Study of the effect of antibodies in the intestinal tract of germ-free baby pigs

The effect of antibodies in the intestinal tract was studied in germ-free baby pigs whose intestinal barrier was closed to macromolecules by the peroral administration of modified cow's milk for the first 72 hours after birth. They were then all contaminated with the pathogenic strainEscherichia coli 055 in amounts of 10⁹ bacterial cells per animal. The controls, which were not given any antibodies, all died within 24 hours. All the experimental animals given 12.5–50ml immune colostrum or serum survived, while of those given 50ml normal serum or colostrum containing natural antibodies reacting with theEscherichia coli test strain, 50% survived. No circulating antibodies were found in the serum of the experimental animals after the administration of serum or colostrum. The antibodies present in colostrum thus appear to protect the newborn organism directly in the intestinal tract, which is the first site of bacterial invasion, as well as after infiltration into the blood stream.     

Studies on the phagocytic activity of the reticuloendothelial system (RES) to rough and smoothEscherichia coli in young conventional and germfree guinea pigs

The functional (phagocytic) capacity of the reticuloendothelial system (RES) of young conventional and germfree guinea pigs was studied using thein vivo blood clearance test of living bacteria (rough and smoothEscherichia coli). It was found that as previously shown in newborn germfree piglets, the smooth strain was taken up from the blood stream of germfree guinea pigs very slowly whereas roughEscherichia coli was phagocytosed effectively. The inability of the RES of germfree guinea pigs to phagocytose the smooth strain is not due to a functional incapability of phagocytic cells, but it reflects rather the lack of serum opsonins to this strain. This was demonstrated in experiments in which smooth bacteria, sensitized prior to injection into the blood circulation with specific antiserum, were phagocytosed as effectively as the rough strain. It is assumed that effective phagocytosis of rough strain is due to the presence of non-specific opsonins (e.g. components of the complement system). In young conventional guinea pigs both strains,i.e. smooth and rough, were taken up from the blood stream very effectively thus indicating that sufficient levels of serum opsonins for both strains were present. This fact could be correlated with the finding that in sera of conventional guinea pigs haemagglutinating antibodies to both strains ofEscherichia coli could be detected, whereas in sera of young germfree guinea pigs, no antibodies to usedEscherichia coli strain were found. The importance of serum opsonins for effective phagocytosis of bacteria by RE cellsin vivo is discussed.     

Stimulating effect ofStreptococcus faecalis on phagocytosis in gnotobiotic piglets

The concentration of active phagocytes in peripheral blood remained in germfree pigs up to the age of one year approximately at the same level as found at the age of 7 d and did not exceed 0.3×10⁹/L of blood, whereas a steady increase was established in conventional pigs. Monoassociation of gnotobiotic piglets withStreptococcus faecalis increased during 24 h the concentration of circulating granulocytes and the concentration of active phagocytes. An even more pronounced effect was obtained when formolizedS. faecalis cells were applied intraperitoneally to germfree piglets. This treatment elevated the phagocytosis index also in conventional piglets, as well as in germfree piglets previously given cyclophosphamide.Escherichia coli O 83 or a mixture of anaerobic bacteria did not cause any serious changes in the activity of phagocytosis in gnotobiotic piglets.S. faecalis seems to be a natural factor stimulating both the release of granulocytes from their depots as well as their phagocytic activity.     

Changes in the level and character of colostral immunoglobulins during the lactation period in sows

A profound decrease in the concentration of colostral proteins (among immunoglobulins primarily IgG) during the first three days of lactation is accompanied by changes in the molecular heterogeneity of IgA. In the course of lactation, the amount of 7S IgA increases in relation to 10S IgA. The total amount of IgA, after the initial decrease during the first three days, maintains a gradual increase. In addition, a transient increase of natural haemagglutinating antibodies toEscherichia coli (serotype O55) was found in fractions corresponding to IgA and IgG during lactation.     

Studies on Immunoglobulins and Trypsin Inhibitor in Colostrum and Milk from Sows and in Serum of Their Piglets

The concentrations of IgG, IgM, IgA and the specific sow colostrum trypsin inhibitor (SCTI) were measured by radial immunodiffusion in colostrum and milk samples from sows and in serum samples from their offspring during the suckling period. A clear time dependence was found for all the measured variates in both whey and serum. Statistically significant positive correlations were found between, on the one hand, concentrations of IgG and IgA, but not IgM, in sera from 39 suckling piglets 1 and 3 days old, and, on the other hand, concentrations of the same immunoglobulins and of the trypsin inhibitor in maternal colostrum (n = 7). Multiple regression analyses showed that at day 1 and day 3 the levels of both IgG and IgA in serum samples from the suckling piglets were positively influenced by both the SCTI and the IgG or IgA contents in maternal colostrum.     

