45 鼠伤寒沙门氏菌脂多糖对肉鸡的影响

Effects of Salmonella typhimurium Lipopolysaccharide on Broiler Chickens 1

H.Xie,*,?N.C.Rath,*,2G.R.Huff,*W.E.Huff,*and J.M.Balog**USDA-ARS-PPPSRU and ?Department of Poultry Science,Poultry Science Center,

University of Arkansas,Fayetteville,Arkansas 72701

ABSTRACT The effects of Salmonella typhimurium lipo-polysaccharide (LPS)on the physiology of 3-wk-old broiler chickens were studied at 12,24,and 48h after a single intravenous injection of saline or LPS.Lipopolysac-charide elevated cloacal temperature by 3h after injection,induced a diuretic response,and decreased BW gain.An increase in the relative liver weight was evident in LPS-treated birds at all time intervals,whereas a decrease in the relative weight of bursa of Fabricius was observed only at the 48-h time point.The plasma interleukin (IL)-6and the blood heterophil concentrations were elevated at 12and 24h following LPS administration.These changes were not observed in control chickens or in LPS-treated chickens at 48h.A decrease in the blood glucose concentration in LPS-treated birds at 12h was accompa-(Key words :chicken,lipopolysaccharide,interleukin-6,liver,heterophil,acute phase protein)

2000Poultry Science 79:33–40

INTRODUCTION

Lipopolysaccharides (LPS)are cell wall components of Gram-negative bacteria that cause in?ammation and sickness (Verdrengh and Tarkowski,1997).The sickness is caused by a variety of neuroendocrine and immunolog-ical changes that activate different hematopoietic and nonhematopoietic cells (Fox and Rajaraman,1980;Mor-rison,1983;MacKay and Lester,1992;Berczi,1998;Naka-mura et al.,1998;Takahashi et al.,1998).These cells in turn produce several pro-and anti-in?ammatory hor-mones and cytokines that regulate different metabolic responses and cause behavioral changes,fever,in?am-mation,cachexia,and ?nal recovery (Abbas et al.,1997).Studies on the effects of LPS on poultry are limited com-pared with studies using mammals because it was gener-

Received for publication March 4,1999.Accepted for publication August 11,1999.1

Mention of a trade name,proprietary product,or speci?c equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.2

To whom correspondence should be addressed:nrath@https://www.wendangku.net/doc/ff3525215.html,.3

Celox Laboratories,Hopkins,MN 55343.4

Bio-Rad Laboratories,Hercules,CA 94547.5

Boehringer Mannheim Co.,Indianapolis,IN 46250.

33

nied by an elevation in the blood phosphate level.An increase in total plasma protein concentration was ob-served only at 24and 48h after LPS https://www.wendangku.net/doc/ff3525215.html,para-tive SDS-PAGE analysis of plasma proteins from these birds under nonreducing conditions showed some quan-titative differences in four bands of proteins between sa-line and LPS-treated chickens.A protein corresponding to an approximate molecular weight (MW)of 65kDa increased in LPS-treated chickens,and three other pro-teins with MW of approximately 39,49,and 56kDa showed reductions in concentration compared with sa-line-treated controls.These results show that LPS induces a number of physiological changes that may be responsi-ble for the regulation of the acute phase response in chickens.

ally considered that birds may be relatively more resistant to endotoxins than mammals (Roeder et al.,1989).How-ever,in recent years many studies on the effects of LPS on chickens have been reported using different in vivo and in vitro parameters.Those studies suggest that birds also show many similar patterns of response to endotox-ins as mammals (Curtis et al.,1980;Jones et al.,1983;Klasing and Peng,1987;Miller and Qureshi,1992;Zhang et al.,1995;Koh et al.,1996;Sunwoo et al.,1996).Neverthe-less,there is a paucity of information on the effects of LPS on cytokines and the acute phase response in birds and their relationship with in?ammation and homeosta-sis.The current study was undertaken to investigate the effects of LPS on different physiological parameters of 3-wk-old male broiler chickens at different time intervals from 12to 48h after a single intravenous injection of either saline or LPS.

MATERIALS AND METHODS

Chemicals,Reagents,and Cells

RPMI-1640medium,3protein determination kit,β-mer-captoethanol,4recombinant human interleukin (IL)-6,5

Abbreviation Key:AGP =α1-acid glycoprotein;APP =acute phase protein;FBS =fetal bovine serum;H:L =heterophil:lymphocyte;IL =interleukin;LPS =lipopolysaccharide;MTT =3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide;MW =molecular weight;RBC =red blood cells;WBC =white blood cells.

