Stimulatory effect of avian influenza virus on chicken lymphocytes

A study was conducted to examine the effect of avian influenza virus (AIV) on chicken lymphocyte activation. Unprimed or Brucella abortus antigen (Ag)-primed lymphocytes were incubated with various doses of the T-cell mitogen concanavalin A (Con A) or Ag, respectively, plus serial dilutions of inactivated AIV for 72 hr, and cell proliferation was measured via uptake of tritiated thymidine. AIV enhanced the proliferative response to Con A or Ag by 150% or better, and the enhancement decreased in a viral dose-dependent manner. The effects were more readily observed in cells that had not been maximally activated by the Con A or Ag. The enhanced response was observed in lymphocytes from both white rock and white leghorn breeds of chicken and in mature peripheral blood lymphocytes or immature thymocytes. The viral activity could be abrogated by pre-treatment of the viral preparation with AIV-specific antisera or prior adsorption of the AIV with chicken erythrocytes. These results indicate that AIV can interact with and modify the in vitro activity of chicken lymphocytes and may exert modulatory effects on the avian immune system.     

Counting absolute number of lymphocytes in quail whole blood by flow cytometry

In a previous study, we reported a new method for counting quail blood cells. After quail blood cells were stained with fluorescent lipophilic dye (DiOC6(3)), absolute counts of erythrocytes, granulocytes, and monocytes were obtained by means of flow cytometry (FC). The FC method has the potential for application to avian blood cells count; however, the method was unable to distinguish between lymphocytes and thrombocytes. In the present study, we improved the FC method to obtain separate counts of lymphocytes using DiOC5(3). After quail blood cells were stained with DiOC5(3), the cells were measured with FC. Each blood cell type was distinguished by means of their typical FL-1 (green fluorescence) and SSC (side scatter). Absolute numbers of erythrocytes, granulocytes, monocytes and lymphocytes in whole blood were obtained. The improved FC analysis worked equally well with chicken (Gallus gallus) and goose (Anser cygnoides) blood.     

安徽省合肥地区家禽交易市场禽流感疫情定点监测情况分析

为了了解安徽省合肥地区家禽禽流感免疫及带毒情况,2011年4—8月,在合肥地区家禽交易市场,采集不同来源、不同品种家禽的血清和同份棉拭子样品（咽喉、泄殖腔双份）603份（实测589份）,分别进行高致病性禽流感免疫抗体和病原学检测,结果表明,全部样品平均免疫抗体合格率为70.80%。对监测结果进行分析发现,不同品种、不同养殖规模、不同月份的高致病性禽流感免疫抗体存在差异,其中,蛋鸡、种鸡高致病性禽流感免疫抗体合格率达80%以上,而肉鸡及水禽的免疫抗体水平则较差;饲养规模在1000羽以上的养禽场家禽高致病性禽流感平均免疫抗体合格率均达到70%以上,而饲养规模在1000羽以下的养禽场和农村散养家禽免疫抗体合格率分别为27.45%和32.84%,尚未达到农业部规定标准;在6月采集的样品,其平均免疫合格率偏低,其他月份差异不显著。病原学检测结果表明,全部检测样品高致病性禽流感病原检测结果均为阴性。     

白头翁及皂苷对鸡免疫功能的影响

为考察白头翁及白头翁皂苷对鸡免疫功能的影响,将AA肉鸡于15日龄添加白头翁及白头翁皂苷饮水,以黄芪组为阳性对照、未加药为阴性对照。通过测定免疫器官增重、白细胞吞噬率与吞噬指数等,考察白头翁及白头翁皂苷对鸡免疫功能的影响。结果表明：白头翁及其皂苷组比对照组在体重和免疫指数方面有明显的优越性,尤其在中性细胞吞噬白色葡萄球菌方面与对照组有极显著差异（P〈0.01）。白头翁及白头翁皂苷对鸡免疫有一定的促进作用,试验为白头翁生产应用提供了参考数据。     

Characterization of porcine peripheral blood leukocytes by light-scattering flow cytometry.

