Molecular detection and characterization of Anaplasma platys and Ehrlichia canis in dogs from the Caribbean.

Anaplasma platys is a tick-transmitted rickettsial pathogen, which is known to be the etiologic agent for cyclic thrombocytopenia in its primary canine host. Infections with this pathogen are also reported in cats, cattle and people. Similarly, Ehrlichia canis is another tick-borne rickettsial pathogen responsible for canine monocytic ehrlichiosis and is also reported to cause infections in people. We describe infections in dogs with these two pathogens on the Caribbean island of Grenada, West Indies by detection using molecular methods. We utilized a 16S rRNA gene-based PCR assay to detect both Ehrlichia and Anaplasma species by screening 155 canine blood samples from asymptomatic dogs. We found 18.7 % of the dogs to be positive for A. platys and 16.8 % for E. canis. Samples that tested positive for A. platys were further assessed by sequence analysis targeting 16S rRNA, 23S rRNA, citrate synthase (gltA) and heat shock protein (groEL) genes. Phylogenetic analysis revealed high correlation of A. platys 16S rRNA and gltA gene sequences with the geographic origins, while 23S rRNA and groEL gene sequences clustered independent of the geographic origins. This study represents an important step in defining the widespread distribution of active rickettsial infections in Caribbean dogs with no apparent clinical signs, thus posing a high risk for canine health and to a lesser extent to humans, as most dogs in the Caribbean are free-roaming.

Initial development and preliminary evaluation of a multiplex bead assay to detect antibodies to Ehrlichia canis, Anaplasma platys, and Ehrlichia chaffeensis outer membrane peptides in naturally infected dogs from Grenada, West Indies.

Tick-borne bacteria, Ehrlichia canis, Anaplasma platys, and Ehrlichia chaffeensis are significant pathogens of dogs worldwide, and coinfections of E. canis and A. platys are common in dogs on the Caribbean islands. We developed and evaluated the performance of a multiplex bead-based assay to detect antibodies to E. canis, A. platys, and E. chaffeensis peptides in dogs from Grenada, West Indies, where E. canis and A. platys infections are endemic. Peptides from outer membrane proteins of P30 of E. canis, OMP-1X of A. platys, and P28-19/P28-14 of E. chaffeensis were coupled to magnetic beads. The multiplex peptide assay detected antibodies in dogs experimentally infected with E. canis and E. chaffeensis, but not in an A. platys experimentally infected dog. In contrast, the multiplex assay and an in-house enzyme-linked immunosorbent assay (ELISA) detected A. platys antibodies in naturally infected Grenadian dogs. Following testing of 104 Grenadian canine samples, multiplex assay results had good agreement with commercially available ELISA and immunofluorescent assay for E. canis antibody-positive dogs ( K values of 0.73 and 0.84), whereas A. platys multiplex results had poor agreement with these commercial assays ( K values of -0.02 and 0.01). Prevalence of seropositive E. canis and A. platys Grenadian dogs detected by the multiplex and commercial antibody assays were similar to previous reports. Although the multiplex peptide assay performed well in detecting the seropositive status of dogs to E. canis and had good agreement with commercial assays, better antigen targets are necessary for the antibody detection of A. platys.

Correlation of pre-veterinary admissions criteria, intra-professional curriculum measures, AVMA-COE professional competency scores, and the NAVLE.