XIE ET AL. 34

and fetal bovine serum(FBS)6were purchased.Reagents used for clinical chemistry and differential cell counts were bought from Ciba Corning Diagnostics Corp.7and Abbott Diagnostics Corp.8,respectively.The B9hybrid-oma cells were kindly provided by L.Aarden9.All other reagents and chemicals,including Salmonella typhimurium LPS,sodium pyruvate,nonessential amino acids,antibi-otic/antimycotic solution,gentamicin,and3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT), were obtained from Sigma Chemical Co.10

Chickens

One-day-old male broiler chickens(Cobb×Cobb)that had been vaccinated in ovo were obtained from a local hatchery and housed in starter batteries with ad libitum access to water and commercial feed that met NRC recom-mendations.At3wk of age,the chickens were randomly divided into six groups of seven birds each with an aver-age BW of0.69±0.03kg.For each time interval study, two groups of chickens were used.One group was in-jected with saline,and the other was injected with LPS. The birds were killed at12,24,and48h after injection. The chickens received weighed amounts of feed at the beginning of injection,and the feed weight was measured at the termination of the experiment to assess the amount of feed consumed during the period. Lipopolysaccharide Administration and Temperature Measurement

The chickens were injected intravenously with either 0.2mL sterile saline(control)or LPS dissolved in saline at an approximate dose of5.0mg/kg BW.The cloacal temperature of each bird was measured before and at3 h after injection using a thermocouple rectal probe ther-mometer.11Subsequently,the temperature of each group of chickens was measured prior to bleeding and euthana-sia.General behavioral changes,such as agility and feed-ing patterns of these birds following treatments,were noted up to4h.

Blood Collection

Respective groups of birds were bled by cardiac punc-ture at12,24,and48h after injection before euthanasia, and blood was collected using heparinized tubes.Ali-quots of heparinized blood were used for differential cell counts.The rest of the blood was used to obtain plasma by centrifugation at1,500×g for15min.The plasma was used for blood chemistry determination,IL-6bioassay, and SDS-PAGE analysis of plasma proteins.

6Hy Clone Laboratories,Inc.,Logan,UT84321.

7Norwood,MA02062.

8Abbott Park,IL60064.

9The Netherlands Red Cross,1006AD Amsterdam,The Netherlands.

10St.Louis,MO63178-9916.

11Physitemp Instruments,Inc.,Clifton,NJ07013.

12Spectramax,Molecular Devices Corp.,Sunnyvale,CA94089.Body and Organ Weights

The BW of each bird was measured before injection (initial BW)and before bleeding(?nal BW).The change in BW caused by saline or LPS treatment was expressed relative to initial BW as a percentage of initial BW and calculated as follows:(?nal BW/initial BW)×100.The birds were killed by cervical dislocation,and selected organs,including liver,spleen,heart,and bursa of Fab-ricius,were removed and cleaned of adherent tissues. The weight of each organ was measured and expressed relative to?nal BW:(organ weight/?nal BW)×100. Differential Cell Counts

The Cell-Dyne3500System8was used for differential cell counts.The system has been standardized for differ-ential counts of poultry blood.Hematologic measure-ments of heparin anticoagulated blood included total counts of white blood cells(WBC),heterophils,lympho-cytes,monocytes,eosinophils,basophils,red blood cells (RBC),hemoglobin concentration,hematocrit,and mean corpuscular hemoglobin.

Blood Chemistry

Concentrations of Ca,P,cholesterol,total protein,albu-min,glucose,alkaline phosphatase,triglycerides,creatine kinase,and Mg in plasma were determined using a clini-cal chemistry analyzer7according to the manufacturer’s recommended procedures.The protein concentration of each plasma sample was measured using a dye binding protein assay kit4for SDS-PAGE analysis. Interleukin-6Bioassay

The IL-6levels of plasma samples were measured using an IL-6dependent murine hybridoma B9cell bioassay (Helle et al.,1988;Rath et al.,1995).Plasma,diluted with RPMI1640medium(1:10;vol/vol),was assayed using proliferation of B9cells as a measure of IL-6activity. Eleven microliters of diluted plasma were added to cell cultures at a concentration of104cells/100μL per well in a96-well plate for72h.Cell proliferation was assessed using the reduction of MTT as an end point.Following 4h of incubation with MTT,the cells were centrifuged at330×g for10min to remove supernatant and then solubilized according to Hansen et al.(1989).The extent of reduction of MTT,which corresponds to the number of viable cells,was measured at570nm using a microplate reader.12The concentrations of IL-6in plasma samples were calculated from a standard curve obtained using recombinant human IL-6.Results were reported as nano-grams of IL-6/mL of plasma.

