IN VITRO ASSAY FOR PHAGOCYTIC ACTIVITY BY CANINE NEUTROPHILS FOR Escherichia coli 1 2 2

INTRODUCTION (Gershwin et al., 1995). A deficiency or a malfunction at any stage of neutrophil function can impair the ability of The Innate immunity is the first line of defense in the an animal to overcome an infection (Gershwin et al., body which includes physical and chemical barriers; 1995). Severe sepsis in diseases may alter innate circulating effector cells [phagocytes and natural killer immune defenses, dis rup t microb ial clearance cells (NK)], complement proteins and cytokines (Abba, mechanisms, and complicate the pathophysiology of 2007). Intact skin and its secretions are impermeable to sepsis. Septic shock itself has significant effects upon most infectious agents and act as major physical barriers the innate and adaptive host immune responses gene ra ti ng the fi rs t li ne of de fense. T hose contributing to a state of sepsis-induced immune microorganisms that evade the epithelial barrier get dysregulation (Opal and Cross, 2005). Although killed by natural antimicrobial peptides, but many get congenital or inherited immunodeficiency disorders are engulfed and killed by phagocytes (Tizard, 2009; Roitt, uncommon in the dog, immunodeficiency secondary to 1988). Phagocytosis is an active process in phagocytic a wide range of causes (e.g., age, drug therapy, and leukocytes , namely, neut roph il s, eosinoph il s, chronic neoplastic or infectious disease) is relatively monocytes, and macrophages. Neutrophils are the first common in adul t dogs . Most canine immune responders that phagocytize and kill invading deficiencies are breed-related. Aged Beagle dogs have pathogens (Abba, 2007). Phagocytosis of cytokine decreased neutrophil phagocytosis and neutrophilactivated neutrophils and their microbicidal activities related gene expression compared to younger dogs are the first line of cellular defense against invading (Hall et al., 2010) pathogens, and are a major part of innate immunity Neutrophil phagocytosis can be measured by several (Silva et al. 1988). methods by allowing bacteria, yeast, or other particles Phagocytic activity is a continuous process involving and neutrophils to interact, and then to measure the discrete stages occurring in a sequential manner, number of neutrophils that have ingested particles, as namely, cell activation, chemotaxis, adherence, well as the phagocytic index determined by the average ingestion, and destruction (Tizard, 2009). The factors number of organisms within a phagocyte (Kaneko and influencing phagocytic properties of cells include cell Harvey, 1997; Gershwin et al., 1995). A phagocytosis age, energy (ATP), and integrity of cellular skeleton assay of canine blood neutrophils for bacteria is a long such as microfilaments and microtubules (Jain, 1986). felt need to identify immune compromised dogs. The The important properties of bacteria influencing objective of this research was to establish an in vitro phagocytos is ar e su rf ace charges, ce ll wa ll phagocytic assay for blood neutrophils isolated from composition, capsule, and toxins. The environmental clinically healthy dogs. factors required are presence of opsonins, temperature 0 (3740 C), pH (6-8), osmolarity, ionic composition and MATERIALS AND METHOD interfacial tension. To evaluate the efficiency of the phagocyte system, it is necessary to examine the Study group number of phagocytes, their adhesion, chemotaxis, Blood was drawn via cephalic venepunture into an phagocytosis, and intra cellular killing mechanisms EDTA tube and a tube without anticoagulant from SUMMARY: This study investigates the in vitro phagocytic activity of blood neutrophils of dogs in Sri Lanka for Escherichia coli. The study sample comprised of 70% mongrel and 30% cross bred dogs of both sexes. The leukocyte isolation method described yielded up to 12,930/ìl viable leukocytes of which 61-90% was neutrophils, with viability ranging from 90-99% (mean 96%). The mean percent phagocytosis (MPP) of canine blood neutrophils was significantly higher at 30 minutes incubation (89%) than at 15 minutes incubation (70 %) with E.coli. The findings support the fact that innate immunity can be measured qualitatively and quantitatively by the technique described which will be useful for the identification of immunocompromised dogs.


