to horses. M-protein migration in horses is variable relative to dogs and cats, yet immunofixation can still be used to identify equine IgG M-protein isotypes. The unique clinical presentation in this case also serves as a reminder to consider neoplasia in horses with unusual or nonspecific clinical signs.

Concurrent chronic lymphocytic leukemia and primary hyperparathyroidism in a mule.

A 26-year-old mule gelding was evaluated for chronic weight loss and decreased appetite. The mule had been losing weight and intermittently hypophagic for approximately 7 months. Laboratory analysis of whole blood and plasma identified severe total hypercalcemia, marked hypophosphatemia, markedly increased parathyroid hormone concentration, and marked lymphocytosis. A sestimibi scan intended to identify parathyroid gland tissue was nondiagnostic. Results of flow cytometry and PCR for antigen receptor rearrangement (PARR) were consistent with a B cell lymphoproliferative disorder, likely chronic lymphocytic leukemia (CLL). Although not previously described concurrently, these conditions may sometimes arise together, complicating definition of the underlying mechanism for weight loss and hypercalcemia in aged equids.

Serum biochemical changes in cats with naturally acquired feline cytauxzoonosis.

OBJECTIVE: The geographical distribution of feline cytauxzoonosis is expanding in the US. Clinical signs of feline cytauxzoonosis, including lethargy, anorexia, and icterus, are similar to hepatic lipidosis and cholangiohepatitis. Hematologic and serum biochemical abnormality patterns may assist practitioners in prioritizing feline cytauxzoonosis as a differential diagnosis over hepatic lipidosis and cholangiohepatitis. SAMPLE: Hematology and serum biochemical profiles of cats with naturally acquired feline cytauxzoonosis, hepatic lipidosis, or cholangiohepatitis. PROCEDURES: Retrospective search and analysis of the Kansas State Veterinary Diagnostic Laboratory or Kansas State University Veterinary Health Center records between January 2007 and June 2018 for cats with cytauxzoonosis, hepatic lipidosis, or cholangiohepatitis. RESULTS: Patients with acute feline cytauxzoonosis presented with frequent nonregenerative anemia (20/28 [71%]), leukopenia (23/28 [82%]), thrombocytopenia (23/23 [100%]), hyperbilirubinemia (27/28 [97%]), hypoalbuminemia (26/28 [93%]), reduced (18/28 [64%]) or low normal (10/28 [36%]) serum ALP activity, and hyponatremia (23/28 [82%]). Reduced ALP activity was unique to cats with feline cytauxzoonosis relative to hepatic lipidosis and cholangiohepatitis. No correlation between the severity of anemia and the magnitude of hyperbilirubinemia was identified in feline cytauxzoonosis patients. CLINICAL RELEVANCE: The combination of nonregenerative anemia, leukopenia, thrombocytopenia, hyperbilirubinemia, and reduced serum ALP activity in icteric cats may increase the clinical suspicion, but is not pathognomonic, for acute feline cytauxzoonosis. Hematologic and serum biochemical abnormalities of naturally acquired feline cytauxzoonosis are like those reported with feline bacterial sepsis. Blood smear evaluation for intraerythrocytic Cytauxzoon felis piroplasms, tissue aspirates for schizont-laden macrophages, and/or molecular testing are required to diagnose feline cytauxzoonosis.

Antibody Response to Rotavirus C Pre-Farrow Natural Planned Exposure to Gilts and Their Piglets.

A longitudinal study was conducted to investigate the dynamics of genotype-specific (G6 and P[5]) antibody response to different doses (3, 2 and 1) of rotavirus C (RVC) natural planned exposure (NPE) in gilt serum, colostrum/milk and piglet serum, and compare with antibody response to rotavirus A NPE (RVA genotypes G4, G5, P[7] and P[23]). G6 and P[5] antigens of RVC were expressed in mammalian and bacterial cells, and used to develop individual indirect ELISAs. For both antigens, group 1 with 3 doses of NPE resulted in significantly higher IgG and IgA levels in colostrum compared to other groups. In piglet serum, group 1 P[5] IgG levels were significantly higher than other study groups at day 0 and 7. Piglet serum had higher IgA levels for group 1 piglets compared to other groups for both antigens. A comparison of colostrum antibody levels to rotavirus A (RVA) and RVC revealed that colostrum RVC IgG and IgA titers were lower than RVA titers irrespective of the G and P-type. Next generation sequencing (NGS) detected same RVC genotypes (G6 and P[5]) circulating in the piglet population under the window of lactogenic immunity. We conclude that the low RVC load in NPE material (real-time PCR Ct-values 32.55, 29.32 and 30.30) failed to induce sufficient maternal immunity in gilts (low colostrum RVC antibody levels) and passively prevent piglets from natural RVC infection in the farrowing room. To the best of our knowledge, this is the first study comparing differences in antibody response to porcine RVA and RVC in a commercial setting.