SDS-PAGE of Plasma Proteins

SDS-PAGE was performed using a miniprotean II sys-tem.4Twelve percent polyacrylamide gels,prepared ac-

LIPOPOLYSACCHARIDE EFFECTS ON CHICKEN

35

FIGURE 1.Effects of saline or lipopolysaccharide (LPS)injection on cloacal temperature.Asterisks indicate differences from the respective saline-treated control groups:*P <0.01;**P <0.005.

cording to Laemmli (1970),were used to separate plasma proteins under nonreducing conditions.Plasma samples from three birds in each experimental group were used.The protein content of the plasma samples was deter-mined using a dye-binding assay kit obtained from Bio-Rad laboratories.4The plasma was diluted to 2.4mg/mL using PBS (pH 7.4)and then diluted 1:1(vol/vol)with nonreducing sample buffer for electrophoresis.The changes in protein pro ?les between saline and LPS-treated birds were compared at each time point.A broad-range molecular weight (MW)standard (4to 250kDa)4was used as a reference.For comparison,chicken serum albumin,human α1-acid glycoprotein (AGP),and chicken Ig were used as additional standards in the same gel.Electrophoresis was conducted at 100V constant.The gels were stained with Coomassie brilliant blue,destained,air-dried on cellulose acetate membrane,and scanned using a Geldoc imaging densitometer.4A standard curve for protein markers was plotted with log values of MW of each marker against its relative mobility.The apparent MW of each plasma protein band of interest was calcu-lated from the standard curve and then rounded to the nearest hundred to give an approximate MW.The relative changes in the protein bands of interest were calculated from the percentage of area under the peak and reported with respect to the controls at that time interval.

Statistical Analysis

The comparisons were made between saline-and LPS-treated birds throughout the experiment.Data were ana-

TABLE 1.Effects of Salmonella typhimurium lipopolysaccharide (LPS)on BW and selective organ weights 1

Time postinjection

12h

24h

48h

Organ

Saline LPS

Saline LPS

Saline LPS

BW,%initial BW 102.08±0.8690.76±0.60*105.98±0.8795.63±2.57*112.84±1.71106.16±1.47*Liver,g/100g BW 2.89±0.14 3.48±0.05* 3.03±0.13 3.82±0.16* 3.11±0.07 3.63±0.10*Bursa,g/100g BW

0.29±0.02

0.25±0.02

0.24±0.04

0.22±0.02

0.31±0.01

0.19±0.02*

1

Values were calculated from seven individual birds per group and expressed as means ±SEM.*Difference (P <0.05)from their respective saline-treated control groups.

lyzed by two-way ANOVA with SAS ?General Linear Models procedure (SAS Institute,1988).Comparisons were made between saline-and LPS-treated birds within a single time point.Signi ?cance was de ?ned at P ≤0.05.

RESULTS

Clinical Changes

Lipopolysaccharide produced symptoms of drowsi-ness,lethargy,and withdrawal from feed and water within 1h of injection;the symptoms persisted at least up to 4h.These chickens also exhibited ruf ?ed feathers and moderate diarrhea.These clinical signs were absent in birds treated with saline alone.The cloacal temperature of LPS-treated birds was elevated at 3h posttreatment (Figure 1).At later time periods,12and 24h,there were no differences in the cloacal temperature between saline-and LPS-treated birds.In the 48-h groups,the saline-treated birds showed a modest increase in cloacal temper-ature (Figure 1)compared with LPS-treated birds but did not appear to be sick.The LPS-treated birds appeared normal by 24h as judged by their feeding behavior and agility.The feed consumption in LPS-treated birds was reduced during the experimental period (data not shown).

Body and Organ Weights

The percentage change in BW of LPS-treated birds was lower than that of saline-treated controls (Table 1).The relative liver weights of all LPS-treated birds were in-creased at all time points compared with their respective controls.Lipopolysaccharide injection also resulted in the reduction of relative bursal weights only at the 48-h treat-ment period (Table 1).There were no changes in spleen and heart weights at any time point (data not shown).

Blood Differential Counts

An increase in total WBC concentration was seen in LPS-treated chickens at 48h post-treatment (Table 2).A similar trend was observed in the blood lymphocyte concentration of LPS-treated birds.The heterophil con-centration was elevated at 12and 24h in LPS-treated birds but was similar to controls in treated birds at 48h.The heterophil to lymphocyte ratio (H:L)was signi ?cantly

XIE ET AL.