Antimicrobial Effect of Human Serum IgA

Serum IgA, IgG and colostrum secretory IgA prepared from specimens pooled from a large number of human beings were shown to have measurable levels of antibodies against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, poliovirus, Coxsackie B virus, echovirus and influenza virus. Serum IgA exerted a bacteriostatic effect in vitro on E. coli and P. aeruginosa, which increased in the presence of the iron-binding proteins lactoferrin and transferrin. This bacteriostasis was reduced when the iron-binding proteins were saturated with iron. Similar results were obtained with IgG and secretory IgA. The bacteriostatic effect of serum IgA was also shown in vivo, in the peritoneal cavity of mice. The effect was suppressed by iron. Iron-chelating substances, siderophores, excreted by E. coli diminished the cosoperative bacteriostatic effect of serum IgA and transferrin. Siderophore production by E. coli was inhibited in the presence of serum IgA, but not when serum IgA was deprived of specific antibody by absorption with E. coli. These results indicate that serum IgA has a potent bacteriostatic effect in cooperation with transferrin or lactoferrin because of the inhibitory effect of the specific antibody on siderophore production by E. coli.     

Course of the post-irradiation syndrome after irradiation with lethal doses in newborn conventional,germ-free andEscherichia coli-monoassociated piglets

The influence of whole-body irradiation with lethal doses of ionizing radiation (⁶⁰Co) was studied in conventional, germ-free andEscherichia coli-monoassociated newborn piglets. The dose 1,200 R produced an acute intestinal death (i.e. within 3–4 days) in conventional animals, whereas survival was three times as long in their germ-free counterparts. Artificial colonization of the intestinal tract of germ-free piglets with non-pathogenic strain ofEscherichia coli, prior to irradiation with the same dose, produced the conventionalization of these animals and reduction in the survival time almost to the level of conventional animals. In conventional animals, profound focal regressive changes of the epithelium accompanying the denudation of intestinal villi were found already on the 2nd–3rd day after irradiation with 1,200 R. On the other hand, the intestinal epithelium of germ-free piglets, irradiated with 1,200 R, was found to be intact on the 7th–9th day of post-irradiation, and the first signs of damage started to occur around the 9–10th days. The morphological characteristics of the intestinal mucous membrane ofEscherichia coli-monoassociated piglets were comparable to those of conventional, irradiated piglets. The role of the presence of the microbial factor for the turnover and radiosensitivity-resistance of enterocytes, and for the survival-death rate of animals irradiated with doses producing the post-irradiation gastro-intestinal syndrome, is discussed.     

Interaction ofEscherichia coli O55 hybrids with bacteriophage lambda. A new type of host specificity betweenEscherichia coli O55 and urinaryEscherichia coli

Escherichia coli O55 hybrids able to adsorb the lambda phage were obtained by mating anEscherichia coli O55 recipient with anEscherichia coli K12HfrC donor. λ mutants, capable of forming plaques on these hybrids, were not isolated. A new type of host specificity betweenEscherichia coli O55 and urinaryEscherichia coli J was established. For efficient reduction of the phage plating ability more growth steps on the new strain are required in this type. Host specificities O55 and J proved to be different from K specificity.     

The phagocytic activity of reticuloendothelial system of newborn precolostral pigs to smooth and roughEscherichia coli

The fate of smooth and rough strains ofEscherichia coli in the blood stream of newborn, germ-free, colostrum deprived piglets was studied using blood clearance test with live and radioisotope-labelled (P³²) bacteria. Smooth strains are eliminated at an extremly slow rate (hours) whereas rough strains are taken up within 30 minutes. Specific immune serum accelerates rapidly the clearance rate of smooth strains after bothin vitro andin vivo opsonisation of bacteria. Clearance of smooth liveEscherichia coli from the blood stream cannot be stimulated by an endotoxin treatment of newborn piglets. The possible role of complement system as a nonspecific opsonin in phagocytosis of rough strains is discussed. The results demonstrate that RES cells of newborn precolostral piglets are functionally competent as to the capacity to remove gramnegative bacteria from the blood stream provided specific and/or nonspecific opsonins are available.     