Data consisting of preadmission criteria scores, annual and final cumulative grade point averages (GPAs), grades from individual professional courses, American Veterinary Medical Association Council on Education (AVMA-COE) Competency scores, annual class rank, and North American Veterinary Licensing Exam (NAVLE) scores were collected on all graduating DVM students at Kansas State University in 2009 and 2010. Associations among the collected data were compared by Pearson correlation. Pre-veterinary admissions criteria infrequently correlated with annual GPAs of Years 1-3, rarely correlated with the AVMA-COE Competencies, and never correlated with the annual GPA of Year 4. Low positive correlations occurred between the NAVLE and the Verbal Graduate Record Examination (GRE) (r=.214), Total GRE (r=.171), and the mean GPA of pre-professional science courses (SGPA) (r=.236). Annual GPAs strongly correlated with didactic course scores. Annual GPAs and final class rank strongly correlated (mean r=-.849), and both strongly correlated with the NAVLE score (NAVLE: GPAs mean r=.628, NAVLE: final class rank r=-.714). Annual GPAs at the end of Years 1-4 weakly correlated or did not correlate with the AVMA-COE Competencies. The AVMA-COE Competencies weakly correlated with scores earned in didactic courses of Years 1-3. AVMA-COE Competencies were internally consistent (mean r=.796) but only moderately correlated with performance on the NAVLE (mean r=.319). Low correlations between admissions criteria and outcomes indicate a need to reevaluate admission criteria as predictors of school success. If the NAVLE remains the primary discriminator for veterinary licensure (and the gateway to professional activity), then the AVMA-COE Competencies should be refined to better improve and reflect the NAVLE, or the NAVLE examination should change to reflect AVMA-COE Competencies.

Intermittent single-agent doxorubicin for the treatment of canine B-cell lymphoma.

Canine B-cell lymphoma is a highly treatable disease, but cost and logistical factors may hamper an owner's ability to pursue treatment of their pet with this disease. The authors evaluated the use of single-agent doxorubicin in an intermittent fashion for efficacy in the treatment of this disease. Morphologic and clinical data were analyzed for prognostic significance. Eighteen dogs with B-cell lymphoma, all with multicentric disease, were enrolled. The overall complete response (CR) rate was 78%, median total doxorubicin remission time (TDR) was 80.5 days, and median overall survival (OS) was 169.5 days. The median number of doxorubicin doses administered was 4.5. First remission times were significantly affected by clinical stage and substage of disease. Outcome for the dogs in this study were similar to those previously reported for single-agent doxorubicin treatment. Additionally, the intermittent nature of the treatments made the described protocol more feasible for the owners who enrolled their pets in this study. Intermittent single-agent doxorubicin is not a substitute for multiagent chemotherapy protocols in the treatment of canine lymphoma; however, it is a reasonable alternative if the cost and time commitments are limiting factors for an owner.

Erythrocyte-bound immunoglobulin isotypes in dogs with immune-mediated hemolytic anemia: 54 cases (2001-2010).

OBJECTIVE: To identify erythrocyte-bound immunoglobulin (Ig) isotypes in dogs with primary immune-mediated hemolytic anemia (IMHA). DESIGN: Retrospective case series. Animals-54 dogs with IMHA. PROCEDURES: Medical records of dogs with IMHA diagnosed between January 2001 and April 2010 were examined. Immunoglobulin isotype (tested via direct immunofluorescence by flow cytometry to identify erythrocyte-bound Ig), Hct, serum bilirubin concentration, presence of autoagglutination, degree of spherocytosis, duration of hospitalization, and 90-day outcome were recorded. RESULTS: The Hct on admission was significantly lower in dogs with IgG and IgM isotypes bound to erythrocytes, compared with dogs with a single Ig isotype, and the degree of spherocytosis was greater in dogs with IgG and IgM bound to erythrocytes, compared with dogs that only had IgM. Dogs with only IgM were not more likely to have autoagglutination, compared with dogs that only had IgG on the erythrocyte surface. Although Ig isotype was not associated with survival time, initial serum total bilirubin concentration was higher in nonsurvivors. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that dogs with IMHA with ≥ 2 Ig isotypes bound to erythrocytes, particularly IgG and IgM, are likely to have a more severe degree of anemia, spherocytosis, and autoagglutination.

Principles and applications of flow cytometry and cell sorting in companion animal medicine.

