Cloning and radiation hybrid mapping of bovine toll-like receptor-4 (TLR-4) signaling molecules

Toll-like receptor (TLR)-4 is a transmembrane receptor for lipopolysaccharide, a highly pro-inflammatory component of the outer membrane of Gram-negative bacteria. To date, molecules of the TLR-4 signaling pathway have not been well characterized in cattle. The goal of this study was to clone and sequence the full-length coding regions of bovine genes involved in TLR-4 signaling including CASP8, IRAK1, LY96 (MD-2), TICAM2, TIRAP, TOLLIP and TRAF 6 and to position these genes, as well as MyD88 and TICAM1, on the bovine genome using radiation hybrid mapping. Results of this work indicate differences with a previously published bovine sequence for LY96 and a predicted sequence in the GenBank database for TIRAP based on the most recent assembly of the bovine genome. In addition, discrepancies between actual and predicted chromosomal map positions based on the Btau_2.0 genome assembly release were identified, although map positions were consistent with predicted locations based on the current bovine-human comparative map. Alignment of the bovine amino acid sequences with human and murine sequences showed a broad range in conservation, from 52 to 93%. Overall, this work should assist in the assembly and annotation of the bovine genome sequence, the identification of variations in genes critically involved in host innate immunity, and facilitate the study of TLR-4 signaling pathways in cattle.     

奶牛乳腺炎源性大肠杆菌csgC基因的克隆及载体构建

根据GenBank上发表的curli菌毛csgC基因序列设计了1对特异性引物.从患乳腺炎的奶牛乳汁中分离出致病性大肠杆菌,经生物学鉴定后,提取全基因组DNA为模板,PCR扩增出csgC基因,连入pMD18-T克隆载体,测序,结果表明,扩增片段含有333个核苷酸,编码111个氨基酸的成熟蛋白,与已报道的大肠杆菌W3110的全基因组DNA中的csgC基因序列最相近,氨基酸序列同源性为99.7%.该基因与原核表达载体pET32a+相连,转入BL21(DE3)感受态细胞.挑选阳性菌落经PCR鉴定和酶切鉴定后表明成功构建原核表达载体pET32a+/csgC.     

Hyperacute Escherichia coli mastitis of cattle in the immediate post-partum period

The pathology of hyperacute coliform mastitis was studied in 5 post-parturient cows. In all infected quarters infiltration of neutrophils was negligible. In all except one case there was severe damage to the ductular and secretory system, involving most areas of the gland. Bacteria were dense in infected alveoli, and there was evidence of substantial phagocytosis of bacteria by the secretory epithelium. The exception showed a large lesion in the middle of the gland from which the spread was ductular; other infections were consistent with spread via the teat canal. The organisms were largely confined to the ductular/secretary lumen and there was little invasion of the parenchyma. The severity of the disease was considered due to the absence of the inflammatory response seen in mid lactation.     

Progressive pathology of severe Escherichia coli mastitis in dairy cows

An investigation was made using light and electron microscopy of the progressive pathological changes in nine experimental and two natural cases of severe Escherichia coli mastitis in dairy cows. The duration of infection varied from 18 hours to 13 days. Epithelial lesions were not found in glands which had been infected for more than 24 hours. However, the epithelia of the sinuses and large ducts became hyperplastic after 60 hours of infection and by six days hyperplasia was extensive on the crests of folds. The leucocyte response in the lumina of the glands and subepithelial tissue showed a progressive change from an acute neutrophil reaction to a chronic mononuclear cell infiltration within the first 36 hours of infection. The only changes affecting the secretory tissue occurred after six days of infection and were typical of mammary gland involution which was probably a direct consequence of anorexia.     

Relationship between serologic recognition of Escherichia coli 0111:B4 (J5) and clinical coliform mastitis in cattle

Serum IgG1 ELISA titers recognizing gram-negative core antigens (Escherichia coli [J5]) were studied at a large dairy in central California. Population mean log10 titer was 2.7357 (equivalent to 1:544) with a SE of 0.03843. Titers increased with increased lactation number (unstandardized regression coefficient = 0.06733). Changes in lactation number accounted for only 6.77% of titer variation. Titers less than 1:240 were associated with 5.33 times the risk of clinical coliform mastitis. Also, older cattle were at greater risk to develop clinical coliform mastitis. These factors apparently affect incidence in a nonlinear fashion, with greatly increased risk associated with titers less than 1:240 and with fourth or greater lactations.     

