In vitro adhesion of K88acEscherichia coli to Peyer's patch and peripheral blood lymphocytes, buccal and rectal epithelial cells or intestinal epithelial brush borders of weaned pigs

Escherichia coli adhesion assays were conducted using isolated porcine peripheral blood lymphocytes, Peyer's patch lymphocytes, rectal epithelial cells or brush borders, buccal epithelial cells and brush borders from small intestinal epithelial cells. The cells and brush borders were tested for their ability to bind K88-piliated exterotoxigenic E. coliStrain M1823B (K88ac) and E. coli Strain 1476 (K-12, K88ac). Comparison of adhesive phenotypes of 37 weaned pigs as determined by the adhesion assay with small intestinal brush borders and the adherence of K88ac⁺ enterotoxigenic E. coli to peripheral blood lymphocytes, Peyer's patch lymphocytes and rectal epithelial cells or brush borders, revealed no correlation. In vitro adhesion of K88ac-bearing E. coli was always negative with buccal epithelial cells. K88ac strains varied in their ability to adhere to lymphocytes and rectale pithelial cells or brush borders, indicating that the mechanism of adherence is unrelated to K88-mediated adhesion observed in animals that had the receptors on small-intestinal epithelial-cell brush borders. The non-piliated control E. coli Strain 123 adhered to fresh peripheral blood lymphocytes, and less intensively to frozen-thawed peripheral blood lymphocytes or Peyer's patch lymphocytes. It was concluded that none of the cell types or brush borders, except small-intestinal epithelial-cell brush borders, could be used as targets for phenotyping pigs for the presence of the K88 receptors that have been associated with adhesion and colonization of K88⁺ enterotoxigenic E. coli in the porcine small intestine.     

Application of indomethacin as a potentiator of lymphocyte blastogenesis in Brucella abortus exposed cattle

Lymphocytes from $\text{Brucella abortus}$ field strain infected, strain 19 vaccinated, non-exposed and field strain infected, but immunologically unresponsive cattle were incubated with $\text{B. abortus}$ antigen and indomethacin. There were significant increases (P < 0.005) in the blastogenic responses, as measured by [³H] thymidine uptake, in cultures with indomethacin as compared to cultures without indomethacin. Lymphocyte blastogenic responses to $\text{B. abortus}$ antigen were potentiated by indomethacin in both $\text{B. abortus}$ exposed and non-exposed cultures. However, potentiation of sensitized lymphocyte blastogenic responses by indomethacin was significantly greater (P < 0.005) than that in non-exposed lymphocytes. Additionally, indomethacin significantly potentiated Brucella-induced lymphocyte blastogenic responses in lymphocytes from anergic cattle.     

Oral immunization of sows: anti-K88 antibodies in serum and milk of the sow and in serum of the piglets

Pregnant sows were immunized by oral application of live E. coli. The effect of immunization was demonstrated by measuring the titers of anti-K88 antibodies in sow serum, colostrum and milk as well as in piglet sera using enzyme-linked immunosorbent assays. The isotype involved in anti-K88 reactivity was found to be IgA. By comparing IgA-titers in colostrum and milk, the local production of this Ig-class in the mammary gland is suggested.     

Lack of reactivity of sera from Theileriaparva-infected and recovered cattle against cell membrane antigens of Theileria parva transformed cell lines

Sera from $\text{Theileria}$$\text{parva}$ infected, recovered and rechallenged cattle were tested in complement-dependent cytotoxicity, membrane immunofluorescence and antibody-dependent cellular cytotoxicity assays for the presence of antibodies against cell membrane antigens of $\text{T}$. $\text{parva}$ transformed cell lines.In the complement-dependent antibody-mediated cytotoxicity assay, sera from lethally infected animals were negative. Some recovered cattle showed a positive reaction, but such reactions were also observed when an eland cell line infected with $\text{T}$. $\text{taurotragi}$, and bovine lymphoblastoid cells were used as targets. Reaction was less against Ig-negative peripheral blood lymphocytes.Evidence is presented that these reactions could be evoked by attachment of immune complexes to Fc-receptors. It is concluded that cattle exposed to $\text{T}$. $\text{parva}$ infection do not develop antibodies against specific $\text{T}$. $\text{parva}$ (or $\text{T}$. $\text{parva}$-induced) cell surface antigens.     