Flow cytometry measures multiple characteristic of single cells using light scatter properties and fluorescence properties of fluorescent probes with specificity to cellular constituents. The use of flow cytometry in the veterinary clinical laboratory has become more routine in veterinary diagnostic laboratories and institutions (http://www.vet.k-state.edu/depts/dmp/service/immunology/index.htm), and reference laboratories. The most common applications in small animal medicine includes quantitation of erythrocytes and leukocytes in automated hematology instruments, detection of antibodies to erythrocytes and platelets in cases of immune-mediated diseases, immunophenotyping of leukocytes and lymphocytes in immunodeficiency syndromes, or leukemias and lymphomas. DNA content analysis to identify aneuploidy or replicating cells in tumor preparations has not gained routine acceptance because of the variability of prognostic results. Other applications including cell sorting and multiplexing using microspheres are potential assays of the future once they become validated and the instrumentation footprint becomes more and more compact, less expensive, and easier to use.

The effect of differing Audience Response System question types on student attention in the veterinary medical classroom.

The purpose of this study was to evaluate the ability of specific types of multiple-choice questions delivered using an Audience Response System (ARS) to maintain student attention in a professional educational setting. Veterinary students (N=324) enrolled in the first three years of the professional curriculum were presented with four different ARS question types (knowledge base, discussion, polling, and psychological investment) and no ARS questions (control) during five lectures presented by 10 instructors in 10 core courses. Toward the end of the lecture, students were polled to determine the relative effectiveness of specific question types. Student participation was high (76.1%+/-2.0), and most students indicated that the system enhanced the lecture (64.4%). Knowledge base and discussion questions resulted in the highest student-reported attention to lecture content. Questions polling students about their experiences resulted in attention rates similar to those without use of ARS technology. Psychological investment questions, based on upcoming lecture content, detracted from student attention. Faculty preparation time for three ARS questions was shorter for knowledge base questions (22.3 min) compared with discussion and psychological investment questions (38.6 min and 34.7 min, respectively). Polling questions required less time to prepare (22.2 min) than discussion questions but were not different from other types. Faculty stated that the investment in preparation time was justified on the basis of the impact on classroom atmosphere. These findings indicate that audience response systems enhance attention and interest during lectures when used to pose questions that require application of an existing knowledge base and allow for peer interaction.

Acute megakaryoblastic leukemia in a German Shepherd dog.

An 11-year-old spayed-female German Shepherd dog was presented to the Veterinary Medical Teaching Hospital at Kansas State University with a history of weight loss, anorexia, depression, and lethargy for 2-3 weeks. Radiographic examination revealed a mass in the spleen and several round radiodense foci in the liver. CBC results included normocytic normochromic anemia, marked thrombocytopenia, and low numbers of neoplastic cells that frequently had cytoplasmic projections or blebs. A bone marrow aspirate contained about 80% neoplastic megakaryoblasts with the same microscopic features as those observed in peripheral blood. Using flow cytometry, cells of large size were identified in peripheral blood that expressed CD41/61, CD45, CD61, and CD62P (P-selectin) and were negative for markers of T cells, B cells, monocyte/macrophages, and dendritic cells. Because of the poor prognosis, euthanasia and subsequently necropsy were performed. On histopathologic examination, neoplastic megakaryoblasts were identified in spleen, liver, mesenteric lymph node, and the pulmonary vasculature. Using immunohistochemistry, the neoplastic megakaryoblasts weakly expressed von Willebrand factor. Based on microscopic and immunophenotypic findings, a diagnosis of acute megakaryoblastic leukemia (AMegL) was made. To our knowledge, this is the first report of AMegL in a domestic animal in which immunophenotyping by flow cytometry and a panel of antibodies against CD41/61, CD61, and CD62P were used to support the diagnosis.

An insight into the transcriptome and proteome of the salivary gland of the stable fly, Stomoxys calcitrans.

