Circulating Foamy Macrophages in the Golden Syrian Hamster (Mesocricetus auratus) Model of Leptospirosis.

Leptospirosis is a world-wide zoonotic disease caused by pathogenic Leptospira and can be asymptomatic or can cause clinical signs ranging from influenza-like to multi-organ failure and death in severe cases. While species and strain specificity can play a major role in disease presentation, the hamster is susceptible to most leptospiral infections and is the model of choice for vaccine efficacy testing. During evaluation of blood smears from hamsters challenged with different species and strains of Leptospira, a circulating population of large, mononuclear, lipid-filled cells, most similar to foamy macrophages (FMs), was detected. Circulating FMs were identified by Giemsa staining and verified by scanning and transmission electron microscopy. FMs were found in the circulating blood of all Leptospira-challenged hamsters, indicating that the finding was not species or strain specific, although higher numbers of FMs tended to correlate with severity of disease. The unique finding of circulating FMs in the hamster model of leptospirosis can yield additional insights into the pathogenesis of leptospirosis and other diseases that induce circulating FMs.

Circulating foamy macrophages in the golden syrian hamster (Mesocricetus auratus) model of Leptospirosis

Leptospirosis is a world-wide zoonotic disease caused by pathogenic Leptospira and can be asymptomatic or can cause clinical signs ranging from influenza-like to multi-organ failure and death in severe cases. While species and strain specificity can play a major role in disease presentation, the hamster is susceptible to most leptospiral infections and is the model of choice for vaccine efficacy testing. During evaluation of blood smears from hamsters challenged with different species and strains of Leptospira, a circulating population of large, mononuclear, lipid-filled cells, most similar to foamy macrophages (FMs), was detected. Circulating FMs were identified by Giemsa staining and verified by scanning and transmission electron microscopy. FMs were found in the circulating blood of all Leptospira-challenged hamsters, indicating that the finding was not species or strain specific, although higher numbers of FMs tended to correlate with severity of disease. The unique finding of circulating FMs in the hamster model of leptospirosis can yield additional insights into the pathogenesis of leptospirosis and other diseases that induce circulating FMs.

Heterophil functional responses to dietary immunomodulators vary in genetically distinct chicken lines.

The effect of dietary supplementation of immunomodulators on in vitro chicken heterophil function was investigated using three diverse genetic lines of chickens (broiler, Fayoumi, and Leghorn). Dietary supplementation with ß-glucan, ascorbic acid, and corticosterone was fed from 8 to 11 weeks of age. Heterophil function was evaluated weekly during supplementation using phagocytosis, bacterial killing, and heterophil extracellular traps (HETs)-DNA release. Fayoumis fed the basal diet had significantly higher HETs-DNA release (P=0.002) than Leghorns and broilers. Both genetic line and immunomodulator diet supplementation had significant effects on bacterial killing (line and diet effect: P<0.001) and HETs-DNA release (line: P<0.001; diet: P=0.043). Dietary supplementation with immunomodulators, therefore, shows potential to affect and augment heterophil function in chickens. The current results also suggest the important role of genetics in innate immune responses.

Genetic control of chicken heterophil function in advanced intercross lines: associations with novel and with known Salmonella resistance loci and a likely mechanism for cell death in extracellular trap production.

Heterophils, the avian polymorphonuclear leukocyte and the counterpart of mammalian neutrophils, generate the primary innate response to pathogens in chickens. Heterophil performance against pathogens is associated with host disease resistance, and heterophil gene expression and function are under genetic control. To characterize the genomic basis of heterophil function, heterophils from F(13) advanced intercross chicken lines (broiler × Leghorn and broiler × Fayoumi) were assayed for phagocytosis and killing of Salmonella enteritidis, oxidative burst, and extracellular trap production. A whole-genome association analysis of single nucleotide polymorphisms at 57,636 loci identified genomic locations controlling these functional phenotypes. Genomic analysis revealed a significant association of extracellular trap production with the SAL1 locus and the SLC11A1 gene, which have both been previously associated with resistance to S. enteritidis. Fine mapping supports SIVA1 as a candidate gene controlling SAL1-mediated resistance and indicates that the proposed cell-death mechanism associated with extracellular trap production, ETosis, likely functions through the CD27/Siva-1-mediated apoptotic pathway. The SLC11A1 gene was also associated with phagocytosis of S. enteritidis, suggesting that the Slc11a1 protein may play an additional role in immune response beyond depleting metal ions to inhibit intracellular bacterial growth. A region of chromosome 6 with no characterized genes was also associated with extracellular trap production. Further characterization of these novel genes in chickens and other species is needed to understand their role in polymorphonuclear leukocyte function and host resistance to disease.