As a basis for other experiments using flow cytometry of porcine peripheral blood leukocytes, cell fractions were isolated by various methods and analyzed by forward angle light scatter and 90 degree light scatter. Cytospin smears of cell samples were also studied by leukocyte differential counts and nonspecific esterase staining. Three main populations of peripheral blood leukocytes [lymphocytes, monocytes, and granulocytes (primarily neutrophils)], were defined in the log 90 degree light scatter by forward angle light scatter histogram. Partial overlap was observed between lymphocyte and monocyte, and between monocyte and granulocyte domains. Correlation between leukocyte differential counts and flow cytometric quantification based on bitmap statistics of appropriate domains was between r = 0.872-0.892 for lymphocyte and granulocyte. Percoll density gradients were used for subfractionation of leukocyte populations, especially for the enrichment of granulocytes. The specific densities were calculated for lymphocytes (1.0585-1.0819 g/cc), monocytes (1.0585-1.0702 g/cc), granulocyte (1.0819-1.0936 g/cc), and erythrocytes (greater than 1.0952 g/cc). We suggest that light scatter characterization is a basis for future studies of porcine blood by flow cytometry.     

Immune status of PIC Camborough-15 sows, 25% Meishan sows, and their offspring kept indoors and outdoors

Newer genetic lines of pigs are being used in indoor and outdoor production systems. The objectives of Exp. 1 were to describe the effects of the maternal sow line genotype, environment (indoor vs outdoor), and the genotype x environment interactions on blood hemoglobin (Hb), immunoglobulin G (IgG) concentrations, white blood cell (WBC) numbers, lymphocyte transformation/blastogenesis (LTA), natural killer (NK) cell activity, neutrophil chemotaxis, cortisol concentrations, and leukocyte differentials. Studies were performed using two genotypes: PIC Experimental-94 (Exp-94, an experimental line containing 25% Meishan) and PIC Camborough-15 (C-15). The Exp-94 sows had lower LTA at 0.2 microg/mL mitogen than the C-15 sows, whereas Exp-94 sows had higher NK cytotoxicity than the C-15 sows. When indoors, the two genotypes showed similar neutrophil chemotaxis. When outdoors, the C-15 genotype had higher (P < .01) neutrophil chemotaxis than the Exp-94 sows. The other immune measures were statistically similar for the two genotypes for each environment and for the genotype x environment interaction of sows. Experiment 2 sought to determine the effects of genotype on the immune system of nursery-age offspring of the experimental lines. Each sow line was bred to a common PIC 405 boar line. The Exp-94 x 405 pigs had elevated WBC numbers than C-15 x 405 pigs. The social status of the Exp-94 x 405 or the C-15 x 405 pigs showed no effect on any of the immune measures studied. The other immune measures were statistically similar for the two lines of pigs. The Exp-94 line had marginally increased NK activity but reduced lymphocyte blastogenesis and neutrophil chemotaxis compared with the C-15 line.     

Flow cytometric patterns in blood from dogs with non-neoplastic and neoplastic hematologic diseases using double labeling for CD18 and CD45

BACKGROUND: In dogs, flow cytometry is used in the phenotyping of immunologic cells and in the diagnosis of hemic neoplasia. However, the paucity of specific antibodies for myeloid cells and B lymphocytes and of labeled antibodies for multicolor techniques limits the ability to detect all leukocyte subpopulations. This is especially true for neoplastic and precursor cells. CD18 and CD45 are expressed on all leukocytes and are involved in cell activation, and together could be useful in helping determine cell lineage. OBJECTIVES: The purpose of this study was to double label canine blood for CD18 and CD45 and to use the differential expression of antigens to identify leukocyte populations in dogs with non-neoplastic and neoplastic hematologic diseases. METHODS: A template was developed using blood samples from 10 clinically healthy dogs and a back-gating technique. Differential leukocyte counts obtained with the template were compared with those obtained by manual and automated methods on blood samples from 17 additional healthy dogs. Blood samples obtained from 9 dogs with non-neoplastic (reactive) hematologic diseases and 27 dogs with hemic neoplasia were double stained for CD18 and CD45 using mouse anticanine CD18 monoclonal antibody (mAb) plus phycoerythrin-conjugated rat anticanine CD45 mAb and fluorescein isothiocyanate-conjugated rabbit antimouse IgG. Hemic neoplasms were diagnosed by cell morphology, and immunophenotypic and cytochemical markers. RESULTS: With the double label, neutrophils, eosinophils, monocytes, and T- and B-lymphocytes were identified. In reactive disorders, a population of activated neutrophils with high CD45 and CD18 expression was detected. In hemic neoplasia, cell lineage was easily determined, even in acute leukemia. CONCLUSIONS: Double labeling for CD18/CD45 may be useful as a screening method to evaluate hematologic diseases and help determine cell lineage, and to aid in the selection of a panel of antibodies that would be useful for further analysis.     