Adult stable flies are blood feeders, a nuisance, and mechanical vectors of veterinary diseases. To enable efficient feeding, blood sucking insects have evolved a sophisticated array of salivary compounds to disarm their host's hemostasis and inflammatory reaction. While the sialomes of several blood sucking Nematocera flies have been described, no thorough description has been made so far of any Brachycera, except for a detailed proteome analysis of a tabanid (Xu et al., 2008). In this work we provide an insight into the sialome of the muscid Stomoxys calcitrans, revealing a complex mixture of serine proteases, endonucleases, Kazal-containing peptides, anti-thrombins, antigen 5 related proteins, antimicrobial peptides, and the usual finding of mysterious secreted peptides that have no known partners, and may reflect the very fast evolution of salivary proteins due to the vertebrate host immune pressure. Supplemental Tables S1 and S2 can be downloaded from http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T1/Sc-tb1-web.xls and http://exon.niaid.nih.gov/transcriptome/S_calcitrans/T2/Sc-tb2-web.xls.

Canine elliptocytosis due to a mutant beta-spectrin.

A 5-year-old, spayed female, mixed-breed dog with persistent elliptocytosis was evaluated at the Veterinary Medical Teaching Hospital at Kansas State University. The elliptocytosis was asymptomatic and was detected during the evaluation of lameness. When subjected to shear stress in an ektacytometer, the dog's erythrocytes had reduced cellular deformability and erythrocyte membranes had decreased mechanical stability. Analysis of erythrocyte membrane spectrin by nondenaturing gel electrophoresis revealed an increased amount of spectrin dimers, indicating a defect in spectrin self-association. DNA analysis detected a beta-spectrin mutation in codon 2110 in which threonine was replaced by methionine. This mutation likely altered the molecular structure of the erythrocyte membrane, leading to impaired spectrin self-association and elliptocyte formation.

Molecular cloning and characterization of equine Toll-like receptor 9.

Innate immunity relies on a series of germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), to detect conserved microbial components. TLR9 is typically expressed intracellularly in immune cells such as dendritic cells and recognizes unmethylated bacterial or viral cytosine-phosphate-guanine DNA (CpG-DNA). To investigate innate immune responses through TLR9 signaling pathway in horses, we cloned and characterized equine TLR9. Protein sequence analysis shows that equine TLR9 has a typically conserved cytosolic Toll/interleukin-1 receptor (TIR) domain, three leucine-rich repeat (LRR) motifs, with greater than 82% identity to human, monkey, bovine, canine, feline, porcine and ovine orthologs. Equine TLR9 mRNA expression was characterized for spleen, lymph node, and peripheral blood leukocyte samples. Flow cytometric analysis of equine TLR9 expression using a cross-reactive TLR9 mAb identified high constitutive expression of equine TLR9 in PMNs, CD4(+) and CD8(+) T-lymphocytes as well as other leukocytes; similar to human TLR9 expression. The conservation of equine TLR9 and high expression profile in leukocytes suggests that equine TLR9 is a frequent target for unmethylated CpG-DNA, an essential mechanism for the activation of innate immunity.

Comparison of two commercial radial immunodiffusion assays for detection of bovine immunoglobulin G in newborn calves.

Bovine failure of passive transfer (FPT), defined as inadequate transfer of colostral immunoglobulins from the dam to the calf, has been associated with increased risk in neonatal mortality. Currently, radial immunodiffusion (RID) assay is considered to be the gold standard in determining FPT in serum samples from calves. There are 2 commercial RID assays routinely used for serodiagnosis of FPT in calves: VET-RID and SRID. Discrepancies between results of these RID assays were observed in the authors' laboratory. The objective of this study was to compare 2 commercial RID assays by testing a paired panel of 30 blood samples collected from newborn Holsteins at birth before, and 24 hr after, ingestion of colostrum, a commercial bovine reference serum, and a panel of different concentrations of 2 purified bovine immunoglobulin G (IgG) products. Overall, the results of this study showed a high level of discrepancy and poor agreement between the 2 RID kits. The interassay precision study revealed lower between-run coefficients of variation for the VET-RID kit compared with the SRID kit. The spiking and recovery study using purified bovine IgG products demonstrated that the VET-RID kit more closely approximates the expected concentrations of the purified bovine IgG products, whereas the SRID kit consistently overestimates the concentration of purified bovine IgG products. It was concluded that this may be due to inaccuracies in the internal standards of the SRID kit.