36TABLE 2.Effects of saline or Salmonella typhimurium lipopolysaccharide (LPS)on the concentrations of various blood cell types 1

Time postinjection

12h

24h

48h

Cell parameters 2Saline LPS

Saline LPS Saline LPS WBC,103/μL

22.03±2.5429.41±2.1431.48±4.1231.23±4.1625.52±2.8134.68±2.92*Heterophil,103/μL 6.02±1.5715.48±1.71* 5.76±0.2910.53±2.83* 5.85±0.92 5.33±0.41Lymphocyte,103/μL 13.69±1.4211.21±1.9323.12±4.3618.71±2.6917.63±1.9026.93±3.16*Eosinophil,103/μL 0.004±0.0020.039±0.015*0.004±0.0040.002±0.0010.005±0.0020.014±0.004RBC,106/μL

2.13

±0.17

2.46

±0.10*

2.29

±0.08

2.34

±0.08

2.29

±0.07

2.37

±0.06

1Values were calculated from seven individual birds per group and expressed as means ±SEM.2

WBC =White blood cells;RBC =red blood cells.

*Difference (P <0.05)from the respective saline-treated control birds.

increased only in LPS-injected chickens at 12h (Figure 2).Both RBC and eosinophil concentrations showed an increase in birds at 12h following LPS injection.No changes were observed in the levels of basophil concen-tration,hemoglobin concentration,and hematocrit in LPS-treated groups at any time point.

Blood Chemistry

Lipopolysaccharide elevated the plasma level of P at 12h and induced hypocholesterolemia at 12and 24h.Plasma glucose concentration decreased at 12h,and the protein concentration was elevated at both 24-and 48-h time points in LPS-injected birds (Table 3).Lipopolysac-charide treatment did not affect plasma levels of Ca,Mg,alkaline phosphatase,triglycerides,or creatine kinase (data not shown).

Interleukin-6

Interleukin-6levels were minimal to undetectable in saline-treated birds at all time points.In LPS-treated birds at 12and 24h,the plasma IL-6was signi ?cantly elevated.At 48h,LPS-treated chickens had very little IL-6activity that was comparable with that of saline-treated chickens (Figure 3).The changes in plasma IL-6activity showed a strong correlation with the blood heterophil levels at all three time points after LPS injection (R 2=

0.97).

FIGURE 2.Effects of saline or lipopolysaccharide (LPS)injection on the ratios of heterophils to lymphocytes.Values were calculated from seven individual birds per group and expressed as means ±SEM.Aster-isk indicates a difference (P <0.01)from the respective saline-treated control groups at that time period.

Plasma Protein,SDS-PAGE Pro?les

Following electrophoretic separation,the total number of plasma protein bands ranged from 10to 12in both saline-and LPS-treated birds (Figure 4).Four bands of plasma proteins in the range of 39to 65kDa of MW showed consistent changes in LPS-treated chickens as compared with saline-treated birds during the same pe-riod (Table 4).A quantitative increase in a 65-kDa MW protein persisted in LPS-treated birds at 12-,24-,and 48-h time periods.A decrease in the intensity of a 56-kDa MW protein was evident at both 24and 48h in LPS-injected birds.A reduction in the plasma level of a 49-kDa MW protein was observed only at time points of 12and 24h in LPS-administered birds.Chicken serum albumin and human AGP showed similar migration in the gel and corresponded to the 49-kDa MW protein band.A 39-kDa MW protein that was initially present as a faint band at 12h in both saline-and LPS-treated birds appeared to decrease in LPS-treated chickens at 48h com-pared with control birds (Table 4).Although this protein constituted only ～1%of total protein,the differences were discernible.

DISCUSSION

The results of this study showed that LPS induced signi ?cant physiological and behavioral changes,which included the elevation of cloacal temperature,depression,lethargy,diarrhea,and avoidance of feed,within a few hours of injection.Endotoxin-induced clinical and behav-ioral changes have been known in several species includ-ing birds (Emery et al.,1991;MacKay and Lester,1992;Johnson et al.,1993;Koh et al.,1996;Berczi,1998).A sig-ni ?cant reduction in BW was also noted in birds treated with LPS,which was probably due to reduced feed intake and diarrhea during early periods of treatment.A de-crease in BW gain in LPS-treated chickens was also ob-served by Koh et al.(1996).Induction of fever by endo-toxin is known in many species including chickens (Jones et al.,1983).The febrile response to S.typhimurium LPS administration in 3-wk-old chickens was studied by Jones et al.(1983),who observed a maximum febrile response at 1to 3h following LPS administration.Our results also showed an elevation of cloacal temperature at 3h after