Protecting animal and human health and the nation's food supply through veterinary diagnostic laboratory testing.

The current detection system for animal diseases requires coordination between veterinarians; veterinary medical laboratories; and state, federal, and international agencies, as well as associated private sector industries. Veterinary clinical pathologists in clinical and governmental laboratories often have responsibilities and expertise in one or more laboratory disciplines involved in diagnosing zoonotic and/or emerging diseases and diseases exotic to the United States that are important to animal and human health and the nation's food supply. The knowledge and roles of all veterinary laboratory professionals are vital to detect, monitor, and confirm diseases and conditions that affect animal and human health and the nation's animal food supply.

Proportion of circulating chicken heterophils and CXCLi2 expression in response to Salmonella enteritidis are affected by genetic line and immune modulating diet.

Genetic line and diet affect chicken heterophil activity and gene expression, and the combination of these factors can enhance disease resistance. This study evaluated the effects of immune modulating diets on heterophil/lymphocyte (H/L) ratio and heterophil chemokine expression in distinct genetic lines. Fayoumi and Leghorn chickens were fed a basal diet or immune modulating diets enhanced with ß-glucans, ascorbic acid, or corticosterone. H/L ratios and heterophil gene expression in response to in vitro stimulation with Salmonella enteritidis (SE) were evaluated on days 1, 3, 7, and 21 of diet treatment. The stress-mimicking corticosterone diet influenced H/L ratio in the Leghorn line, but not the Fayoumi line, suggesting resistance to stress-induced immunosuppression in the Fayoumi line. Leghorn line H/L ratios were increased on days 1 and 3 of corticosterone diet treatment, but not days 7 or 21. Expression of CXCLi2 by SE stimulated heterophils was higher in the Leghorn line, suggesting that Leghorns rely more heavily on inflammatory response than do Fayoumis. Corticosterone diet was associated with reduced CXCLi2 expression in heterophils from both lines. Dietary ß-glucan or ascorbic acid did not affect H/L ratio or CXCLi2 expression, suggesting that benefits of these immunomodulators may not be evident in healthy birds.

Use of rapid cytochemical staining to characterize fish blood granulocytes in species of special concern and determine potential for function testing.

Studies of innate immunity in fish species of special concern are essential for better understanding of their health status during hatchery rearing conditions. The cytochemical and morphological characterizations of blood granulocytes have been used to provide information about phylogenetic differences and determine the potential use of neutrophil function assays. Rapid, simple, cytochemical staining kits used routinely for staining mammalian granulocytes have been used to characterize granulocytes from blood of four fish species: Arctic grayling, cutthroat trout, June sucker, and shovelnose sturgeon. Blood smears were stained with Peroxidase 391 (myeloperoxidase, MPO), alkaline phosphatase (AP), Periodic Acid Schiff (PAS) and Diff-quick stain; examined using bright field and differential interference contrast microscopy. Granulocytes on blood smears were evaluated based on the cell morphology, and presence or absence of the specific chromogen. Presence of lymphocytes, monocytes, platelets/thrombocytes and granulocytes was determined in all fish species. Arctic grayling, June sucker, and cutthroat trout had MPO positive granulocytes, while shovelnose sturgeon heterophils had positive reaction for leukocyte AP, but not MPO. Presence of MPO indicated potential to measure oxidative burst and degranulation of neutrophil primary granules in Arctic grayling, cutthroat trout and June sucker. Absence of MPO in shovelnose sturgeon suggested use of different enzyme marker (AP) in degranulation assay for this species. Standardization of cytochemical techniques allowed for rapid screening of leukocyte types, reducing the number of fish, time and effort to select adequate neutrophil function assays to be used in studies of health status in species of special concern.

Chicken heterophils from commercially selected and non-selected genetic lines express cytokines differently after in vitro exposure to Salmonella enteritidis.