INTRODUCTION
. A deficiency or a malfunction at any stage of neutrophil function can impair the ability of The Innate immunity is the first line of defense in the an animal to overcome an infection (Gershwin et al., body which includes physical and chemical barriers; 1995).Severe sepsis in diseases may alter innate circulating effector cells [phagocytes and natural killer imm une def ens es, dis rup t mic rob ial cle ara nce cells (NK)], complement proteins and cytokines (Abba, mechanisms, and complicate the pathophysiology of 2007).Intact skin and its secretions are impermeable to sepsis.Septic shock itself has significant effects upon most infectious agents and act as major physical barriers the innate and adaptive host immune responses ge ne ra ti ng th e fi rs t li ne of de fe ns e. T ho se contributing to a state of sepsis-induced immune microorganisms that evade the epithelial barrier get dysregulation (Opal and Cross, 2005).Although killed by natural antimicrobial peptides, but many get congenital or inherited immunodeficiency disorders are engulfed and killed by phagocytes (Tizard, 2009; Roitt, uncommon in the dog, immunodeficiency secondary to 1988).Phagocytosis is an active process in phagocytic a wide range of causes (e.g., age, drug therapy, and le uk oc yt es , na me ly, ne ut ro ph il s, eo si no ph il s, chronic neoplastic or infectious disease) is relatively monocytes, and macrophages.Neutrophils are the first comm on in adul t dogs .Most cani ne immu ne resp onde rs th at ph agoc ytiz e and kill inva ding deficiencies are breed-related.Aged Beagle dogs have pathogens (Abba, 2007).Phagocytosis of cytokine decreased neutrophil phagocytosis and neutrophilactivated neutrophils and their microbicidal activities related gene expression compared to younger dogs are the first line of cellular defense against invading (Hall et al., 2010) pathogens, and are a major part of innate immunity Neutrophil phagocytosis can be measured by several (Silva et al. 1988).
methods by allowing bacteria, yeast, or other particles Phagocytic activity is a continuous process involving and neutrophils to interact, and then to measure the discrete stages occurring in a sequential manner, number of neutrophils that have ingested particles, as namely, cell activation, chemotaxis, adherence, well as the phagocytic index determined by the average ingestion, and destruction (Tizard, 2009).The factors number of organisms within a phagocyte (Kaneko and influencing phagocytic properties of cells include cell Harvey, 1997;Gershwin et al., 1995).A phagocytosis age, energy (ATP), and integrity of cellular skeleton assay of canine blood neutrophils for bacteria is a long such as microfilaments and microtubules (Jain, 1986).felt need to identify immune compromised dogs.The The important properties of bacteria influencing objective of this research was to establish an in vitro ph ag oc yt os is ar e su rf ac e ch ar ge s, ce ll wa ll phagocytic assay for blood neutrophils isolated from composition, capsule, and toxins.The environmental clinically healthy dogs.factors required are presence of opsonins, temperature 0 (37-40 C), pH (6-8), osmolarity, ionic composition and MATERIALS AND METHOD interfacial tension.To evaluate the efficiency of the phagocyte system, it is necessary to examine the Study group number of phagocytes, their adhesion, chemotaxis, Blood was drawn via cephalic venepunture into an phagocytosis, and intra cellular killing mechanisms EDTA tube and a tube without anticoagulant from SUMMARY: This study investigates the in vitro phagocytic activity of blood neutrophils of dogs in Sri Lanka for Escherichia coli.The study sample comprised of 70% mongrel and 30% cross bred dogs of both sexes.The leukocyte isolation method described yielded up to 12,930/ìl viable leukocytes of which 61-90% was neutrophils, with viability ranging from 90-99% (mean 96%).The mean percent phagocytosis (MPP) of canine blood neutrophils was significantly higher at 30 minutes incubation (89%) than at 15 minutes incubation (70 %) with E.coli.The findings support the fact that innate immunity can be measured qualitatively and quantitatively by the technique described which will be useful for the identification of immunocompromised dogs.