大肠杆菌MM—3基因工程活菌苗免疫母猪血清及乳清中抗K_(88ac)及LTB抗体动态

以MM-3基因工程活菌苗口服免疫妊娠73d、83d、103d母猪各4头,口服及肌注免疫妊娠92d母猪各5头,用间接BA-ELISA法检测血清及乳清中IgG、IgA和IgM型抗K_(88ac)及LTB抗体的动态变化.结果表明,无论口服还是肌注免疫母猪,抗K_(88ac)及抗LTB抗体均极显著增高.肌注以IgG增加为主,口服以IgA增加为主.肌注免疫IgG型抗K_(88ac)抗体滴度极显著地高于口服免疫(P<0.01);口服免疫IgA型抗LTB抗体滴度显著高于肌注免疫(P<0.05).口服及肌注免疫乳清中抗K_(88ac)及抗LTB抗体滴度很高,达1:3200～204800,但在1周内即降低,约减少一半(约3～5个滴度),2～3周内各型抗体下降缓慢,维持在对照组周内水平,4周后达最低水平.妊娠不同时期口服免疫,其血清中抗K_(88ac)抗体的动态变化相似,但乳汁中抗体则差异非常明显,妊娠83d和92d免疫组IgG、IgA和IgM均显著增加,且高于103d组(P<0.01)和73d组(P<0.05).口服免疫的最佳时间为分娩前26～30d,肌注免疫最佳免疫时间为分娩前21d.     

Immunity to Escherichia coli in pigs: Antibody Secretion by the Mammary Gland after Intramammary or Intramuscular Vaccination with an E. coli Vaccine

Indirect hemagglutinating antibody titres in individual gland samples of colostrum and milk from 13 sows were measured. Five of the sows were vaccinated via a mammary gland and five by the intramuscular route with a live formalinised Escherichia coli vaccine and three remained as non-vaccinated controls.

Antibody titres were higher in colostral and milk whey from the vaccinated sows than from non-vaccinated groups. The inoculated gland in the group of sows given vaccine by the intramammary route secreted milk containing markedly more antibodies to the vaccine E. coli strain than did the non-vaccinated glands. Milk from the vaccinated gland did not contain higher titres to heterologous E. coli O antigens than milk from non-vaccinated glands. Serum titres were the same or higher than the titres in colostrum from non-vaccinated glands.

     

Survey on the prevalence of diarrhoea in pre-weaning piglets and on feeding systems as contributing risk factors in smallholdings in Central Vietnam

A cross-sectional survey on the prevalence of diarrhoea in pre-weaning piglets, and on management and feeding systems under farm conditions was carried out in Thua Thien Hue Province. Faecal samples were collected from 63 piglets without, and 90 piglets with diarrhoea to determine the occurrence of Salmonella, Escherichia coli (E. coli) and different E. coli antigens (K88, K99 and 987P). The prevalence of diarrhoea was higher in the rainy season than in the dry season (33% vs 18%) and the results indicated differences in prevalence between areas. Salmonella and E. coli were found to the same extent in faeces from piglets without and with diarrhoea. All E. coli antigens were isolated from piglets without and with diarrhoea. However, the frequency was much higher in piglets suffering from diarrhoea. In piglets with diarrhoea antigen K88 was found in 26% and 20% of the samples, antigen K99 in 37% and 24% of the samples, and antigen 987P in 31% and 32% of the samples collected in the dry and rainy seasons, respectively. Nutrient supply for sows and for piglets was low in comparison with feeding standards, which may be a contributory factor to the high incidence of diarrhoea in piglets. Thus, the nutrition of sows as well as piglets could be important components in the aetiology of the disease and needs to be studied further.     