Resistance to pathogens such as Salmonella enteritidis (SE) is a heritable trait important in maintaining the health of chickens and reducing bacterial contamination of poultry products. In chickens, heterophils act as the first responders to bacterial infections and are, therefore, responsible for initiating the immune response against SE challenge. This study measured mRNA expression of several immune response genes [interleukin-6 (IL-6), IL-10, transforming growth factor-beta4 (TGF-beta4), granulocyte macrophage-colony stimulating factor (GM-CSF), and Toll-like receptor-4 (TLR-4)] by heterophils from broiler, Leghorn, and Fayoumi chickens, either non-stimulated or stimulated in vitro with SE using quantitative reverse-transcriptase PCR. We found that heterophils of commercially selected broiler and Leghorn birds had differing early heterophil responses to SE in comparison with the native Fayoumi line. Heterophil stimulation with SE in vitro increased expression of pro- (IL-6 and GM-CSF) and anti-inflammatory cytokine mRNA (IL-10 and TGF-beta4) in the Fayoumi line, while the broiler and Leghorn line heterophils had decreased or no changes in the cytokine gene expression levels. The unique response of the Fayoumi line is in contrast to the lines with a history of genetic selection to increase growth or reproduction, a process which may favor reduced or suppressed inflammatory responses. The findings illustrate the potential value of native lines to provide biodiversity to enhance innate health in commercially selected poultry.

Chicken heterophil extracellular traps (HETs): novel defense mechanism of chicken heterophils.

Recent findings in mammals and fish have revealed that neutrophil nuclear material associated with cytoplasmic granular content is released in the form of neutrophil extracellular traps (NETs) that can trap and kill invading microorganisms in vitro and in vivo. To determine if a similar mechanism is present in chicken heterophils, hydrogen peroxide (H(2)O(2)) and phorbol myristate acetate (PMA) were used for stimulation of blood-derived heterophils. Stimulated heterophils produced structures that were characterized using immunocytochemistry and confocal microscopy as heterophil extracellular traps (HETs). Released HETs contained DNA, histone-DNA complex and elastase from heterophil cytoplasmic granules. HETs released from chicken heterophils are structurally similar to NETs found in mammalian and fish neutrophils. Extracellular DNA released from heterophils was quantified by Picogreen assay. Stimulation with PMA or H(2)O(2) significantly increased the HET-DNA release index in vitro compared to non-stimulated heterophils (1.11+/-0.04 and 1.55+/-0.10, respectively), and H(2)O(2) stimulation induced significantly higher HET-DNA release than PMA (P<0.001). Thus, HETs are now characterized as an important heterophil-mediated defense mechanism in chickens.

Evaluation of the use of subcutaneous implantable vascular access ports in feline blood donors.

OBJECTIVE: To compare the ease and effects of collecting blood from cats by use of subcutaneous totally implantable vascular access ports (VAPs) with collection via conventional jugular phlebotomy. DESIGN: Prospective randomized experimental study. ANIMALS: 8 healthy cats. PROCEDURES: Cats in the port group (n = 4) underwent monthly blood donation by use of VAPs and manual restraint, and cats in the nonport group (4) underwent monthly blood donation by use of conventional jugular phlebotomy and sedation, for 6 months. RESULTS: Postsurgical VAP-related complications developed in 3 cats and included port erosion (n = 1), disconnection of the port from the catheter (1), and seroma formation (1). Blood was successfully collected 24 of 24 and 20 of 20 times in the nonport and port groups, respectively. Results of bacterial culture of blood were negative in 22 of 24 and 15 of 20 nonport and port collections, respectively. No differences in RBC morphology were observed between groups. Mean blood collection and total donation times were significantly longer for the nonport group. Collection time was more variable in the nonport group, and cats were less tolerant of handling during venipuncture, compared with cats in the port group. Blood collection required a mean of 2.4 persons for the nonport group and 2.1 persons for the port group. CONCLUSIONS AND CLINICAL RELEVANCE: Positive results for blood collections via VAPs were increased donor acceptance, decreased number of personnel required, and decreased collection time. Drawbacks included contamination of blood products and port-related complications.

Zebrafish (Danio rerio) whole kidney assays to measure neutrophil extracellular trap release and degranulation of primary granules.

The zebrafish (Danio rerio) is an excellent model system for studies in developmental biology, genetics, and toxicology, and is increasingly gaining importance in the field of immunology. The use of whole zebrafish kidneys as source of neutrophils for degranulation assays and detection of neutrophil extracellular traps is described for the first time. Neutrophils from zebrafish kidneys released neutrophil extracellular traps (NETs) and myeloperoxidase (MPO) upon stimulation with calcium ionophore, phorbol myristate acetate, and beta-glucan. Immunocytochemical study of zebrafish kidney cells revealed that NETs are made of DNA fibers associated with neutrophil granular proteins, but not with cytoskeleton. Rapid, direct MPO and extracellular DNA detection assays were developed to quantify NET release and degranulation of neutrophil primary granules from whole zebrafish kidneys. The assays were used to measure the effects of acute crowding and handling stress on neutrophils, and to examine the potential for use of zebrafish whole kidney assays in evaluation of neutrophil function under different conditions in vivo. The whole kidney NET release and degranulation assays are quantitative, can rapidly measure a large number of samples, and are capable of detecting inhibition of neutrophil activity in stressed fish, overcoming the limitations that prevented use of zebrafish in the investigations of cellular innate immune function. The assays can be used as a new research model to study effects of stress, immunomodulators, toxicants, and diseases on fish neutrophil biology.