Clinical evaluation of the CA530-VET hematology analyzer for use in veterinary practice

BACKGROUND: The CA530-VET is a completely automated impedance cell hematology analyzer, which yields a 16-parameter blood count including a 3-part leukocyte differential. OBJECTIVES: The aim of this study was to examine the operational potential of the CA530-VET and its value for use in veterinary practice. METHODS: The analyzer was tested for blood carry-over, precision, and accuracy. Comparison methods included the CELL-DYN 3500, microhematocrit centrifugation, manual platelet (PLT) counting for feline and equine species, and a 100-cell manual WBC differential. Blood samples for comparison of the methods were obtained from 242 dogs, 166 cats, and 144 horses. RESULTS: The carry-over ratio (K) was 0.28% for RBC, 0.59% for PLT, 0.32% for WBC, and 0.18% for hemoglobin (HGB) concentration. Coefficients of variation (CVs) for within-batch precision and duplicate measurement of blood samples were clearly within the required limits, except for duplicate platelet counts in cats (8.7%) and horses (9.5%). The WBC count was in excellent agreement for dogs and horses and RBC count was in excellent agreement for horses. The accuracy of feline WBC counts was not acceptable, with the exception of values at the high end of the range. RBC counts in dogs and cats, and HGB concentration and MCV in all 3 species were sufficiently accurate. The CA530-VET HCT results were in excellent agreement with microhematocrit results in horses but exceeded the maximum allowed inaccuracy for cats and dogs. In all species, PLT counts established mechanically and manually were not in adequate agreement. Large differences were found between the CA530-VET and the manual differential percentage for lymphocytes and "mid-sized cells" (monocytes and basophilic granulocytes). CONCLUSIONS: The CA530-VET can be considered useful for routine canine, feline, and equine blood cell analyses. It should not be considered accurate, however, for PLT counts, feline total WBC counts in the subnormal and normal range, and leukocyte differentials, except for granulocytes.     

Differential leukocyte counts determined in chicken blood using the Cell-Dyn 3500

Background: Automated hematology instruments commonly are used for mammalian blood analysis, but there is a lack of accurate automated methods available for avian leukocyte analysis. Objective: The aim of this study was to validate differential leukocyte counts in blood from chickens using the Cell-Dyn 3500 hematology system and avian-specific software.
Methods: Blood samples were collected in lithium-heparin tubes from 2 groups (n = 84 and n = 139) of laying hens. Manual 200-cell differential counts were done on routinely-stained blood smears, and manual total granulocyte counts (heterophils and eosinophils) were done using an eosinophil stain in a counting chamber. Automated differential counts were done using VET 2.3, a research and development version of avian-specific software for the Cell-Dyn 3500. Results were analyzed using Pearson's correlation and difference plots.
Results: Automated granulocyte counts from the Cell-Dyn were in good agreement with manual granulocyte counts ( r = 0.93 and 0.80 for the 2 study groups). No correlation was found between automated and manual lymphocyte counts. Correlation coefficients for monocyte counts were 0.70 and 0.43. Conclusion: Automated leukocyte results from the Cell-Dyn using VET 2.3 software were not fully accurate. Total granulocyte counts may be of clinical usefulness, but results obtained for other parameters were unreliable.     