Development of immune responsiveness to Ascarissuum antigens in pigs vaccinated with ultraviolet-attenuated eggs

Pigs fed $\text{Ascaris}$$\text{suum}$ eggs attenuated by short wave ultraviolet-radiation developed resistance to challenge infection. Per os inoculation of pigs on three successive weeks with 10,000 eggs irradiated to total exposures of 150,100 and 75 μW-min/cm², respectively, resulted in an 88% reduction in the number of larvae recovered from the lungs 7 days after challenge with 10,000 infective eggs. Peripheral blood lymphocytes (PBL) from vaccinated pigs were specifically stimulated in vitro to incorporate tritiated thymidine by egg hatching fluid (Ea) and by excretory-secretory products obtained from cultures in defined-media of second-stage larvae (L2) developing to third-stage (L2–3) and from cultures of third-stage larvae developing to fourth-stage (L3–4). PBL were also specifically stimulated by living L2. Ea and L2 stimulated pig PBL significantly at 7 days after the first inoculation; responses to L2–3 and L3–4 developed 7 days after a second inoculation. The antigen-responsive cells in the PBL population were non-immunoglobulin-bearing lymphocytes. Antibodies to Ea and L2–3 were not detected in the serum of vaccinated pigs, and only 3 of 7 pigs had low concentrations of serum antibodies to L3–4.     

一株母猪产道益生性粪肠球菌的分离鉴定

产道作为母猪生殖系统的重要器官组织,与繁殖性能密切相关。本试验无菌采集江西省某养殖场窝产14头母猪产道中段阴道分泌物进行传统细菌分离培养,初步筛选出4株细菌,挑选其中一株通过生化反应和16S rDNA序列分析方法进行鉴定,同时进行抗生素抵抗能力试验和牛津杯法体外抑制试验。生化鉴定结果表明该菌可降解果糖、乳糖、半乳糖等,该菌代谢产物与葡萄糖酸盐无反应,葡萄糖产气试验说明该菌产酸不产气。分离菌株通过NCBI同源性比对为粪肠球菌（登录号EU794735.1）。分离菌对青霉素、头孢哌酮等高度敏感,对红霉素、庆大霉素等耐药。体外抑制试验中分离菌对大肠杆菌k88和变形杆菌具有良好的抑制效果,可以作为候选益生菌株进行深入研究。这为今后益生菌代替抗生素及产道菌群移植防治母猪产道炎症提供理论基础。     

Phenotypic expression of K88 adhesin alone or simultaneously with K99 and/or F41 adhesins in the bovine enterotoxigenic Escherichia coli strain B41

F41-positive and F41-negative derivatives of bovine enterotoxigenic Escherichia coli strain B41 carrying K88 or K88 and K99 plasmids were investigated for stability and expression of genes for their fimbrial antigens. Either K88 plasmid alone or both K88 and K99 plasmids could be maintained in these strains though stability could depend on culture medium. K99 antigen could be detected in each strain bearing K99 plasmid. Clones that produced K88 antigen or clones that did not produce this antigen could be isolated from each strain, except from the strain that possessed K99 plasmid in the strain that did not possess the ability to produce F41 antigen. Strains possessing K88 plasmid in the strain able to produce F41 antigen produced clones expressing either both K88 and F41 antigens, (also F41 appeared strongly expressed in some clones) or clones that produced only F41 antigen or no antigen at all. Clones that produced only K88 antigen or others that did not produce this antigen could be produced from a strain bearing only K88 plasmid and that did not possess the ability to produce F41 antigen. None of these strains bearing K88 plasmid alone or additionally K99 plasmid produced mannose-resistant hemagglutination of horse or sheep erythrocytes at 20°C as found for K99 and F41 ETEC natural strains, respectively. These results suggested that the structures of pili when several genetic determinants were present simultaneously may not be identical to those of original strains. In this study, clones expressing either one, two or three adhesin bearing antigens could be obtained from the strain B41.     