LIPOPOLYSACCHARIDE EFFECTS ON CHICKEN 37

TABLE 3.Effects of saline and Salmonella typhimurium lipopolysaccharide (LPS)on blood chemistry of 3-wk-old male broiler chickens 1

Time postinjection

12h

24h

48h

Variables

Saline LPS

Saline

LPS Saline LPS P,mg/dL

6.82±0.26

7.87±0.31* 6.28±0.14 6.56±0.21 6.41±0.247.00±0.23Cholesterol,mg/dL 110.17±6.0285.43±4.06*129.60±5.7592.43±4.59*123.4±5.32131.20±4.66Total protein,g/dL 3.08±0.14 3.23±0.06 2.78±0.18 3.34±0.10* 3.02±0.15 3.53±0.09*Glucose,mg/dL

233.17

±7.41

199.29

±7.30*

233.40

±8.03

230.71

±9.18

235.40

±5.27

225.70

±3.46

1

Values were calculated from seven individual birds per group and expressed as means ±SEM.*Difference (P <0.05)from the respective saline-treated control groups.

LPS treatment.There were no differences in cloacal tem-perature between control and LPS-treated birds at 12-and 24-h time points.However,the increase in cloacal temperature of saline-treated birds at 48h remains intri-guing because these birds showed no signs of sickness and were normal with respect to BW,agility,and feeding.The increase in the relative weights of liver in LPS-treated chickens underscores the importance of liver in the acute phase response.The liver is the site for the synthesis of acute phase proteins and detoxi ?cation of xenobiotics.The changes in liver metabolism caused by endotoxin treatment or live bacterial challenge have been observed in both mammals and birds (Curtis et al.,1980;Kushner,1982;Sherwin and Sobenes,1996;Bayyari et al.,1997).It is likely that the apparent changes in liver weight may be due to an increase in its metabolic function cou-pled with loss of BW.The weights of both heart and spleen remained unaffected in saline-and LPS-treated groups at all time points,whereas a decrease in the rela-tive weight of the bursa of Fabricius at 48h post-treatment was seen in LPS-treated birds.It is likely that there was an equivalent decrease in the weights of spleen and heart in LPS-treated birds,although no changes in the relative weights of these organs were evident.Stress-induced bur-sal atrophy has been suggested to be caused by an in-creased corticosteroid production (Riddell,1987).Lipo-polysaccharide is an immunological stressor that in-creases the production of corticosteroids,which may account for this effect.The increase in the blood

concen-

FIGURE 3.Plasma levels of interleukin (IL)-6activity following saline or lipopolysaccharide (LPS)injection.Values were calculated from seven individual birds per group and expressed as means ±SEM.Asterisk indicates a difference (P <0.001)from the respective saline-treated con-trol groups at that time period.

trations of eosinophils,heterophils,and the H:L was evi-dent in LPS-treated birds at 12h.Although the heterophil concentration remained elevated in LPS-treated birds at 24h,the H:L was similar to that of the controls.Increased H:L is considered an indicator of stress in birds (Gross and Siegel,1983;Maxwell and Robertson,1998).Hetero-phils are parts of natural immunity and cellular defense against microbial infections.Therefore,it is not surprising that the blood level of heterophils is elevated early in response to an endotoxin challenge.At later times,their migration to in ?ammatory sites results in a decrease in their blood level (Harmon,1998;Rath et al.,1998).On the other hand,WBC and lymphocyte concentrations were elevated in LPS-treated birds at 48h,suggesting that the change in lymphocyte concentration is a late stage response to LPS and may be related to adaptive im-munity.

The elevation of plasma P levels in LPS-treated birds at the 12-h time period could be related to reduced

P

FIGURE 4.The typical gel patterns of plasma samples derived from chickens at 48h after saline (left)or lipopolysaccharide (LPS;right)ad-ministration.

XIE ET AL.

38

TABLE4.Relative changes of SDS-PAGE resolved plasma proteins following saline and lipopolysaccharide(LPS)treatment1

Time postinjection

12h24h48h

Apparent MW2Saline LPS Saline LPS Saline LPS

65kDa,% 6.87±0.9910.72±0.40*7.21±0.3211.47±0.29* 5.61±0.128.90±0.79* Relative change,%100±14156±6100±4159±4100±2159±14 56kDa,% 3.35±0.00 2.99±0.00 4.19±0.11 3.24±0.21* 3.42±0.08 2.77±0.07* Relative change,%100±089±0100±377±5100±281±2

49kDa,%53.80±1.8248.75±1.36*41.38±0.5937.38±0.64*44.76±1.4439.77±0.92 Relative change,%100±391±3100±190±2100±389±2

39kDa,%BD3BD 1.64±0.09 1.35±0.00 1.14±0.000.82±0.09* Relative change,%BD BD100±582±0100±072±8

1Diluted plasma containing equal quantities of proteins were separated on12%SDS-PAGE gels under nonreducing conditions,processed as described in Materials and Methods,and scanned using a Geldoc imaging densitometer.Values were calculated from three individual plasma samples per group and expressed as means±SEM.

