Melinda S. Camus

Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro.

The macrocyclic lactones are the only anthelmintics used to prevent heartworm disease, but it is very difficult to reproduce their in vivo efficacy against Dirofilaria immitis larvae in experiments in vitro. These assays typically measure motility, suggesting that paralysis is not the mode of action of the macrocyclic lactones against D. immitis. We isolated peripheral blood mononuclear cells (PBMC) and neutrophils from uninfected dogs and measured their adherence to D. immitis microfilariae in the presence of varying concentrations of ivermectin. We found that adherence of PBMC to the microfilariae was increased in the presence of ivermectin concentrations ≥100 nM and adherence of neutrophils was increased in drug concentrations ≥10 nM. Up to 50% of microfilariae had adherent PBMC in the presence of the drug, and binding was maximal after 40 h incubation. Neutrophil adherence was maximal after 16 h, with approximately 20% of the microfilariae having at least one cell adhered to them. Adherent neutrophils showed morphological evidence of activation. These results are consistent with a model in which the macrocyclic lactones interfere with the parasites ability to evade the hosts innate immune system.

Cytologic Criteria for Mast Cell Tumor Grading in Dogs With Evaluation of Clinical Outcome

A 2-tiered histologic grading scheme for canine cutaneous mast cell tumors (MCTs) is based on morphologic characteristics of neoplastic cells, including karyomegaly, multinucleation, nuclear pleomorphism, and mitotic figures. Aspirates from MCTs may provide the same information more quickly, inexpensively, and less invasively. This study used these criteria to develop a cytologic grading scheme for canine MCTs to predict outcome. Three anatomic pathologists graded histologic samples from 152 canine MCTs. Three clinical pathologists evaluated aspirates from these masses using similar criteria. A cytologic grading scheme was created based on correlation with histologic grade and evaluated with a kappa statistic. Survival was evaluated with Kaplan-Meier survival curves. Cox proportional hazards regression was used to estimate hazard ratios for tumor grades and individual grading components. Simple logistic regression tested for relationships between risk factors and mortality. The cytologic grading scheme that best correlated with histology (kappa = 0.725 ± 0.085) classified a tumor as high grade if it was poorly granulated or had at least 2 of 4 findings: mitotic figures, binucleated or multinucleated cells, nuclear pleomorphism, or >50% anisokaryosis. The cytologic grading scheme had 88% sensitivity and 94% specificity relative to histologic grading. Dogs with histologic and cytologic high grade MCTs were 39 times and 25 times more likely to die within the 2-year follow-up period, respectively, than dogs with low grade MCTs. High tumor grade was associated with increased probability of additional tumors or tumor regrowth. This study concluded that cytologic grade is a useful predictor for treatment planning and prognostication.

ASVCP quality assurance guidelines: external quality assessment and comparative testing for reference and in‐clinic laboratories

The purpose of this document is to educate providers of veterinary laboratory diagnostic testing in any setting about comparative testing. These guidelines will define, explain, and illustrate the importance of a multi-faceted laboratory quality management program which includes comparative testing. The guidelines will provide suggestions for implementation of such testing, including which samples should be tested, frequency of testing, and recommendations for result interpretation. Examples and a list of vendors and manufacturers supplying control materials and services to veterinary laboratories are also included.

Livestock abundance predicts vampire bat demography, immune profiles, and bacterial infection risk

Human activities create novel food resources that can alter wildlife–pathogen interactions. If resources amplify or dampen, pathogen transmission probably depends on both host ecology and pathogen biology, but studies that measure responses to provisioning across both scales are rare. We tested these relationships with a 4-year study of 369 common vampire bats across 10 sites in Peru and Belize that differ in the abundance of livestock, an important anthropogenic food source. We quantified innate and adaptive immunity from bats and assessed infection with two common bacteria. We predicted that abundant livestock could reduce starvation and foraging effort, allowing for greater investments in immunity. Bats from high-livestock sites had higher microbicidal activity and proportions of neutrophils but lower immunoglobulin G and proportions of lymphocytes, suggesting more investment in innate relative to adaptive immunity and either greater chronic stress or pathogen exposure. This relationship was most pronounced in reproductive bats, which were also more common in high-livestock sites, suggesting feedbacks between demographic correlates of provisioning and immunity. Infection with both Bartonella and haemoplasmas were correlated with similar immune profiles, and both pathogens tended to be less prevalent in high-livestock sites, although effects were weaker for haemoplasmas. These differing responses to provisioning might therefore reflect distinct transmission processes. Predicting how provisioning alters host–pathogen interactions requires considering how both within-host processes and transmission modes respond to resource shifts. This article is part of the theme issue ‘Anthropogenic resource subsidies and host–parasite dynamics in wildlife’.