Proteome profiles of mucosal immunoglobulin uptake in inflamed porcine gut

Acquisition of passive immunity by endocytosis of intact immunoglobulins (Ig) from colostrum is critical for prevention of intestinal and systemic diseases in neonatal mammals. We compared proteome patterns of healthy and inflamed gut tissues from pre-term piglets to investigate the effect of inflammation on acquisition of passive immunity. A clear difference in the two-dimensional gel electrophoresis protein patterns between healthy and inflamed intestinal tissues was observed, suggesting that inflamed tissues failed to absorb and transfer Ig from colostrum to epithelial cells. We have mapped and identified the Ig proteins that are taken up by healthy intestinal tissues, and found that isoforms of the IgA and IgG heavy chain and Ig kappa and lambda light chains were internalized. Our results indicate that colostrum protein uptake in the porcine gut is a selective process that is obstructed in inflamed pre-term gut.     

Early antibodies to human serum albumin formed in germfree piglets

Immunization of germfree piglets with HSA at the time of birth results in formation of antibodies of IgM, IgG and IgA classes in spite of the fact that prior to immunization only IgG and IgA immunoglobulins are detectable in their sera. The possible significance of this finding is discussed. Early IgG antibodies formed in these piglets differ from late and adult pig antibodies by the presence of lower molecular weight constituents and their molecular heterogeneity corresponds to that of IgG of nonimmunized newborn precolostral piglets suggesting that small amounts of immunoglobulins formed in piglets during the intrauterine life are of antibody nature. Dedicated to Prof. F. Patočka on the occasion of his 70th birthday The work was supported by a WHO grant.     

Antibody repertoire development in fetal and neonatal piglets. VIII. Colonization is required for newborn piglets to make serum antibodies to T-dependent and type 2 T-independent antigens

Cesarean-derived piglets were reared for 5 wk under germfree conditions or monoassociated with a benign Escherichia coli (G58-1) or a enterohemorrhagic strain (933D) derived from O157:H7, and immunized i.p. with the T-dependent (TD) Ags fluorescein-labeled (FL) keyhole limpet hemocyanin or trinitrophenylated (TNP) keyhole limpet hemocyanin and the type 2 T-independent Ags TNP-Ficoll or FL-Ficoll. Only colonized piglets showed an increase in serum IgG, IgA, and IgM and had serum Abs to FL, TNP, and colonizing bacteria. While serum Abs to FL or TNP appeared following colonization alone, secondary responses were restricted to piglets immunized using TD carriers. While animals colonized with 933D had significantly higher total serum IgG and IgM levels and specific IgG Abs than those colonized with G58-1, no differences were seen in serum IgA levels, B cell diversification in the ileal Peyer's patches, and specific activity (ELISA activity per micrograms of Ig) of pre-boost serum IgG and IgM anti-TNP and anti-FL Abs. Serum IgA Abs to TNP, FL, or bacteria were not detected. Ag-driven responses, as measured by an increase in specific Ab activity, were only observed in secondary responses to TD Ags and to colonizing, pathogenic E. coli. We propose that germline-encoded, isotype-switched B cells in newborn piglets differentiate to Ab-secreting cells 1) after stimulation by bacteria-activated APCs or 2) through direct stimulation by bacterial products. We further propose that Ag-driven systemic responses require both bacterial colonization and TD Ags translocated to the peritoneum.     

Selective decontamination,induced colonization resistance and connected immunological changes in piglets

14-d-old conventional piglets were picked from normal piggery, washed with disinfectants, placed into isolators suitable for germfree work, fed a sterile diet and treated with peroral antibiotics (nalidixic acid, kanamycin, and nystatin). Beginning with day 5 or 7, Enterobacteriaceae were not found in feces. The absence of these bacteria was proved by inoculation of germfree newborn piglets with caecal content. In selectively decontaminated piglets, the white blood cell count in blood had fallen to 6 X 10(9)/L; this decrease was due to an extremely low number of granulocytes (to 0.8 X 10(9)/L). On day 35, IgG-positive cells almost disappeared from the spleen, whereas IgA cells were found in an unusually great amount. Corresponding changes in serum levels were established. The colonization resistance effect in Enterobacteriaceae-deprived piglets was confirmed; settling of introduced various E. coli strains did not occur or was delayed.     