2MW=Molecular weight.

3BD=Below detection limit.

*Difference(P<0.05)from the respective saline-treated control groups.

excretion resulting from some transient impairment of kidney function that has been noted during acute phase reaction(First,1996).The LPS-induced hypocholesterole-mia at the12and24-h time points may be attributed to changes in cholesterol and lipoprotein metabolism in the liver that are known to occur during acute phase response (Kushner,1982),although this effect has not been studied in birds.Increased levels of blood protein concentration in LPS-treated birds may be due to an altered production of proteins related to the acute phase response as known in other species(Schreiber et al.,1989;Aldred and Schreiber,1993).Similarly,the hypoglycemia observed in LPS-treated chickens at12h could be due to increased insulin production that is known to occur during early time points of LPS-induced endotoxemia(Berczi,1998). Interleukin-6is a pro-in?ammatory cytokine secreted by many different cell types including macrophages,en-dothelial cells,and connective tissue cells.Along with tumor necrosis factor and IL-1,IL-6is one of the primary mediators of the acute phase response(Lotz et al.,1989; Whicher et al.,1989;MacKay and Lester,1992;Abbas et al.,1997;Klasing,1998).An elevation of plasma levels of IL-6caused by LPS treatment was observed at12-and 24-h time periods in the current study.Recently,Naka-mura et al.(1998)also showed an increased blood level of IL-6in broiler chickens following Esherichia coli LPS administration,which persisted up to48h.In this study, the IL-6activity was almost undetectable by48h.The difference between our result and that of Nakamura et al.(1998)could possibly be due to LPS types.It is known that the ef?cacy and virulence of LPS from different bacte-ria is related to factors such as serotypes of the bacteria and constituents of their lipid A moeity(Berczi,1998). Nevertheless,the plasma levels of IL-6showed a strong correlation with the concentrations of blood heterophils. One of the major events in many in?ammatory proc-esses is the change in the concentrations of certain plasma proteins known as acute phase proteins(APP).These proteins are produced in the liver and are released into the blood stream(Aldred and Schreiber,1993;Sherwin and Sobenes,1996).The APP are responsible for damage control and exhibit a variety of anti-in?ammatory,antimi-crobial,and cytotoxic properties(Berczi,1998).Although the role of these APP are scantily de?ned,it is well-known that the levels of some of these proteins in plasma show profound changes during acute phase reactions(Kushner, 1982;Whicher et al.,1989).There are a number of APP in mammals that have been used as markers of in?amma-tion(Sherwin and Sobenes,1996).However,much less is known about these proteins in birds,although the evi-dence of the presence of a variety of APP in birds is accumulating(Delers et al.,1983;Grieninger et al.,1986; Amrani et al.,1986;Tohjo et al.,1995;Klasing,1998).There is,however,no detailed characterization of these proteins in https://www.wendangku.net/doc/ff3525215.html,ing SDS-PAGE under nonreducing condi-tions to compare qualitative and quantitative changes of plasma proteins of saline-and LPS-treated birds,we observed four bands of plasma proteins showing quanti-tative differences.Both39-and56-kDa MW protein bands showed a reduction by20to30%at24and48h after LPS administration compared with saline-treated chickens.It was not,however,possible to determine the identities of these proteins because of the lack of authentic protein standards of avian origin.The other two proteins that showed changes were49-and65-kDa proteins.Recently, AGP has been used as an APP marker in chickens(Taka-hashi et al.,1995,1998;Nakamura et al.,1998).Because of the reported similarities in MW of avian and human AGP(Charlwood et al.,1976),we used a commercially available human AGP as a standard.However,this stan-dard comigrated with the49-kDa MW protein that corres-ponded to the MW of chicken serum albumin.In mam-mals,albumin behaves as a negative APP because its plasma concentration signi?cantly decreases during the acute phase response(Grieninger et al.,1986;Aldred and Schreiber,1993).In the present study,the49-kDa band showed a consistent decrease in birds treated with LPS. Similarly,hemopexin is another chicken APP that is in-