Rotavirus Infection in Swine: Genotypic Diversity, Immune Responses, and Role of Gut Microbiome in Rotavirus Immunity.

Rotaviruses (RVs) are endemic in swine populations, and all swine herds certainly have a history of RV infection and circulation. Rotavirus A (RVA) and C (RVC) are the most common among all RV species reported in swine. RVA was considered most prevalent and pathogenic in swine; however, RVC has been emerging as a significant cause of enteritis in newborn piglets. RV eradication from swine herds is not practically achievable, hence producers' mainly focus on minimizing the production impact of RV infections by reducing mortality and diarrhea. Since no intra-uterine passage of immunoglobulins occur in swine during gestation, newborn piglets are highly susceptible to RV infection at birth. Boosting lactogenic immunity in gilts by using vaccines and natural planned exposure (NPE) is currently the only way to prevent RV infections in piglets. RVs are highly diverse and multiple RV species have been reported from swine, which also contributes to the difficulties in preventing RV diarrhea in swine herds. Human RV-gut microbiome studies support a link between microbiome composition and oral RV immunogenicity. Such information is completely lacking for RVs in swine. It is not known how RV infection affects the functionality or structure of gut microbiome in swine. In this review, we provide a detailed overview of genotypic diversity of swine RVs, host-ranges, innate and adaptive immune responses to RVs, homotypic and heterotypic immunity to RVs, current methods used for RV management in swine herds, role of maternal immunity in piglet protection, and prospects of investigating swine gut microbiota in providing immunity against rotaviruses.

The Utility of Bile Acids for the Diagnosis of Liver Disease in Exotic Animals.

The synthesis of bile acids occurs during the degradation of cholesterol in hepatocytes. Thus, this analyte is expected to be a sensitive indicator of hepatocellular dysfunction or alterations in portal circulation. Bile acids can be quantified via an enzymatic reaction to a highly conserved moiety across species. The evidence for the clinical utility of bile acids for the diagnosis of liver disease is strongest in birds and ferrets with equivocal evidence in rodents, rabbits, and reptiles. Current limitations to the interpretation of bile acids in exotic animal species include a paucity of species-specific reference intervals and incomplete understanding of bile acid metabolism in nonmammalian species and the diversity of bile acids synthesized by vertebrates.

Elective ovariohysterectomy or castration can proceed in shelter dogs despite mild to moderate presurgical leukocytosis.

OBJECTIVE: To determine whether shelter dogs presenting for elective ovariohysterectomy or castration have leukocytosis, whether leukocytes are associated with age and infection, and whether leukocytosis precludes progression to surgery. ANIMALS: 138 dogs (from 13 regional shelters) presented for ovariohysterectomy or castration between October 7 and December 6, 2019. PROCEDURES: For this prospective study, each dog underwent presurgical physical examination, CBC, and tests for Dirofilaria immitis antigen and Anaplasma phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies, with additional tests performed as needed. Dogs were aged by dentition as juvenile (< 3 or ≥ 3 to ≤ 6 months) or adult (> 6 months). Leukogram results were compared across age groups with recognized infections and parasitism and with dogs' progression to surgery. RESULTS: There were 34 dogs < 3 months old, 22 dogs ≥ 3 to ≤ 6 months old, and 82 > 6 months old. Sixty-three of 138 (45.6%) dogs had leukocytosis (median, 16,500 cells/µL; range, 13,700 to 28,300 cells/µL). Dogs < 3 months of age had higher median leukocyte and lymphocyte counts (14,550 cells/µL and 3,700 cells/µL, respectively) than dogs > 6 months of age (12,500 cells/µL and 2,400 cells/µL, respectively). Only 1 dog had a stress leukogram. Forty-seven dogs had recognized infection, but there was no association with leukocytosis. Surgery proceeded successfully for all dogs with leukocytosis. CLINICAL RELEVANCE: Mild to moderate leukocytosis is common before elective surgery in shelter dogs, but surgery can proceed safely. A CBC should be reserved for ill-appearing dogs rather than as a screening test, and age-specific reference intervals should be considered.

Feline sarcomatoid renal cell carcinoma with peritoneal carcinomatosis and effusion.