禽白血病p27抗原检测阳性与病毒分离的相关性比较

本试验以80只300日龄的A品系蛋鸡为试验对象,分5个日龄段按翅号采集蛋清、泄殖腔棉拭子,无菌抗凝血和血清.用ALV p27抗原检测试剂盒检测蛋清和泄殖腔棉拭子.将无菌抗凝血分离血浆接种DF-1细胞,培养一周后用同样方法检测上清收集液,分析该群鸡只在不同日龄段泄殖腔棉拭子阳性、蛋清样本阳性和病毒分离阳性之间的相关性.用ALV-Ab抗体试剂盒检测各日龄段血清的抗体水平.此外,选取某一日龄段蛋清和泄殖腔拭子样本用4个不同厂家的ALV p27抗原检测试剂盒进行检测比较.结果表明,5个日龄段泄殖腔棉拭子平均阳性率为61％,蛋清样本平均阳性率为72.6％,病毒分离平均阳性率为48.8％.5个日龄段ALV的抗体阳性率一直为零;4个厂家的ELISA试剂盒对同一批样本的检测结果表明,IDEXX试剂盒的敏感度最高.本试验为外源性鸡白血病病毒检测及鸡白血病净化其方法的应用、试剂盒的选择、减少判定的误差、提高净化效果提供了一定的科学依据.     

Changes in peripheral blood leukocyte populations in pigs with naturally occurring exudative epidermitis

The objective of the study was to analyze changes in peripheral blood leukocyte subsets in cases of naturally occurring exudative epidermitis (EE) in pigs. Five of ten piglets developed the chronic clinical form of EE 2-5 days after weaning (PW). Blood samples were obtained at 7, 14 and 21 days from both normal and clinically affected piglets for routine haematology and for the determination of CD45, CD21, CD4, CD8 and gammadeltaTCR cell markers by flow cytometry. When compared with clinically normal piglets EE affected pigs showed significantly decreased values of monocytes at 14 and 21 days PW, and increased numbers of neutrophils and leukocytes at 21 days PW. The EE affected pigs also had an early significant CD4(+) and CD8(high+) T lymphocyte proliferative response at 7 days PW. However affected pigs had a significantly reduced number of B (CD21(+)) and gammadeltaTCR(+) T lymphocytes in blood at 21 days PW. Although all values remained within the normal range, the significant differences in some peripheral blood leukocyte subsets between the two groups of piglets suggest that the generalised cutaneous infection with Staphylococcus hyicus is severe enough to induce a systemic inflammatory and immune responses.     

Evaluation of piglet blood utilizing the Technicon H*1

The analytic precision of an automated blood analyzer, the Technicon H*1(R), was evaluated utilizing blood samples collected from 20 piglets at 1 and 14 days of age. The effect of storing the blood samples at 4 degrees C for 24 and 48 hours also was determined. Blood samples were analyzed twice on the first day and once on each of the subsequent tow days. Within-sample coefficient of variation was approximately 1% for hemoglobin concentration, erythrocyte count, hematocrit, mean cell volume, erythrocyte distribution width and hemoglobin distribution width (HDW); and approximately 5% for total leukocyte (WBC), neutrophils and lymphocyte counts. Mean HDW and automated differential WBC counts changed during storage to a degree that could be of clinical importance. Manual determination of differential WBC counts were compared with those obtained from the automated analyzer. Results correlated well for neutrophils (r=0.92 in 1-day-old and r=0.93 in 14-day-old piglets, P<0.001) and lymphocytes (r=0.85 in 1-day-old and r=0.93 in 14-day-old piglets, P<0.001). Other WBC values were too low to compare reasonably.     

Flow cytometric analysis of feline reticulocytes.

Hemolytic anemia was induced in five Domestic Shorthair cats (four adult males and one spayed female obtained from a breeding colony at Colorado State University, CO), and blood samples were analyzed from five other cats (two castrated male Domestic Shorthairs, one castrated male Domestic Longhair, one castrated male Persian, and one spayed female Siamese presented to the Veterinary Teaching Hospital at Colorado State University for miscellaneous problems). Blood samples taken from these cats had percentages of aggregate reticulocytes that ranged from 0% to 14.5% as determined by manual counting and were used to identify the best technique for staining cat reticulocytes for flow cytometric analysis. The best technique was mixing a blood sample (1/2,000 dilution) with 0.2 micrograms thiazole orange in 1 ml of diluent and incubating the mixture in the dark at room temperature for 30 to 60 minutes. The percentage of reticulocytes determined by flow cytometry correlated well (r = 0.88) with manually determined aggregate reticulocyte percentages; no significant differences were observed between the two techniques (P > 0.05). For the conditions used, punctate reticulocytes were not detected by flow cytometry. Samples with very high platelet numbers and very low packed cell volumes may show falsely elevated percentages of reticulocytes as determined by flow cytometry. The reproducibility of the flow cytometric technique was good; the coefficient of variation ranged from 4.8% to 17.9% in two samples with two different times of incubation. Staining of cat aggregate reticulocytes with thiazole orange and use of flow cytometric quantification is a reproducible technique that has a good correlation with the manual reticulocyte counting method.     