Measurement of serum antinuclear antibody titer in dogs with and without systemic lupus erythematosus: 120 cases (1997-2005).

OBJECTIVE: To determine serum antinuclear antibody (ANA) titers in dogs with systemic lupus erythematosus (SLE) and in dogs with related clinical and clinicopathologic findings. DESIGN: Retrospective case series. ANIMALS: 120 dogs. PROCEDURES: Information that was evaluated included signalment, clinical signs, results of routine laboratory testing, ANA titer, and diagnosis. RESULTS: The most common clinical signs were arthralgia, myalgia, and stiffness (n = 41 [34.2%]); the most common clinicopathologic abnormality was thrombocytopenia (30 [25%]). Serum ANA titer was < 160 (seronegative) in 89 dogs (74.2%), 160 in 14 dogs (11.7%), 320 in 5 dogs (4.2%), and > or = 640 in 12 dogs (10%). Immune-mediated disease was confirmed in 40 dogs, 18 of which fulfilled the criteria for a definitive or probable diagnosis of SLE. Only 1 of 47 dogs with no major signs compatible with SLE had immune-mediated disease, compared with 26 of 57 dogs with 1 major sign and 13 of 16 dogs with > or = 2 major signs. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that measurement of ANA titer was not a useful diagnostic test in dogs without any major clinical or clinicopathologic abnormalities suggestive of SLE. In contrast, there was a good chance that results of the ANA assay would be positive and that the dog would be found to have immune-mediated disease if at least 2 major signs were evident. Findings suggest that it would be reasonable to limit the use of the ANA assay to those dogs that have at least 1 major sign compatible with a diagnosis of SLE.

Blood smear from a cat: features to "dys"cover.

A 4-year-old, spayed female, domestic shorthair cat was presented for lethargy, nonregenerative anemia, and inappetence. Results of a CBC included macrocytic, normochromic, nonregenerative anemia and a glucocorticoid-associated leukogram. On blood smear examination, neutrophils had abnormal features including hyposegmentation and a diffuse chromatin pattern with nuclear filament formation and nuclear blebbing. Microscopic examination of a roll preparation of bone marrow revealed hypolobulated megakaryocytes with asynchronous maturation of nuclei. The granulocytic to erythrocyte (G:E) ratio was 76. Segmented neutrophils had asynchronous maturation and dysplastic features. The entire erythroid lineage was markedly decreased for the degree of anemia and rare dysplastic features were noted in erythroid precursor cells. The interpretation of bone marrow findings was erythroid hypoplasia, megakaryocytic dysplasia, and granulocytic hyperplasia with dysplasia. Histopathologic examination of a bone marrow core sample also revealed myeloid hyperplasia and erythroid hypoplasia. The result of a direct immunofluorescence assay for FeLV performed on the bone marrow roll preparation was positive. A diagnosis of dysmyelopoiesis associated with FeLV infection was made. This case was unique in that the dysplastic changes occurred in cell lines that did not have associated cytopenias. The dysmyelopoiesis most closely resembled myelodysplastic syndrome with refractory cytopenia (MDS-RC); however, secondary dysmyelopoiesis could not be ruled out.

Detection of cellular immunity to rabies antigens in human vaccinees.

A nonradioactive multi-parameter flow cytometry assay was developed to identify antigen-specific lymphocytes in human subjects previously vaccinated against rabies virus and was subsequently compared to the standard tritiated thymidine method. A cell tracking dye, carboxyfluorescein succinimidyl ester, was used in combination with surface label for CD4 and CD8 cells in order to determine the response of lymphocytes to killed rabies virus in an antigen recall assay. The rabies virus-specific lymphocyte response was compared to the humoral immune response in each of ten vaccinated and five non-vaccinated subjects. Lymphocyte responses to rabies virus were observed in all ten vaccinated subjects; some noted as early as 3 days after stimulation while others were not until 7 days after stimulation. There was good agreement between the proliferation index of the CFSE assay and the simulation index of the [3H]thymidine assay (kappa statistic=0.73). An inverse relationship was detected between the level of rabies virus neutralizing antibody (RVNA) and the lymphocyte response to inactivated rabies virus in the vaccinated subjects. The association between cytokines production and level of humoral and cellular response was investigated in four representative subjects. Two vaccinated subjects with high proliferation indices and low RVNA titers produced Th1 type cytokines to rabies virus stimulation, whereas two vaccinated individuals with low proliferation indices and high RVNA titers responses did not produce these cytokines.