Partial budget analysis of vaccinating dairy cattle against coliform mastitis with an Escherichia coli J5 vaccine

Partial budget analysis of clinical coliform mastitis prevention supported vaccination of dairy cows with an Escherichia coli J5 vaccine. Increased profits of $57/cow lactation were predicted using a computer spreadsheet derived partial budget with generalized herd input data. Herd vaccination programs were predicted to be profitable when greater than 1% of cow lactations resulted in clinical coliform mastitis. Herd vaccination programs were predicted to be profitable at all herd milk production levels.     

Shiga toxin-producing Escherichia coli (STEC) isolated from healthy dairy cattle in southern Brazil

Over a period of 1 year, the production of verotoxin was investigated in 1127 Escherichia coli isolated from 243 dairy cattle from 60 small farms in southern Brazil. Vero cell assay was used to detect toxins in culture supernatants from E. coli isolated from bovine feces. Shiga toxin-producing E. coli (STEC) detection rates were 95% (57 of 60) for farms and 49% (119 of 243) for cattle. Prevalence of STEC-positive cattle in the farms ranged from 0 to 100%. Ninety-six percent (315 of 327) of the STEC isolates did not react in the panel of sera used for typing. Twelve isolates, all non-motile, belonged to serogroups previously associated with human diseases, and 67% (8 of 12) were of only two serotypes (O91:H- and sorbitol-fermenting O157:H-). These results indicate that dairy cattle from the region surveyed may be a source of STEC potentially pathogenic for humans.     

Experimental udder infections with Escherichia coli. 4. Discussion of the pathogenesis of acute mastitis

The correlations among findings obtained from experimental Escherichia coli udder infection and reported in the three previous communications are discussed in this paper. The defence capabilities elucidated included specific and unspecific activities of non-epithelial cells, primarily neutrophilic granulocytes, macrophages, lymphocytes, plasma cells, and mast cells, as well as the capability of epithelial cells proper of absorbing pathogenic material from lumens of lactiferous ducts and milk cisterns and of storing such material within intracytoplasmic vacuoles. Hence, alterations in the course of pathogenesis of acute coli mastitis were found to be of complex nature and could be properly followed up by their morphological patterns.     

Characterisation and clonal relationships of Shiga-toxigenic Escherichia coli (STEC) isolated from Australian dairy cattle

A total of 136 Shiga toxin-producing Escherichia coli (STEC) isolated during a longitudinal survey of three Australian dairy farms were examined to determine their virulence factors, serotype and genomic relationships. This study aimed to assess the potential of these STEC to cause disease in humans and to analyse the on-farm ecology of STEC. Virulence factors (stx, eae, ehxA) were used as determinants of potential to be enterohaemorrhagic E. coli (EHEC) and were examined using polymerase chain reaction (PCR). Among the cattle groups tested, calves, both before and during weaning, shed the most putative EHEC and were the main source of serotypes commonly associated with human disease. E. coli O157:H7 and E. coli O26:H11 represented 9.4 and 7.8% of cattle STEC isolates respectively, with other putative EHEC serotypes reported for the first time from cattle. Based on serotype and virulence factors, 20% of STEC were putative EHEC. Pulsed-field gel electrophoresis (PFGE) was used to compare the genomic profiles of STEC from dairy farms. Isolates common to cattle and the farm environment were identified. Multiple strains of STEC with high clonal turnover were detected in the faeces of cattle, and isolates appeared to be specific to individual farms. To fully assess the pre-slaughter EHEC risk factors on-farm, examination of STEC virulence is as important as determination of STEC prevalence.     