Erythrocyte rosettes--a marker for bovine T cells.

Many species of erythrocytes were investigated for their ability to form spontaneous rosette with bovine peripheral blood leukocytes and fetal thymocytes. Only sheep and chicken red blood cells gave rosettes. Using conditions shown optimum for the demonstration of human rosette forming cells, only low numbers of bovine rosettes were demonstrable. By changing culture conditions to include 100% fetal calf serum, neuraminidase treated erythrocytes and/or lymphocytes and optimizing the incubation times and temperature, up to 38% of peripheral blood leukocytes and 52% of thymocytes formed rosettes. A thymic origin of rosetting cells was ascribed to T cells for the following reasons: 1) thymocytes gave higher numbers than did peripheral blood leukocytes, 2) rosette forming cell numbers were increased in peripheral blood leukocyte subpopulations enriched in T cells by nylon column separation and 3) only very few rosette forming cells had surface immunoglobulin, a marker of B lymphocytes. The reasons why all T cells were not detected by the technique were discussed.     

Use of a prestorage leukoreduction filter effectively removes leukocytes from canine whole blood while preserving red blood cell viability

Leukoreduction of blood products is a technique used to prevent leukocyte-induced transfusion reactions. Filters currently used for human blood products achieve at least a 99.9% reduction in leukocyte numbers per unit (450 mL) of blood. Goals of this study were to determine if a prestorage leukoreduction filter could effectively achieve leukoreduction of canine blood and to determine if viability of the leukoreduced red blood cell (RBC) product could be maintained after 35 days of storage. Blood collected from each dog was filtered through a leukoreduction filter at either room temperature or after cooling (4 degrees C) for 4 hours. Filtration efficacy was determined by measurement of pre- and postfiltration leukocyte counts. In vitro viability of RBCs was determined by comparing RBC adenosine triphosphate concentration and percent hemolysis before and after the storage period. In vivo viability of stored cells was determined using a biotin-streptavidin-phycoerythrin labeling technique and flow cytometry. Blood filtered within 30 minutes of collection versus blood filtered after cooling had mean reductions in leukocyte numbers of 88.90 and 99.99%, respectively. The mean ATP and hemoglobin concentrations from the in vitro analysis were comparable to those obtained in previously for canine RBC adequately stored for 35 days. The mean in vivo 24-hour survival of the stored RBC was 84.7%. The leukoreduction filter used did not adversely affect in vitro or in vivo viability of canine RBCs. The filter effectively removed leukocytes from blood, with maximal efficiency of filtration achieved with use of cooled blood.     

Canine differential leukocyte counting with the CellaVision DM96Vision, Sysmex XT-2000iV, and Advia 2120 hematology analyzers and a manual method

Background: For differential leukocyte counts, automated blood smear evaluation systems have been too slow or inaccurate to replace or supplement the manual differential count. The CellaVision DM96Vision (DM96V), a new instrument, is an automated image analysis system that is rapid and accurate enough to be used for enumerating human leukocytes and may be useful for analysis of canine blood. Objectives: The aims of this study were to evaluate the performance of the DM96V in differential counting of canine leukocytes, to compare its performance with that of other methods, and to analyze interoperator variability. Methods: Four methods of determining the leukocyte differential count of 108 canine blood samples were compared based on agreement, precision, and errors as well as relative performance. Differential counts were obtained using the DM96V, the manual method, and automated methods performed by the Advia 2120 and Sysmex XT‐2000iV. Results: All leukocyte types were detected by the DM96V and the manual method, and all 4 methods had similar mean and median results in most cases. The automated methods were more precise than either the DM96V or manual method when comparing identification of a single type of leukocyte, especially neutrophils and lymphocytes. However, precision of the automated methods was only fair for monocytes, and the Advia and Sysmex failed to identify basophils. The Advia reported fewer monocytes and eosinophils than did the other methods. Significantly fewer lymphocytes were identified by the manual method than by the Sysmex, Advia, and DM96V. The DM96V occasionally presented duplicate images of the same neutrophils. Conclusions: The CellaVision DM96V is a satisfactory system for facilitating canine differential leukocyte counting. The DM96V differential count was more similar to the manual count than to automated counts, which were more precise but had errors and omissions in detecting some types of leukocytes.     