The irradiation of Babesia bovis. II. The immunogenicity of irradiated blood parasites for intact cattle and splenectomised calves

Intraerythrocytic forms of $\text{B. bovis}$ were exposed to 350 Grays (Gy) γ irradiation and were then injected intravenously into intact two and three year old Hereford steers. One of 15 steers died on initial infection and subsequently six steers were given a virulent heterologous challenge three weeks after recovery; all six animals were highly immune. The remaining eight animals were kept under quarantine conditions for 10 months and were then challenged with a different virulent heterologous strain of $\text{B. bovis}$. Seven of eight were highly immune, but one animal died. Subsequently a further 12 steers were injected intravenously with 1 × 10⁸ irradiated organisms. All showed only mild transient clinical signs. After 12 months quarantine in a tick-free area these animals were then challenged with a virulent heterologous strain and all 12 were shown to be highly immune. Irradiation reduced the infective dose from 1 × 10⁸ to 2.5 × 10³ parasites. These parasites multiplied at the same rate, and achieved the same maximum parasitaemia as the parent non-irradiated strain, but the disease produced by them was not severe. A dose of 2.5 × 10³ non-irradiated paasites was lethal to all of the four animals which received it. It was concluded that irradiation had produced a predominantly avirulent parasite population.     

Inhibition of adhesion of Escherichia coli K88 antigen by mammary secretions of susceptible and resistant sows

Anti-adhesive activities of colostrum and milk from genetically susceptible sows, which protected their susceptible offspring in an outbreak of neonatal diarrhoea caused by K88-positive Escherichia coli, were compared with the activities in mammary secretions of resistant dams that did not protect their susceptible progeny. There was significantly more anti-adhesive antibody in the secretions of susceptible sows than in resistant sows, both during the disease period, and 1 year later. Fractionation of colostrum by gel filtration and ion-exchange chromatography led to identification of the anti-adhesive antibodies as including both IgA and IgM.     

Response of Gnotobiotic Pigs to Escherichia coli

In a study of the response of gnotobiotic pigs to coliform infections, 45 one-week-old germfree pigs were divided into five groups and each group was inoculated orally with a different strain of Escherichia coli. Three of these were enteropathogenic swine strains, P307[08:K87(B), K88 a,b (L):H19]; P570 [0138:K81]; P568[0141:K85a,b(B), K88a,b(L):H4], one was a virulent human strain, H224, [026:K60(B6)], and one was a non-enteropathogenic swine strain, P581[OX13:K68]. It was attempted to protect a portion of the pigs with orally administered specific antisera and sera from non-immunized specific pathogenfree (SPF) pigs. Observations were made on the clinical response, bacterial counts of feces and intestinal contents, gross pathological changes, distribution of the organisms in organs and serum hemagglutinin titers.

Infection with E. coli P307 resulted in diarrhea, dehydration and death, unless the pig was protected with specific antiserum. The pigs infected with E. coli P570 had a transient diarrhea but retained their appetites and recovered. Those infected with the other three strains remained healthy throughout. No circulating hemagglutinating antibody against the test strains of E. coli could be detected in any of the pigs seven days or earlier post-inoculation.

Relationship could not be established between the numbers of viable E. coli in the feces and the presence of clinical colibacillosis. Orally administered specific antiserum afforded protection against strain P307, but did not reduce the number of E. coli in the gut or alter their distribution in the internal organs. This suggested that the protective effect of specific antibody in the intestine was due to its action on a metabolite (enterotoxin) produced by E. coli P307 rather than the organism itself.