Evaluation of the Positive Predictive Value of Serum Protein Electrophoresis Beta-Gamma Bridging for Hepatic Disease in Three Domestic Animal Species

Beta–gamma bridging (β-γ bridging) on serum protein electrophoresis is touted as being virtually pathognomonic for hepatic disease. However, the criteria for β-γ bridging are not defined, and few publications support a relationship between β-γ bridging and liver disease. The goal of this retrospective study was to evaluate the prevalence of hepatic pathology in animals with β-γ bridging. All serum protein electrophoretograms from clinical patients generated at the University of Georgia between 1994 and 2008 were evaluated for the presence of β-γ bridging, defined as (1) an albumin:globulin ratio below the reference interval; (2) indistinct separation between all β and γ globulin fractions or between the β2 and γ fractions, with a negative shoulder slope of < 5%; and (3) predominance of γ proteins versus β proteins. Of the 237 electrophoretograms examined, 25 (11 dogs, 11 cats, 3 horses) met the inclusion criteria for β-γ bridging. Patients were classified into disease categories on the basis of biochemical, cytologic, and/or histologic findings. Positive predictive values of β-γ bridging for hepatic and infectious diseases were determined with a one-sided exact binomial test. Of 25 animals, 8 had evidence for hepatic disease, whereas 9 had infectious diseases. As such, the positive predictive value of β-γ bridging for hepatic disease was 32.0%, with a 95% confidence interval of 15.0% to 53.5% (P < .001), whereas for infectious disease, the positive predictive value was 36.0%, with a similar confidence interval. Beta–gamma bridging is not pathognomonic for liver diseases and is as frequently found with infectious diseases.

Cell cannibalism by malignant neoplastic cells: three cases in dogs and a literature review

Cell cannibalism refers to the engulfment of cells by nonprofessional phagocytic cells. Studies in human medicine have demonstrated a relationship between the presence of cell cannibalism by neoplastic cells and a poor outcome, and have shown a positive correlation with the presence of metastasis at the time of diagnosis. The biologic significance of cell cannibalism is unknown, but it is proposed that it may represent a novel mechanism of tumor immune evasion as a survival strategy in cases of unfavorable microenvironmental conditions. This report describes clinical and morphologic features of 3 cases of dogs with malignant neoplasia in which the presence of cellular cannibalism was observed in cytologic and histologic specimens. In the 1(st) case, a dog with a primary tonsillar squamous cell carcinoma with metastasis to retropharyngeal lymph nodes had neoplastic epithelial cells engulfing neutrophils noted in cytologic examination of the lymph nodes. In the 2(nd) case, neoplastic epithelial cells were seen engulfing each other in fine-needle aspirates from a primary mammary carcinoma with lung metastasis. In the 3(rd) case, poorly differentiated neoplastic mast cells from a recurrent, metastatic grade III mast cell tumor were observed cannibalizing eosinophils. A brief review of the literature describing known cell-into-cell relationships and the possible biologic significance and mechanisms involved in this phenomenon is provided. The relationship between cell cannibalism and distant metastasis should be explored in further studies, as it may prove to be a criterion of malignancy, as it is proposed in human medicine.

Chromatophoroma in a crevice kelpfish (Gibbonsia montereyensis)

A captive adult crevice kelpfish, Gibbonsia montereyensis, developed a cutaneous mass, approximately 9 × 7 mm on the right side of the head in an area of nonscaled skin. Following surgical debulking, examination of both impression smears and histologic sections of the tumor revealed a predominant population of round to spindloid to polygonal cells with a moderate amount of lightly basophilic cytoplasm. The cytoplasm was filled with round, variably-sized reddish-brown granules that often obscured the nucleus. Nuclei were round to ovoid with coarsely granular chromatin. There was minimal anisocytosis and anisokaryosis. The cytoplasmic granules in histologic sections were weakly positive by the Fontana-Masson method, and staining was eliminated with melanin bleach. Immunohistochemical staining was strongly positive with a murine monoclonal antibody for melan A. As the specificity of melan A for melanophores is not clearly defined in nonmammalian species, the tumor was examined by transmission electron microscopy. Melanophores were not detected. Instead, neoplastic cells were filled with numerous intracytoplasmic organelles with triple-limiting membranes composed of concentric lamellae; these structures were most compatible with pterinosomes, which are the pigment-containing organelles of cells called xanthophores and erythrophores. As both of these organelles are ultrastructurally indistinguishable and as kelpfish skin is known to contain both xanthophores and erythrophores, a diagnosis of a mixed pigment cell tumor or chromatophoroma was made. As the tumor was grossly reddish-brown, the possibility of a neoplastic population of only erythrophores could not be excluded. Pigment cell tumors, arising from cells of the embryonic neural crest, are common in reptiles and bony fish.