Trypsin Inhibitor and Immunoglobulins in Porcine Colostrum

The specific trypsin inhibitor in porcine colostrum first described by Laskowski et al. (1957) is assumed to protect maternal antibodies in colostrum during absorption from the gut of the neonatal piglets (Baintner 1973). Investigations of Jensen & Pedersen have shown that the serum levels of IgG and IgA in newborn suckling piglets depend on both the immunoglobulin and the trypsin inhibitor levels in the colostrum of their mothers. Accordingly, the sow colostrum trypsin inhibitor (SCTI) is essential in order to ensure optimal systemic antibody protection to the newborn and young piglets. The secretory IgA in colostrum and milk, which gives local passive immunity to the gastro-intestinal tract of the piglets (Bourne 1973), is assumed in itself to be relatively resistant against proteolytic degradation (Tomasi & Bienenstock 1968).     

Local and systemic antibody response in infants after oral administration of inactivated enteropathogenicE. coli serotype O111 and O55

In ten infants divided into two groups (up to one month of age and at 2–7 months of age) the dynamics and formation of different antibody isotypes produced locally in the intenstine and in serum after orally administered inactivated enteropathogenicE. coli strains O111 and O55 was followed during 30 d after the first and booster dose by using an indirect immunofluorescence method. Infants up to one month of age produced antibodies of IgM isotype in stool together with the IgA isotype after the first and booster dose of the vaccine against both antigens. Serum IgG antibody increased after 2 d following the first and second dose of antigens and remained higher during 5 d. The infants aged 2–7 months expressed predominantly the IgA isotype response in stool after the first and booster dose of antigens. The serum immunoglobulin levels did not change after oral antigen administration.     

The fertility of a smooth strain ofEscherichia coli

Auxotrophic mutants of the potentially pathogenic smooth strain ofEscherichia coli O55: B5 were isolated and used as recipients in mating experiments withEscherichia coli K12. The recombination frequency obtained in mating K12HfrH donor with O55 recipient was about 100 to 1,000 times lower than in K12×K12 cross. The F-prime donor O55 strain when backerossed with the K12 recipient again mated with low efficiency. Crosses with derivatives of O55 which were presumed to carry K12 host specificity genes were inconclusive and we cannot exclude the possibility that O55 restricts K12 DNA. A polyauxotrophic mutant of O55 with better mating ability was isolated. The improved genetic homology in K12 and O55 chromosomes could account for increased frequency of recombinants obtained. This strain was used to establish the location of the loci for O55 and B5 antigens. No close linkage was found between these two antigenic determinants, locus for O55 antigen being located close tohis marker and locus for B5 antigen rather totrp marker.     

苏北地区规模化猪场产肠毒素大肠杆菌的分离鉴定及灭活疫苗效果评估

[目的]产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)是引起仔猪腹泻的重要病原菌,本研究通过调查苏北地区规模化猪场ETEC的流行情况,分析其生物学特性,研制具有免疫保护效果的优势血清型菌株的灭活疫苗,以期对苏北地区ETEC的防控提供参考。[方法]从苏北地区规模化猪场采集3-30日龄的仔猪新鲜粪样、肛拭子及小肠组织样,分离出ETEC,对分离菌株进行血清型鉴定、耐药性测定、小鼠致病力测定;最后通过动物免疫试验研究优势血清型菌株灭活疫苗对小鼠的免疫保护效果。[结果]从21个规模化猪场采集病料562份,通过PCR鉴定及测序得到141株ETEC;血清凝集试验鉴定出85株菌的O抗原血清型,其中08、0101和0128为优势血清型,占定型菌株的61.2%(52/85),其他血清型包括09、03、020、0148、0149等;分析141株ETEC对14种常见抗生素的耐药情况,得出分离株对新霉素、红霉素、四环素、庆大霉素、强力霉素、阿莫西林、甲氧苄啶/磺胺甲恶唑高度耐药,耐药率均高达80%以上;对恩诺沙星敏感性较高,敏感率达50.4%(71/141);对多粘菌素B和头孢噻肟中介耐药,占比分别为66%(93/141)和51.8%(73/141);多重耐药现象严重,其中10重耐药的菌株占比最大,为19%(27/141);小鼠攻毒试验测得08血清型强毒株YC-6的半数致死量(median lethal dose,LD50)为1.4×10^7 CFU/只,最低致死量(minimum lethal dose,MLD)为3×10^7 CFU/只;08血清型强毒株YC-6和0101血清型强毒株LYG-3制备的单价灭活疫苗对小鼠的保护率均达到100%,因此利用08血清型强毒株YC-6和0101血清型强毒株LYG-3研制二价灭活疫苗,结果显示该二价疫苗对感染不同血清型ETEC小鼠的保护率在83%以上。[结论]本研究通过对苏北地区ETEC的流行病学调查,得出其优势血清型,并研制出针对对优势血清型免疫保护效果较好的二价灭活疫苗,给临床ETEC的监测和防控提供参考。     