     

Effects of ionizing radiation on in vitro proliferative responses of porcine peripheral blood lymphocytes

The radiosensitivity of $\text{in vitro}$ proliferative responses of porcine peripheral blood lymphocytes (PBL) was assessed. PBL were stimulated by Con-A, PHA, culture supernates from mitogen-stimulated porcine lymphocytes, or in the case of antigen-primed swine, specific antigens. The resulting levels of proliferation were assessed by a determination of the level of incorporation of tritiated thymidine $\text{in vitro}$, and in some cases by the presence of blast cells in the cultures. Porcine PBL were found to be more radioresistant than either mouse PBL or mouse spleen cells. Irradiation levels of greater than 3000 rads were necessary to arrest Con-A or PHA-induced proliferative responses. Proliferation induced by lymphokines in the form of supernates from mitogen-stimulated lymphocyte cultures was arrested in PBL that had received 3000 rads prior to culture. Antigen-induced proliferative responses in primed porcine PBL populations were the most radiosensitive, in that a previous irradiation with 500 rads was sufficient to completely abolish a secondary $\text{in vitro}$ proliferative response.     

Bovine monoclonal antibodies to the F5 (K99) pilus antigen of E. coli, produced by murine/bovine hybridomas

Lymph node cells from calves immunized with purified pilus antigen of K99+ enterotoxigenic E. coli (ETEC) were fused with mouse myeloma (NSO) cells, and with non-Ig producing mouse/calf hybridomas or with a bovine Ig-producing mouse/calf/calf secondary hybridoma. Lines secreting bovine monoclonal IgG1 specific for K99 pilus antigen in an ELISA were obtained in each case. The two lines derived from xenohybridoma fusion partners have been secreting anti-K99 bovine monoclonal antibody for over one year in continual passage. None of the antibodies cross-reacted with other pilus types including K88, CFAI, CFAII, 987P or CP; they all inhibited agglutination of horse RBC (which have a K99 receptor) in the presence of K99 antigen; they showed positive fluorescence in an indirect binding assay on K99+ ETEC and inhibited K99+ ETEC adhesion to piglet enterocytes. These antibodies have potential prophylactic and therapeutic use in control and treatment of diarrhoea.     

Performance and diarrhoea in piglets following weaning at seven weeks of age: Challenge with E. coli O 149 and effect of dietary factors

Four dietary factors (ad libitum versus feed restriction, control versus protein restriction at ad libitum feeding, control versus inclusion of lupin as a protein source at ad libitum feeding, and control versus extra vitamin E at ad libitum feeding) were tested in four separate experiments for the effect on diarrhoea. To introduce a diarrhoea-like condition, half of the piglets were challenged with an E. coli O 149 dose of 1 × 10⁸ colony forming units on days one and two after weaning (day of weaning = day zero). All piglets were susceptible since the dams were tested mono-zygotic susceptible to the attachment site of E. coli O 149 in the intestines. Each of the four experiments included 32 piglets from 4 sows. The design was a 2 × 2 factorial with dietary factor and E. coli O 149 challenge as the two factors, each at two levels. The piglets were housed individually during the experiment which lasted for 10 days from weaning at 7 weeks of age. The daily recordings included feed intake, weight and faecal score (from 1 = solid and cloddy to 6 = watery and yellow). Faeces from days 1 to 4 were tested for E. coli strains. In addition, blood was sampled and serum was analysed for antibodies to E. coli, IgG and IgM. Generally the E. coli challenge had no effect on growth and feed intake whereas faecal score and number of faecal haemolytic bacteria increased and faecal dry matter decreased. Feed restriction decreased the weight gain while faecal characteristics were unaffected. An analysis including all four experiments revealed that a feed intake of less than 200 g during the first day after weaning seems to be associated with a relatively high incidence of a post-weaning diarrhoea-like condition. Protein restriction decreased faecal score and increased faecal dry matter while weight gain tended to decrease. Inclusion of lupin affected neither weight gain nor faecal characteristics. Extra vitamin E did not affect weight gain while faecal dry matter decreased, and faecal score and number of faecal haemolytic bacteria increased. The dietary treatments had no effect on the measured immunoglobulins. In conclusion, the studied dietary factors could not alleviate a diarrhoea-like condition and at the same time maintain the growth rate. Furthermore, the results indicate that performance can be improved if piglets achieve a daily feed intake of at least 200 g during the first day after weaning.     