A 9-y-old, castrated male, domestic medium-hair cat diagnosed previously with chronic kidney disease developed anorexia and vomiting. Ultrasonography revealed abdominal effusion and a left renal perihilar mass. Cytologic evaluation of the peritoneal fluid and mass identified atypical epithelioid cells suspected to be of renal epithelial or possible mesothelial origin. Immunohistochemical (IHC) evaluation of a formalin-fixed, paraffin-embedded peritoneal fluid cell block indicated both pancytokeratin and vimentin expression in the atypical epithelioid cell population. With scanning electron microscopic evaluation, similar epithelioid cells lacked the cell-surface microvilli expected of mesothelium, supporting an antemortem diagnosis of probable carcinoma. On postmortem examination, the left kidney was effaced by an infiltrative neoplasm with myriad similar nodules throughout the peritoneum. The neoplasm was composed primarily of polygonal-to-spindle-shaped cells with strong vimentin and weak pancytokeratin cytoplasmic immunolabeling. Further IHC characterization with PAX8, CK18, KIT, napsin A, SMA, desmin, CD18, and claudin 5 was performed. Histologic and IHC findings supported a diagnosis of sarcomatoid renal cell carcinoma with peritoneal carcinomatosis. An in vitro cell culture line of neoplastic cells harvested from the primary tumor was successfully established for future research endeavors.

Comparisons of hematologic results for juvenile versus adult shelter dogs presented for ovariohysterectomy or castration.

OBJECTIVE: To compare hematologic results for juvenile versus adult dogs from shelters that outwardly appeared healthy and were presented for ovariohysterectomy or castration. ANIMALS: 138 dogs from 13 regional shelters. PROCEDURES: Each dog underwent a physical examination (including use of a flea comb), age estimation by dental eruption characteristics, PCV, CBC, and tests for Dirofilaria immitis antigen and Anaplasma phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies. Additional diagnostic tests were performed as needed. Dogs were grouped by age as < 3, ≥ 3 to ≤ 6, or > 6 months of age, with dogs ≤ 6 months of age considered juveniles and dogs > 6 months of age considered adults. Hematologic results were compared across groups. RESULTS: There were 138 dogs, of which 56 were juveniles (34 dogs < 3 months old; 22 dogs ≥ 3 to ≤ 6 months old) and 82 were adults. Juvenile (vs adult) dogs had lower mean calculated Hct and mean PCV whether dogs with infectious agents or parasites were included or excluded. The mean PCV and mean cell hemoglobin concentration were lower and the reticulocyte count higher for juvenile dogs < 3 months old (35.8%, 33.1 g/dL, and 135,000 reticulocytes/µL) versus adults (44.9%, 34.7 g/dL, and 68,500 reticulocytes/µL). Most (98.6%) dogs underwent surgery as scheduled; 2 dogs had surgery postponed because of thrombocytopenia or parvovirus infection. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings indicated that outwardly healthy-appearing juvenile shelter dogs often have results for PCV and calculated Hct that are lower than those for adult shelter dogs and adult dog reference intervals but rarely require postponement of ovariohysterectomy or castration.

CD4 and CD8 double-negative immunophenotype of thymoma-associated lymphocytes in a dog.

Persistent small-cell lymphocytosis in dogs with a concurrent mediastinal mass has been associated with both thymoma and small-cell lymphoma. In thymomas, neoplastic thymic epithelial cells induce overproduction and release of polyclonal lymphocytes, whereas thymic lymphoma results in thymic effacement by a clonal expansion of neoplastic lymphocytes and subsequent leukemic phase of lymphoma. Flow cytometry has been used to differentiate these 2 entities by immunophenotyping mediastinal mass aspirates. It has been reported that cases with mediastinal masses in which ≥ 10% of the associated small-cell lymphocytes were double positive for CD4 and CD8 were thymomas, whereas masses associated with < 10% were suggestive of lymphoma. We report a unique case of thymoma-associated lymphocytosis lacking the classic CD4+CD8+ immunophenotype. Our findings suggest that there may be more diversity in the thymoma-associated lymphocyte immunophenotype than has been identified previously; immunophenotyping alone might not be sufficient to differentiate thymic small-cell lymphoma from thymoma-associated lymphocytosis. In dogs with mediastinal masses and peripheral lymphocytosis, employing a variety of testing modalities to avoid misdiagnosis is prudent. These modalities include cytologic and/or histologic evaluation, immunophenotyping, and clonality assessment.

A Protocol for the Isolation, Culture, and Cryopreservation of Umbilical Cord-Derived Canine Mesenchymal Stromal Cells: Role of Cell Attachment in Long-Term Maintenance.