LIPOPOLYSACCHARIDE EFFECTS ON CHICKEN39

duced by turpentine oil and that has an apparent MW of 49kDa(Grieninger et al.,1986).Because AGP,hemopexin, and albumin have relatively similar MW and because the former two are less abundant,they could have been masked by albumin,which dominates all other plasma proteins.Therefore,it is dif?cult to discern the magnitude of change in their levels.On the other hand,a signi?cant increase in the concentration of a65-kDa MW protein was observed in LPS-treated birds.The induction of this protein in LPS-treated chickens was48to59%higher than that of saline-treated control birds,and it showed an increase at all time points.The identity of the65-kDa protein is not clear.Tohjo et al.(1995)identi?ed transferrin,an80-kDa protein as an APP in chickens with turpentine oil-induced in?ammation.Other possible avian APP,which have been observed under different experimental conditions,have been recently reviewed by Klasing(1998).The MW of transferrin is reported to be 76kDa in mammals(Kaneko,1989).However,in this experiment,there were no changes in the intensities of proteins in a range of75to85kDa between saline-and LPS-treated birds.

In conclusion,this study shows that S.typhimurium LPS produced in?ammatory responses in chickens character-ized by the elevation in cloacal temperature,feed avoid-ance,loss of BW,an increase in liver weight,the elevation of heterophil counts,an increase in plasma level of IL-6,and selective changes in the concentrations of certain plasma proteins,notably a65-kDa protein band.Further characterization of the acute phase response and APP may be of prognostic value as well as helpful to under-stand the physiology of in?ammation in poultry.

ACKNOWLEDGMENTS

The authors thank D.Horlick,D.Bassi,S.Tsai,and S. Zornes for technical assistance.We also thank G.Erf and D.Barnes for their critical review.

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患儿女，2个月15天，以“发热、腹泻4天”为主诉入院。 病例简介 查体 患儿于入院前4天总有无明显有瘾出现发热，体温最高39.2℃，无寒战及抽搐，口服“对乙酰氨基酚”体温可降至 37.5℃，间隔月6-7小时反复发热，伴有腹泻，每日排7-8次黄绿色粘液变，口服“琥乙红霉素”0.1克，日2次，“电解质泡腾片”随服，“酪酸梭菌二联活菌散”500mg，日2次，治疗4天病情无好转，入院当天大便可见血丝，再次来我院，急诊以“细菌性痢疾”为诊断收入院，患儿病后精神状态一般，有鼻塞，偶有喉中痰鸣，无咳嗽及喘息，人工喂养，配方奶 120ml/次，无呕吐及腹胀，尿量略少 T：36.9℃，P：154次/分，R：44次/分，BP：94/46mmHg,意识清楚，状态反应可，全身皮肤无黄染，皮肤弹性可，无皮疹及出血点，全身浅表淋巴结无肿大。前囟平坦，2.0*2.0cm，对光反射 灵敏，无鼻扇，口唇粘膜略干燥、无发绀，口腔粘膜光滑，眼部粘膜充血，颈软无抵抗，无三凹征，双肺叩诊轻音，双肺呼吸音粗糙，心前区无隆起，心尖搏动范围正常，心浊音界无扩大，心率：154次/分，节律齐，心音有力，心前区可闻及2/6级收缩期杂音，无心包摩擦感，腹部略膨隆，柔软，无压痛，无包块，肝下界在中线肋缘下1cm，质地软，脾肋下未触及。肠鸣音4次/分。 现病史