Fish cast NETs: neutrophil extracellular traps are released from fish neutrophils.

Neutrophil extracellular traps (NETs), which are extracellular DNA structures released from neutrophils, are described and characterized for the first time in fish using fluorescent confocal microscopy. Confocal images of fish neutrophil suspensions stained with 6'-diamino-2-phenylindole, dihydrochloride DNA fluorescent stain (DAPI) revealed the presence of NETs which appeared as fibrous structures connecting several cells. Co-localization of NETs with neutrophil granular proteins and actin was investigated using specific antibodies and probes. Double staining of neutrophils with SYTOX green and DAPI revealed that SYTOX stain applied to living cells stained extracellular DNA, but not nuclei. NETs are actively released from stimulated living cells, associated with granular proteins, but not with cytoskeleton, and are not a product of nuclear degradation seen in late apoptotic stages. Additionally, a fluorometric microtiter plate assay to quantify the release of NETs was adopted for use with fish neutrophils, and the effect of stress on NETs release was studied. This assay detected the inhibition of DNA release during stress conditions. In summary, NETs were released from living fish kidney neutrophils upon stimulation, characterized using fluorescence DNA-binding dyes, specific antibodies and probes, and quantified using a microtiter plate fluorometric assay that can rapidly measure a large number of samples. Detection of NETs can be used as an additional assay to an existing battery of functional tests, and as a new research model to study the effects of stress, immunomodulators, and diseases.

Immunomodulatory effects of beta-glucan on neutrophil function in fathead minnows (Pimephales promelas Rafinesque, 1820).

Stimulatory effects of yeast beta-1,3-1,6-glucans on neutrophils have long been recognized, but effects of glucans on degranulation of primary granules in fish neutrophils have not been previously reported. Neutrophil function was monitored during in vitro and in vivo application of glucans to non- (NS), acute- (AS) and chronically stressed (CS) fish. beta-Glucan proved to be a strong and quick (80%, 2 min) stimulant of degranulation. Dietary glucan increased degranulation in NS fish, and prevented a decrease in AS fish. Degranulation in CS fish returned to NS levels 3 days after the glucan diet was fed. Fathead minnows appear to be a useful model to investigate neutrophil degranulation in fish exposed to different environmental conditions and immunomodulators. Use of beta-glucans in fish diets prior to AS and during chronic stress can enhance neutrophil function, potentially increasing disease resistance and survival rates after transportation or exposure to poor water quality.

Experimental canine leptospirosis caused by Leptospira interrogans serovars pomona and bratislava.

OBJECTIVE: To evaluate gross, histopathologic, and serum biochemical findings caused by Leptospira interrogans serovars pomona and bratislava inoculated in dogs. ANIMALS: Twenty-seven 8-week-old female Beagles. PROCEDURE: Dogs were randomly assigned to challenge or control groups. Challenge groups were conjunctivally inoculated on 3 successive days with 5 x 10(7) L interrogans serovar pomona (n = 12) or serovar bratislava (11). Clinical signs were recorded throughout the experiment, and clinical pathology assays, bacteriologic culture, and necropsies (6 or 7 dogs necropsied at each time point) were done on postinoculation day (PID) 7, 10, 14, and 20. RESULTS: Infection could not be confirmed in any serovar bratislava-inoculated dog, and control dogs remained healthy throughout the experiment. Positive culture and fluorescent antibody test results were confirmed in 11 of 12 serovar pomona-inoculated dogs. Fever and lethargy starting at PID 7 were the most common clinical signs in serovar pomona-infected dogs. On day 10, gross lesions included multifocal renal and pulmonary hemorrhage and perirenal edema. Serovar pomona-inoculated dogs had histopathologic lesions including hepatitis, interstitial nephritis, and pneumonia at PID 7, 10, 14, and 20. Increases in BUN, anion gap, and bilirubin concentration occurred on PID 10, 14, and 20. Platelet counts in dogs with positive results of bacteriologic culture were decreased from baseline values on PID 10, 12, and 14. CONCLUSIONS AND CLINICAL RELEVANCE: Conjunctival inoculation with L interrogans serovar pomona resulted in a high rate of infection with concomitant hemorrhagic and inflammatory lesions of the kidneys, liver, and lungs.