The inhibition of bactericidal activity of sera of newborn germ-free piglets to roughEscherichia coli by yeast mannan

The yeast phosphomannan (PM) derived fromHansenula capsulata strain exerts an inhibitory effect on thein vitro bactericidal activity of fresh sera of newborn, colostrum-deprived germ-free piglets to rough strains ofEscherichia coli (S-16 and Lilly). The experiments presented indicate that the PM function probably takes place at the C1 level. The inhibitory effect of PM does not occur provided bacteria are sensitized by specific antiserum prior to exposure to piglet serum. The antibody which was responsible for removal of PM blockade was of 19S nature, 2-mercaptoethanol-sensitive and can be absorbed by heat inactivated bacteria (roughEscherichia coli) or inhibited by addition of soluble somatic antigen (endotoxin) obtained from the same strain ofEscherichia coli (rough). The possible mechanism of inhibition of bactericidal activity by PM is discussed. This investigation was done in the Laboratory of Dr. M. A. Leon, Pathology Research, St. Luke's Hospital, Cleveland, Ohio, U.S.A.     

Variation of immunoglobulins G,A, and M and bovine serum albumin concentration in Holstein cow colostrum

Immunoglobulins G (IgG), A (IgA), and M (IgM) represent 70–80% of total proteins in cattle colostrum and are essential for the passive transfer of antibodies from the dam to the calf. Considering the practical difficulties of colostrum sample collection and the high cost of analysis, non-genetic sources of variation of the three immunoglobulins fractions have been scarcely studied together on a large scale in dairy cows. In the present study, IgG, IgA, IgM, and bovine serum albumin (BSA) were determined in colostrum samples of Holstein cows through bovine-specific radial immunodiffusion kits; such phenotypes allowed to investigate the effects of parity, herd, and calving season, and interactions. Only the first colostrum was considered in the present study, as the calf was separated from the dam immediately after birth and was not allowed to suckle. The average of IgG (n = 676), IgA (n = 573), IgM (n = 658), total immunoglobulins (n = 525), and BSA (n = 614) was 91.31, 4.20, 105.99, 5.05, and 2.47 g/L, respectively, and all traits positively correlated to each other. Overall, the immunoglobulins were less concentrated in colostrum of first- and second-parity cows than later-parity cows. These findings suggest that colostrum quality, based on Ig, is overall greater in cows that experienced more than two lactations, likely due to a greater experience of the immune system and to a wider immune heritage compared to younger cows. As regards the effect of calving season, the concentration of all Ig tended to be generally greater in colostrum sampled from August to November. Moreover, there were differences in IgG, IgA, and IgM concentration among the nine herds involved. Future studies will investigate the relationships of these traits with yield, and gross and detailed composition of bovine colostrum and will consider their genetic background to evaluate potential selection strategies to improve colostrum quality.     

The Influence of Sow Colostrum Trypsin Inhibitor on the Immunoglobulin Absorption in Newborn Piglets

The influence of sow colostrum trypsin inhibitor (SCTI) on the immunoglobulin absorption from the gut of 16 newborn colostrum-deprived piglets was investigated in a paired feeding experiment. Three times at 1 h intervals the piglets were fed an experimental diet consisting of sow milk, purified swine serum immunoglobulins containing agglutinins against Bordetella bronchiseptica, and purified SGTI (diet I) or saline (diet II). The serum concentrations of IgG, IgM, IgA, and antibodies for B. bronchiseptica were measured by single radial immunodiffusion and by a tube agglutination procedure and used to evaluate the immunoglobulin absorption. Four and 6 h after the first experimental meal, blood samples from the piglets given SGTI in their diet had a generally higher level of IgG, IgA and aggutinins against B. bronchiseptica than blood samples from the piglets d no SGTI. No real differences were found in the IgM levels. Although the piglets fed no SGTI all showed a considerable immunoglobulin absorption, the SCTI was found to have a statistically significant positive influence on the IgG and IgA absorption.