A rapid microtechnique for in vitro stimulation of canine lymphocytes using whole blood

A simple, rapid microtechnique utilizing whole blood has been developed for evaluating the in vitro stimulation of canine lymphocytes. The test uses 5 ul. of heparinized whole blood per culture in a microtiter plate and requires no serum supplement. Cultures are harvested by an automated multiple sample cell harvester. This technique permits large numbers of replicate samples to be tested on individual animals with minimal amounts of blood and minimal technician time.     

Molecular characterization and B cell membrane expression analysis of Fc fragment gene of goose IgY

A novel goose immunoglobulin υ chain (Igυ) Fc fragment gene was cloned from splenic tissue mRNA using RT-PCR. Deduced amino acid sequence data from different vertebrates revealed high similarity to IgY-Fc fragments of duck (91%) and chicken (64%). Molecular characterization showed that the goose IgY-Fc fragment was consistent with the definition of immunoglobulin, and had the same antigenicity to natural IgY. Flow cytometry and laser scanning confocal microscopy showed that the polyclonal antibody against GoυFc reacted with the membrane surface of B lymphocytes in peripheral blood, which indicates that IgY was expressed on the surface of B cells. Analyses of the gene sequence of the goose IgY-Fc fragment and expression of B cell membrane may provide insight into the evolution of the Ig heavy chain gene family and benefit future studies on the avian immune system.     

4型禽腺病毒HLJ1701株灭活疫苗的研制

利用4型禽腺病毒HLJ1701株进行灭活疫苗的研制,并对疫苗的免疫效果进行评价,为家禽4型禽腺病毒的防控提供数据及参考。将HLJ1701株用灭菌生理盐水作10~4倍稀释后,接种9日龄SPF鸡胚,37℃孵育72 h后收获感染鸡胚尿囊液,经甲醛灭活后,加白油佐剂乳化制成油乳剂灭活疫苗,对制备疫苗的性状、安全性、免疫效力等进行检验。结果显示,制备的3批4型禽腺病毒灭活疫苗(HLJ1701株)均为油包水型,黏度均在50 cP以内,对3批疫苗取样,样品经3000 r/min离心15 min,管底无水相析出。安全性试验结果显示,将疫苗按1 mL/只超剂量接种3周龄SPF鸡,试验鸡在观察期内全部健活,未出现局部或全身不良反应,表明疫苗对SPF鸡具有良好的安全性;免疫效力及攻毒保护试验结果显示,用疫苗按0.2 mL/只的剂量免疫接种3周龄SPF鸡1次,免疫接种后21d试验鸡血清中HLJ1701株的抗体平均效价可达2~8以上,使用4型禽腺病毒(HLJ1701株)接种0.2 mL/只(100 LD_(50))对免疫鸡进行攻毒,疫苗对免疫鸡的保护率均为100%。研究表明,实验室条件下研制的4型禽腺病毒(HLJ1701株)灭活疫苗的各项指标均符合标准。     

Causes of erroneous white blood cell counts and differentials in clinically healthy young northern elephant seals (Mirounga angustirostris).

From 1993 to 1995, approximately 10% of the clinically healthy northern elephant seals (Mirounga angustirostris) at The Marine Mammal Center in California exhibited a large unexplained increase in their white blood cell (WBC) count. In these animals, WBC counts ranged from 28,780 to 125,000/mm3, with a mean of 50,087/mm3. Significant correlations between the leukocytosis and weight gain and day of admittance were identified, but no correlation existed between leukocytosis and general state of health, sex, length of stay, or diet. Bone marrow contamination of blood samples, erroneous automated leukocyte counts, and leukogram changes consistent with subclinical inflammation were the major factors contributing to the elevated WBC counts in these apparently clinically healthy animals.