Biochemical reference intervals and pathophysiological changes in Flavobacterium psychrophilum-resistant and -susceptible rainbow trout lines.

Host genetic resistance against disease-causing pathogens can be enhanced through family-based selective breeding. At present, there is an incomplete understanding of how artificial selection of fish alters host physiology and response following pathogen exposure. We previously reported the generation of selectively-bred rainbow trout Oncorhynchus mykiss lines with either increased resistance (ARS-Fp-R) or susceptibility (ARS-Fp-S) to bacterial cold water disease (BCWD). This study (1) determined baseline reference-range intervals for packed cell volume (PCV) and 18 plasma biochemistry analytes, and (2) examined pathophysiological changes following infection between the genetic lines. PCV and biochemistry reference-range intervals did not significantly differ between genetic lines; thus data were pooled into a single reference-range population (n = 85). ARS-Fp-R and ARS-Fp-S line fish were intraperitoneally challenged with Flavobacterium psychrophilum, and plasma was collected on Days 1, 3, 6, and 9 post-challenge. Splenic bacterial load was measured using an F. psychrophilum-specific qPCR assay. In both genetic lines, changes were observed in mean PCV, total protein, albumin, glucose, cholesterol, chloride, and calcium, falling outside the established reference intervals and significantly differing from phosphate-buffered saline challenged fish, on at least 1d post-challenge. Mean PCV, total protein, and calcium significantly differed between ARS-Fp-R and ARS-Fp-S line fish on Day 9 post-infection, with values in the ARS-Fp-S line deviating most from the reference interval. PCV, total protein, cholesterol, and calcium negatively correlated with bacterial load. These findings identify divergent pathophysiological responses between ARS-Fp-R and ARS-Fp-S line fish following laboratory challenge that are likely associated with differential survival.

Comment on Davis and Holcomb: “Intraerythrocytic inclusion bodies in painted turtles (Chrysemys picta picta) with measurements of affected cells”

Dear Editor, We are writing regarding the recently published article by Andrew Davis and Kerry Holcomb (2008) entitled “Intraerythrocytic inclusion bodies in painted turtles (Chrysemys picta picta) with measurements of affected cells”. In our diagnostic pathology services we see intraerythrocytic inclusion bodies similar to those described in this article in multiple reptile species, particularly in turtles. We have understood these structures to be iron organelles, which are phagocytizing ferric iron that is not needed for incorporation in the hemoglobin molecule. In our laboratory these structures stain brightly positive with the Perl’s (Prussian blue) reaction, indicative of ferric iron. Though published findings on this subject are few, we would like to direct the authors’ attention to several articles suggesting that these structures are degenerate organelles, rather than infectious agents, as suggested in the authors’ introduction. Richey et al. (2000) describe similar basophilic, intraerythrocytic structures and demonstrate with transmission electron micrographs that they are degenerate, ferritin-containing organelles. In an article by Alleman et al. (1992), similar intraerythrocytic structures were determined ultrastructurally to represent degenerated organelles, possibly mitochondria. Finally, Clark et al. (2001) described basophilic intraerythrocytic inclusions and used transmission electron microscopy to demonstrate that the inclusions were composed of aggregated endoplasmic reticulum. While Dr. Davis and Dr. Holcomb have excellently quantified the prevalence of inclusions and the size of cells that contain them, we are concerned that neither special stains nor electron microscopy were utilized to support their assumption of a viral etiology. The introduction and references were limited only to infectious causes. The article characterizes the affected cells as more mature erythrocytes, consistent with the hypothesis that these structures are organelles degrading substances, including iron, that are no longer needed for incorporation into hemoglobin. We would be interested in Dr. Davis and Dr. Holcomb’s thoughts on our alternative hypothesis, and are curious regarding the results of iron stains on their blood smears.

ASVCP guidelines: Allowable total error hematology

The purpose of this document is to provide total allowable error (TEa ) recommendations for commonly analyzed hematology measurands for veterinary personnel. These guidelines define relevant terminology and highlight considerations specific to hematology measurands. They also provide reasons and guidelines for using TEa in instrument performance evaluation, including recommendations for when the total observed error exceeds the recommended TEa . Biological variation-based quality specifications are briefly discussed. The appendix describes the derivation of the hematology TEa recommendations and provides resources for external quality assurance/proficiency testing programs and a worksheet for implementation of the guidelines.