Extended‐spectrum beta‐lactamase‐producing Escherichia coli in common vampire bats Desmodus rotundus and livestock in Peru

Antibiotic resistance mediated by bacterial production of extended‐spectrum beta‐lactamase (ESBL) is a global threat to public health. ESBL resistance is most commonly hospital‐acquired; however, infections acquired outside of hospital settings have raised concerns over the role of livestock and wildlife in the zoonotic spread of ESBL‐producing bacteria. Only limited data are available on the circulation of ESBL‐producing bacteria in animals. Here, we report ESBL‐producing Escherichia coli in wild common vampire bats Desmodus rotundus and livestock near Lima, Peru. Molecular analyses revealed that most of this resistance resulted from the expression of bla_CTX‐M‐15 genes carried by plasmids, which are disseminating worldwide in hospital settings and have also been observed in healthy children of Peru. Multilocus sequence typing showed a diverse pool of E. coli strains carrying this resistance that were not always host species‐specific, suggesting sharing of strains between species or infection from a common source. This study shows widespread ESBL resistance in wild and domestic animals, supporting animal communities as a potential source of resistance. Future work is needed to elucidate the role of bats in the dissemination of antibiotic‐resistant strains of public health importance and to understand the origin of the observed resistance.     

The phagocytic cell response of the ovine lung to Pasteurella haemolytica

Conventionally-reared sheep were inoculated with (3.0 ± 0.6 × 10⁷) viable Pasteurella haemolytica type A1 by the intratracheal route and were killed immediately (0-time) or 2, 4, 12, 16, 24, 48 or 72 h later. Lung-wash cells and free bacteria were recovered by pulmonary layage.The number of recoverable bacteria tended to increase between 0-time and 4 h post-in-oculation (p.i.) then decline rapidly over the next 8 h. However, the rate of clearance was extremely variable and viable bacteria were recovered from $\text{3}\text{5}$ animals at 48 h p.i. and from $\text{1}\text{5}$ at 72 h p.i.In parallel with the clearance of the majority of the bacteria, total neutrophil numbers in the lung-wash rose to a peak of (36 ± 6) × 10⁸ cells/lung, which was, on average, 70-fold higher than 0-time levels. Their numbers remained constant from 12 to 24 h p.i. then fell to be 5-fold above 0-time levels at 72 h p.i. Macrophage numbers rose slowly throughout the experiment but most of the increase occurred between 24 and 48 h p.i. They reached a peak of (17 ± 11) × 10⁸ cells/lung at 48 h i.p. which was 3-fold higher than 0-time levels.     

Bacteria in milk from anterior and posterior mammary glands in sows affected and unaffected by postpartum dysgalactia syndrome (PPDS)

Background

The performance of piglet weight gain is strongly dependent on the sow''s ability to meet the demand for adequate milk. Postparturient disorders, especially those subsumed under the term postpartum dysgalactia syndrome (PPDS), can alter or reduce the milk production sensitively, resulting in starving piglets. The aim of this study was to gather further information about the prevalence of different bacterial species in the anterior and posterior mammary glands of sows with respect to the clinical appearance of PPDS.

Methods

In this study, the health status of 56 sows after farrowing was determined with special regard to mastitis and dysgalactia. Pooled milk samples from anterior and posterior glands were taken from both affected and non-affected animals and analysed bacteriologically for the presence of a wide spectrum of different pathogens.

Results

Mainly Escherichia coli, staphylococci and streptococci were detected in high percentages but without significant differences in healthy and diseased animals and anterior and posterior glands. However, the large percentages of coliform bacteria suggested a transmission route via faecal contamination.

Conclusion

In this study, the prevalence of different bacteria in anterior and posterior glands in PPDS positive and negative sows was analysed. No significant differences in bacteria of healthy and diseased sows were assessed. Therefore, the development of clinical PPDS and actual infection seems to be largely dependant on individual resistance in single sows.