clinically healthy dogs of 2-10 years of age presented to corresponding absorbance for bacterial count of 1 x 10 /µl Veterinary Teaching Hospital (VTH), University of was extrapolated from a standard curve plotted with Peradeniya.The study sample comprised of 70% mongrel absorbance against Log bacteria counts.This bacterial 10 and 30% cross bred dogs of both sexes.The EDTA blood concentration was used in all experiments to yield a was subjected to full blood count using MS 9-5V neutrophil:bacteria concentration of 1:50 for optimum Veterinary Hematological counter (Melet Schloesing phagocytosis.One hundred microlitre of the above Laboratories, 2003 France) and the differential leukocyte bacterial suspension was mixed separately and counts were calculated from Leishman stained thin blood individually with 100 µl of serum collected from each 0 smears.Thirty six dogs with cellular parameters within animal, and was incubated at 37 C for 30 min for the normal ranges and negative for blood parasites were opsonization.Tubes were gently mixed at 5min intervals.selected for the study (Silva and Mallawa, 2010).Blood Samples were kept on ice until used for the phagocytosis sample without EDTA was used to obtain fresh serum. assay.

Isolation of neutrophils Assay for phagocytic activity The leukocyte isolation protocol of Carlson and Kaneko
In a single experimental run, 200ml opsonized bacterial (1973), which was modified by increasing the duration for suspension was mixed with the calculated volume of 0 RBC lysis to 50 seconds, based on results of a prior neutrophil suspension and incubated at 37 C. A negative experiment done to measure the minimum time required control was made by substituting the bacterial aliquot with to lyse RBCs in canine blood.The EDTA blood was PBSa.An aliquot of the incubated sample was used to 0 centrifuged at 200 x g for 10 min at 4 C in a Hettich prepare thin smears for microscopic examination at 15 Universal 32 R centrifuge (Germany).The plasma and the and 30 min incubation.buffy coat were decanted, in order to increase the harvest of phagocytes which were lodged in the hematocrit, based Staining, Microscopic Examination and Statistical on preparatory investigations.The remaining packed red Analysis cells were mixed with 6ml of sterile Distilled Water (DW) The above thin air-dried smears of leucocyte-bacteria pH 7.2 for 50 sec to lyse the RBCs.Then 4 ml of sterile were stained with Leishman.The percentage of phosphate buffered saline in 0.0132 M phosphate buffer phagocytically active neutrophils (cells with ingested E. with 2.7% NaCl, pH 7.2 (PBSb) was added to the cell coli) was determined by counting 5000 neutrophils under suspension to restore isotonicity.Tubes were then mixed oil immersion objective (x1000 magnification) of a light 0 gently and centrifuged at 200 x g for 10 min.at 4 C.The microscope.Paired t-test using Minitab release 14 was supernatant was discarded and the pellet was washed by used to observe the statistical significance at (P < 0.05), resuspending in 4 ml of 0.0132M phosphate buffer, pH 7.2 under 95% confidence interval.0 (PBSa) and re-centrifuged at 200 x g for 10 min at 4 C.The washing process was repeated once more to remove RESULTS AND DISCUSSION debris, if any from the cell lysis, and the pellet was suspended in 500 µl sterile PBSa and was kept on ice until The present study was conducted to investigate the in used.The viability of the isolated leukocytes was vitro phagocytic activity of blood neutrophils of dogs in calculated using 0.1% Nigrosin (Cheesbrough, 2006) Sri Lanka for Escherichia coli.The study sample within 3 min of isolation.The differential leukocyte comprised of 70% mongrel and 30% cross bred dogs of counts of isolated leukocytes of each dog were counted on both sexes.The leukocyte isolation method described Leishman stained smears to estimate the neutrophil count yielded 1,170 -12,930/ìl leukocytes of which 61-90% was in each sample to be used in the assay.All samples were neutrophils.The viability of the isolated leukocytes analyzed within one and half hours of collection.