Development and evaluation of a flow cytometry microsphere assay to detect anti-histone antibody in dogs.

Anti-nuclear antibody (ANA) is one of the diagnostic parameters that support a diagnosis of autoimmune disorders in humans, dogs, and horses, particularly the condition systemic lupus erythematosus (SLE). The most commonly used method for detecting ANA in canine serum is the indirect immunofluorescence antibody assay (IFA) that detects dog IgG with reactivity towards mammalian cell nuclei. Interpretation of the IFA results is very subjective and dependent on the source of tissue/cellular substrate. We have developed a flow cytometry based assay to detect canine serum antibodies specific to histones. Histones were chosen as the target antigen because these nuclear proteins are the most common nuclear substrate for ANA in dogs with SLE. Microsphere beads were coated with histones and incubated with canine sera. Bound anti-histone antibodies were detected by FITC-conjugated rabbit F(ab')2 anti-dog IgG. Sera from four groups of dogs (47 dogs total) were tested for anti-histone antibodies and compared with the traditional IFA assay. The groups included 15 healthy dogs, 15 dogs with noninflammatory diseases, 9 dogs with polyarthritis and positive ANA, and 8 German shepherds with perianal fistulas. The microsphere assay results indicated that only one dog in the noninflammatory group and four out of nine dogs in the polyarthritis group had mean fluorescent intensity values above our established cut-off (defined as 2 S.D. above the mean of healthy controls). There was moderate agreement between the anti-histone assay and the traditional ANA (kappa statistic=0.54). Absorption of ANA positive serum with total histones dramatically diminished the fluorescent signal detected by flow cytometry and the speckled nuclear pattern observed by IFA, whereas preabsorption did not change the diffuse nuclear staining pattern. These findings indicate that the anti-histone assay will not replace the ANA test and that other nuclear proteins, such as ribonucleoproteins may contribute to the diffuse ANA patterns.

Afibrinogenemia and a circulating antibody against fibrinogen in a Bichon Frise dog.

A 1.5-year-old female Bichon Frise dog was evaluated for a life-threatening hemorrhagic condition that occurred after ovariohysterectomy, requiring 4 whole-blood transfusions. A hemostatic profile, including activated clotting time (ACT), one-stage prothrombin time (OSPT), activated partial thromboplastin time (APTT), buccal mucosal bleeding time, and specific assays (heat-precipitation microhematocrit method and electroimmunoassay) for fibrinogen, were performed to investigate the coagulopathy. Clotting times for all tests having a fibrin clot endpoint (ACT, OSPT, APTT) and buccal mucosal bleeding time were prolonged. Plasma fibrinogen was not detected by heat-precipitation microhematocrit method or electroimmunoassay. Using the Ellis-Stransky method, a mixture of patient plasma and normal canine plasma with known fibrinogen content yielded substantially less than the calculated fibrinogen concentration, indicating the presence of an interfering substance. The interferent properties of the patient's plasma were retained following heat precipitation at 56 degrees C indicating the absence of a pyroglobulin or an abnormal fibrinogen molecule. Radial immunodiffusion assay using the patient's plasma and activated thrombin confirmed the existence of an inhibitor to the formation of fibrin. Western blot analysis using the patient's plasma identified an IgG antibody that reacted with the Beta- and gamma- but not the Alpha-subunits of canine fibrinogen. Antibody was detected in samples taken 8, 16, and 68 days after the surgery; peak titers were evident at day 16. These results supported a diagnosis of afibrinogenemia with a circulating antibody inhibitor to fibrin clot formation that developed secondary to blood transfusion.