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沙门氏菌病

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八、鼠伤寒沙门氏菌／回复突变试验 Salmonella Typhimurium / Reverse Mutation Assay 1 范围 本规范确定了鼠伤寒沙门氏菌／回复突变试验的基本原则、要求和方法。 本规范适用于化妆品原料及其产品的基因突变检测。 2 规范性引用文件 OECD Guidelines for Testing of Chemicals (No.471,Adopted:21,July 1997)。 3 定义 3.1回复突变（Reverse mutation） 细菌在化学致突变物作用下由营养缺陷型回变到原养型(prototroph)。 3.2基因突变（Gene mutation） 在化学致突变物作用下细胞DNA中碱基对的排列顺序发生变化。 3.3碱基置换突变（Base substitution mutation） 引起DNA链上一个或几个碱基对的置换。 碱基置换有转换(transition)和颠换(transversion)两种形式。 转换是DNA链上的一个嘧啶被另一嘧啶所替代，或一个嘌呤被另一嘌呤所代替。 颠换是DNA链上的一个嘧啶被另一嘌呤所替代，或一个嘌呤被另一嘧啶所代替。 3.4 移码突变（Frameshift mutation） 引起DNA链上增加或缺失一个或多个碱基对。 3.5 鼠伤寒沙门氏菌/回复突变试验（Salmonella typhimurium/reverse mutation assay） 利用一组鼠伤寒沙门氏组氨酸缺陷型试验菌株测定引起沙门氏菌碱基置换或移码突变的化学物质所诱发的组氨酸缺陷型(his-)→原养型(his+)回复突变的试验方法。 3.6 S9 经多氯联苯(PCB混合物)或苯巴比妥钠和β-萘黄酮结合诱导的大鼠制备肝匀浆，在9000g 下离心10min后的肝匀浆上清液。 4 原理 鼠伤寒沙门氏组氨酸营养缺陷型菌株不能合成组氨酸，故在缺乏组氨酸的培养基上，仅少数自发回复突变的细菌生长。假如有致突变物存在，则营养缺陷型的细菌回复突变成原养型，因而能生长形成菌落，据此判断受试物是否为致突变物。 某些致突变物需要代谢活化后才能引起回复突变，故需加入经诱导剂诱导的大鼠肝制备的S9混合液。 5 仪器和设备 培养箱、恒温水浴、振荡水浴摇床、压力蒸汽消毒器、干热烤箱、低温冰箱(-80℃) 或液氮生物容器、普通冰箱、天平(精密度0.1g和0.0001g)、混匀振荡器、匀浆器、菌落计数器、低温高速离心机，玻璃器皿等。

鼠伤寒沙门氏菌哺乳动物微粒体酶试验

鼠伤寒沙门氏菌/哺乳动物微粒体酶试验 1 主题内容与适用范围 本标准规定了Ames试验的基本技术要求。 本标准适用于检测环境有害物质的致突变作用。 2 原理 鼠伤寒沙门氏菌的突变型(即组氨酸缺陷型)菌株在无组氨酸的培养基上不能生长，在有组氨酸的培养基上可以正常生长。但如在无组氨酸的培养基中有致突变物存在时，则沙门氏菌突变型可回复突变为野生型(表现型)，因而在无组氨酸培养基上也能生长，故可根据菌落形成数量，检查受试物是否为致突变物。某些致突变物需要代谢活化后才能使沙门氏菌突变型产生回复突变，代谢活化系统可以用多氯联苯(PCB)诱导的大鼠肝匀浆(S－9)制备的S－9混合液。 3 仪器 3.1 实验室常用设备。 3.2 低温高速离心机，低温冰箱(－80℃)或液氮罐，洁净工作台，恒温培养箱，恒温水浴，蒸气压力锅，匀浆器等。 4 培养基制备及试剂的配制 培养基成分或试剂除说明外至少应是化学纯，无诱变性。避免重复高温处理，选择适当保存温度和期限，如肉汤保存于4℃不超过六个月，其他详见下述各培养基及溶液说明。 4.1 营养肉汤培养基 牛肉膏 2.5g 胰胨(或混合蛋白胨) 5.0g 氯化钠 2.5g 磷酸氢二钾(K2HPO4·3H2O)1.3g 蒸馏水500mL 加热溶解，调PH至7.4，分装后0.103MPa20min灭菌，普通冰箱保存备用，保存期不超过半年。 4.2 营养肉汤琼脂培养基：用作a.基因型鉴定的结晶紫敏感试验，抗氨节青霉素和四环素试验，紫外线敏感性试验。b.细菌活力鉴定。 琼脂粉 1.5g 营养肉汤培养基100mL 加热融化后调pH为7.4，0.103MPa 20 min灭菌。 4.3 底层培养基所需试剂及配制方法如下： 4.3.1 磷酸盐贮备液 磷酸氢钠铵(NaNH4HPO4·4H2O) 17.5g 柠檬酸(C6H8O7·H2O) 10.0g 磷酸氢二钾(K2HPO4) 50.0g 硫酸镁1)(MgSO4·7H2O) 1.0g 加蒸馏水至100mL，0.103MPa 20min灭菌。 注：1)待其他试剂完全溶解后再将硫酸镁缓慢放入其中继续溶解，否则易析出沉淀。 4.3.2 40%葡萄糖溶液 葡萄糖40.0g 加蒸馏水至100mL，0.055MPa 20min灭菌。 4.3.3 1.5%琼脂培养基 琼脂粉 6.0g 蒸馏水 400mL 融化后0.103MPa 20min灭菌。 4.3.4 底层培养基(无菌操作) 趁热(80℃)，在灭菌琼脂培养基中(400mL)依次加入：