A rapid, direct assay to measure degranulation of primary granules in neutrophils from kidney of fathead minnow (Pimephales promelas Rafinesque, 1820).

A direct, rapid, quantitative colorimetric assay to determine neutrophil primary granule degranulation was adapted for use with fathead minnow kidney neutrophils. The assay measures the exocytosis of myeloperoxidase (MPO) using 3,3',5,5'-tetramethylbenzidine as a substrate. The assay was validated by comparing the total myeloperoxidase content of neutrophil populations obtained from adult cattle, as a known positive, and fish; evaluating the effects of calcium ionophore (CaI), phorbol myristate acetate (PMA), aqueous solution of beta-glucan (MGAQ) and zymosan (Z) with and without cytochalasin B (cyto B) as stimulants of degranulation; determining the kinetics of primary granule exocytosis and detecting changes in degranulation when fish were exposed to stress and anaesthesia with MS-222. The MPO assay detected MPO activity in fathead minnow neutrophils that correlated to neutrophil numbers, confirmed that degranulation was increased when CaI was used compared to other stimulants, determined degranulation peak at 60 min and confirmed decreased degranulation after exposure to handling and crowding stress, with and without MS-222. Therefore, the MPO assay is capable of detecting important differences that may occur in degranulation of fathead minnow kidney neutrophil primary granules and in total neutrophil myeloperoxidase content.

Clinical and pathologic comparison of acute leptospirosis in dogs caused by two strains of Leptospira kirschneri serovar grippotyphosa.

OBJECTIVE: To develop a method for inducing acute leptospirosis in dogs. ANIMALS: 31 nine-week-old female Beagles. PROCEDURE: Beagles were randomly assigned to 2 inoculation groups or a control group. Dogs were inoculated on 3 successive days by conjunctival instillation of 5 x 10(7) cells of Leptospira kirschneri serovar grippotyphosa strain 82 (12 dogs) or strain RM 52 (14 dogs). Control dogs (n = 5) were similarly inoculated with sterile leptospiral culture media. Clinical signs, clinicopathologic variables, anti-leptospiral antibody titers, and evidence of leptospires in tissues and body fluids were evaluated. Dogs were euthanatized and necropsied on days 7, 14, 22, or 28 after inoculation or as required because of severe illness. RESULTS: Clinical signs in infected dogs included conjunctivitis, lethargy, diarrhea, dehydration, vomiting, and icterus. Consistent clinicopathologic alterations included azotemia, hyperphosphatemia, increased anion gap, hyperbilirubinemia, and an increase in alkaline phosphatase activity. Leptospires were cultured from the kidneys (11/12), urine (6/9), aqueous humor (9/12), blood (12/12), and liver (12/12) of dogs inoculated with strain 82. Only 3 of 14 dogs became infected after inoculation with strain RM 52. Histopathologic lesions in infected dogs included interstitial nephritis, renal tubular degeneration and necrosis, pulmonary hemorrhage, and hepatic edema and perivasculitis. CONCLUSIONS AND CLINICAL RELEVANCE: Conjunctival exposure to L kirschneri serovar grippotyphosa strain 82 resulted in acute leptospirosis in all inoculated dogs, but only 3 of 14 dogs inoculated with strain RM 52 became infected. This method of infection by serovar grippotyphosa can be used to study the pathogenesis and prevention of leptospirosis in dogs.

Keratinizing ameloblastoma in a 9-month-old llama (Lama glama).

A 9-month-old male llama (Lama glama) was presented because of a rapidly growing mass on the right side of the face. Radiographs revealed a marked expansion of the right caudal face region with bone lysis involving the maxilla and the nasal, lacrimal, zygomatic, and palatine bones. Cytologically, the mass consisted of atypical round to polygonal cells with round nuclei and basophilic cytoplasms that formed acini and rows. Histologically, the mass consisted of anastomosing cords and sheets of neoplastic odontogenic epithelial cells embedded in a loose fibrovascular connective tissue. Single layers of peripheral, polarized, palisading, columnar epithelial cells were seen at the edges of some cords. Within the centers of the cords, epithelial cells showed rapid progression to keratin production. The histologic diagnosis was keratinizing ameloblastoma. Ameloblastomas are neoplasms of odontogenic epithelium that tend to be locally aggressive and can cause substantial destruction of bony structures. Because ameloblastomas do not tend to metastasize, they can be successfully treated by complete surgical excision, performed before extensive bony destruction occurs. Ameloblastoma, although expected to be rare, should be onthe list of differential diagnoses for facial swelling in llamas.