Mesenchymal stromal cells (MSCs) hold great promise in the field of regenerative medicine due to their ability to create a variable localized anti-inflammatory effect in injuries such as Crohn's disease and osteoarthritis or by incorporation in tissue engineered constructs. Currently, the MSC literature uses rodents for preclinical disease models. There is growing interest in using naturally occurring disease in large animals for modeling human disease. By review of the canine MSCs literature, it appears that canine MSCs can be difficult to maintain in culture for extended passages and this greatly varies between tissue sources, compared with human and rodent MSCs, and limited lifespan is an obstacle for preclinical investigation and therapeutic use. Research using canine MSCs has been focused on cells derived from bone marrow or adipose tissue, and the differences in manufacturing MSCs between laboratories are problematic due to lack of standardization. To address these issues, here, a stepwise process was used to optimize canine MSCs isolation, expansion, and cryopreservation utilizing canine umbilical cord-derived MSCs. The culture protocol utilizes coating of tissue culture surfaces that increases cellular adherence, increases colony-forming units-fibroblast efficiency, and decreases population doubling times. Canine MSCs isolated with our protocol could be maintained longer than published canine MSCs methods before senescing. Our improved cryopreservation protocols produce on average >90% viable MSCs at thaw. These methods enable master-bank and working-bank scenarios for allogeneic MSC testing in naturally occurring disease in dogs.

Regulation of proteoglycan production by varying glucose concentrations controls fiber formation in tissue engineered menisci.

Fibrillar collagens are highly prevalent in the extracellular matrix of all connective tissues and therefore commonly used as a biomaterial in tissue engineering applications. In the native environment, collagen fibers are arranged in a complex hierarchical structure that is often difficult to recreate in a tissue engineered construct. Small leucine rich proteoglycans as well as hyaluronan binding proteoglycans, aggrecan and versican, have been implicated in regulating fiber formation. In this study, we modified proteoglycan production in vitro by altering culture medium glucose concentrations (4500, 1000, 500, 250, and 125 mg/L), and evaluated its effect on the formation of collagen fibers inside tissue engineered meniscal constructs. Reduction of extracellular glucose resulted in a dose dependent decrease in total sulfated glycosaminoglycan (GAG) production, but minimal decreases of decorin and biglycan. However, fibromodulin doubled in production between 125 and 4500 mg/L glucose concentration. A peak in fiber formation was observed at 500 mg/L glucose concentration and corresponded with reductions in total GAG production. Fiber formation reduction at 125 and 250 mg/L glucose concentrations are likely due to changes in metabolic activity associated with a limited supply of glucose. These results point to proteoglycan production as a means to manipulate fiber architecture in tissue engineered constructs. STATEMENT OF SIGNIFICANCE: Fibrillar collagens are highly prevalent in the extracellular matrix of all connective tissues; however achieving appropriate assembly and organization of collagen fibers in engineered connective tissues is a persistent challenge. Proteoglycans have been implicated in regulating collagen fiber organization both in vivo and in vitro, however little is known about methods to control proteoglycan production and the subsequent fiber organization in tissue engineered menisci. Here, we show that media glucose content can be optimized to control proteoglycan production and collagen fiber assembly, with optimal collagen fiber assembly occurring at sub-physiologic levels of glucose.

Hydroxyapatite mineral enhances malignant potential in a tissue-engineered model of ductal carcinoma in situ (DCIS).

While ductal carcinoma in situ (DCIS) is known as a precursor lesion to most invasive breast carcinomas, the mechanisms underlying this transition remain enigmatic. DCIS is typically diagnosed by the mammographic detection of microcalcifications (MC). MCs consisting of non-stoichiometric hydroxyapatite (HA) mineral are frequently associated with malignant disease, yet it is unclear whether HA can actively promote malignancy. To investigate this outstanding question, we compared phenotypic outcomes of breast cancer cells cultured in control or HA-containing poly(lactide-co-glycolide) (PLG) scaffolds. Exposure to HA mineral in scaffolds increased the expression of pro-tumorigenic interleukin-8 (IL-8) among transformed but not benign cells. Notably, MCF10DCIS.com cells cultured in HA scaffolds adopted morphological changes associated with increased invasiveness and exhibited increased motility that were dependent on IL-8 signaling. Moreover, MCF10DCIS.com xenografts in HA scaffolds displayed evidence of enhanced malignant progression relative to xenografts in control scaffolds. These experimental findings were supported by a pathological analysis of clinical DCIS specimens, which correlated the presence of MCs with increased IL-8 staining and ductal proliferation. Collectively, our work suggests that HA mineral may stimulate malignancy in preinvasive DCIS cells and validate PLG scaffolds as useful tools to study cell-mineral interactions.