ranged from 90-99% (mean 96%).Neutrophils isolated from fresh blood samples of clinically healthy dogs were Opsonization of bacterial suspension incubated with E.coli which is a facultative gram-negative A pure culture of Escherichia coli (ATCC 25938) in 5 % bacterium found in the normal flora of the gastrointestinal sheep blood agar (Oxoid, UK) was used in the study.One tract.The mean percent phagocytosis (MPP) of canine colony of E. coli was inoculated into 5 ml of brain heart blood neutrophils for a pure culture of E. Coli ATCC 0 infusion broth (Oxoid, UK) and incubated at 37 C for 18 25938, was 70% (63%-73%) and 89 % (78% -92%) at 15 hrs.The broth culture was centrifuged at 2000 x g for 15 minute and 30 minute incubation, respectively.Similar minutes and the resulting pellet was resuspended in 10 ml phagocytic activity had been recorded for blood sterile PBSa.neutrophils of cows for E. coli which were 74% and 84% The optical density of a series of two fold dilutions of at 15 and 30 minutes incubation, respectively (Silva et al., the bacterial sample was recorded at 540 nm wavelength 1989).It was also revealed that MPP of bovine neutrophils using a Jenway 6400 spectrophotometer.The viable was high for E coli, Salmonella sp, Strep.agalactiae (73bacterial count of each two -fold dilution was determined 81%); intermediate for Staph.aureus (64%), and low for by the spread plate method (Harrigan & McCance, 1976).M. bovis and B. abortus (24 -40%) (Silva & Jain 1988).Briefly, a tenfold serial dilution was made for each of the The phagocytic activity of buffalo (Bubalis bubalus) was above two-fold dilutions using sterile PBSa to calculate slightly different in that the MPP was highest for Staph the viable bacterial count in each two-fold dilution.The aureus (82%) at 30 min incubation compared to E. coli (77%) and Strep.agalactiae (72%) (Silva and in the blood, viral infection, such as Distemper, or a Kariyawasam, 1996).When buffalo (Bubalus Bubalus) congenital defect.neutrophils were exposed to mixed bacterial cultures, Products of bacteria activate the expression of the affinity for phagocytizing E. coli was much less in glycoprotein on capillary endothelial surface, tether the presence of Staph.aureus than in the presence of neutrophils and stimulating them to roll along the Strep.agalactiae (Silva and Thattil, 1995).
endothelium (Tizard, 2009;Kaneko, 1997).The platelet The MPP of neutrophils at 15 min incubation was activating factor (PAF) secreted by the endothelial cells, significantly lower than that at 30 minute incubation (P activate the rolling neutrophils to express the adhesive = 0.00), indicating that 30 minute is required to obtain a protein integrin, which allows the neutrophils to bind better phagocytic activity.It has been shown previously strongly to a glycoprotein intercellular adhesion that although phagocytosis was not impaired after 30 or molecule-1 (ICAM-1; CD54) expressed on the 60 minutes the average bactericidal activity incubation endothelial cells.This strong binding allows the rolling diminishes significantly after 30 minutes (Gosset, neutrophils to attach firmly to the vessel wall despite the 1983).However, the phagocytic activity and shearing force of the blood flow.Younger dogs have postphagocytic bactericidal activity of buffalo significantly higher levels of mRNA for interleukin-8 neutrophils improved when incubated up to 60 minutes receptor (IL-8R), l-selectin and interleukin-1beta-(86% for E. coli and 81% for Strep.agalactiae) (Silva converting enzyme (ICE), specific for neutrophil and Kariyawasam, 1996).Quantitative analysis of migration or killing functions.A significant effect of age phagocytosis and oxidative burst in canine neutrophils on percent bacterial killing by neutrophils has been performed by flow cytometry techniques showed that shown in Beagle dogs, with a decline in phagocytic storage of canine PMN for 24 hrs at room temperature ability from 39% to 25% at 4 and 10 years of age, had no negative influence on phagocytosis or oxidative respectively, suggesting that older dogs have depressed burst measurements (Eickhoff et al., 2004).Untreated innate immune responses compared with younger dogs carcinomas and sarcomas in dogs may suppress the (Hall et al., 2010).oxidative burst of neutrophils while sarcomas may The findings support the fact that the innate immunity suppress the phagocytic activity of neutrophils of dogs can be measured qualitatively and quantitatively (LeBlanc et al., 2010).The bactericidal activity during and the technique described can be useful for the the course of the inflammation did not correlate with identification of immunocompromised dogs.neutrophil count, number of toxic neutrophils, or clinical course of the inflammation (Gosset, 1983).

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