Role of prostaglandins in pathogenesis of bovine mastitis induced by Escherichia coli endotoxin

Four doses (5 to 100 micrograms, 1 dose/quarter) of Escherichia coli endotoxin were introduced into lactating mammary glands of 2 cows. There was no effect on milk prostaglandin (PG) E2 concentration, except that the concentration was increased from 200 pg/ml of milk to 1,060 pg/ml at post-treatment hour (PTH) 8 in cow 1 and from 75 to 420 pg/ml at PTH 4 in cow 2 after the highest dose 100 micrograms. Endotoxin caused a dose-dependent increase in milk PGF2 alpha concentrations in both cows. After the highest dose, PGF2 alpha was maximally increased from 200 to 3,500 pg/ml at PTH 4 in cow 1 and from 250 to 2,000 pg/ml in cow 2 at PTH 8. The instillation of 50 micrograms of endotoxin in all 8 quarters of 2 more lactating cows caused no significant (P greater than 0.05) changes in milk PGE2 and thromboxane B2 concentrations, whereas milk PGF2 alpha was significantly increased from the base-line value of 642 to 2,683, 1,189, and 2,281 pg/ml at PTH 4, 8, and 12, respectively. The 6-keto-PGF1 alpha was also significantly increased from the base-line value of 305 to 871, 631, and 600 pg/ml at the corresponding times, respectively. A marked increase in vascular permeability, as judged by high concentrations of serum albumin in the whey, was observed as early as PTH 4 and peaked at PTH 12 followed by a gradual decline, although it remained significantly increased over the control for 48 hours after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of toll-like receptors in the pathogenesis of Streptococcus suis

Streptococcus suis is an important agent of swine and human meningitis. Sequence type (ST) 7 emerged in China and was responsible for the human epidemic caused by S. suis in 2005. The virulence of S. suis ST7 is greater than the wild type pathogenic S. suis, ST1; however, the mechanisms for this increased pathogenicity are unknown. The aim of this study was to determine the role of different toll-like receptors (TLRs) involved in regulating the host response to the S. suis infection and to speculate on differing mechanisms used by ST7 strains to induce disease. Here we compared two ST7 strains isolated in the 2005 Sichuan outbreak to two ST1 strains. Our data show TLR2, 6 and 9 are involved in the recognition of heat-killed S. suis independent of the ST type. We found the TLR-dependent cytokine production differed between the two types of strains using whole cell lysate proteins. TLR6 played a greater role in cytokine production induced by the whole cell lysate proteins from the ST7 strain than in that induced by the ST1 strain lysates. The data suggest that mechanisms of inflammation induced by S. suis strains differ where this will be useful in designing efficient strategies in combating streptococcal toxic shock-like syndrome caused by the S. suis ST7 strains.     

Sortase anchored proteins of Streptococcus uberis play major roles in the pathogenesis of bovine mastitis in dairy cattle

Streptococcus uberis, strain 0140J, contains a single copy sortase A (srtA), encoding a transamidase capable of covalently anchoring specific proteins to peptidoglycan. Unlike the wild-type, an isogenic mutant carrying an inactivating ISS1 insertion within srtA was only able to infect the bovine mammary gland in a transient fashion. For the first 24 h post challenge, the srtA mutant colonised at a similar rate and number to the wild type strain, but unlike the wild type did not subsequently colonise in higher numbers. Similar levels of host cell infiltration were detected in response to infection with both strains, but only in those mammary quarters infected with the wild type strain were clinical signs of disease evident. Mutants that failed to express individual sortase substrate proteins (sub0135, sub0145, sub0207, sub0241, sub0826, sub0888, sub1095, sub1154, sub1370, and sub1730) were isolated and their virulence determined in the same challenge model. This revealed that mutants lacking sub0145, sub1095 and sub1154 were attenuated in cattle. These data demonstrate that a number of sortase anchored proteins each play a distinct, non-redundant and important role in pathogenesis of S. uberis infection within the lactating bovine mammary gland.     

奶牛乳腺炎防治的思考与探讨

从 3个方面分析了奶牛乳腺炎防治失败的原因 ,包括 :抗生素 ,细菌和宿主之间相互作用和自身发生变化 ;不注重隐性乳房炎的治疗 ;挤奶技术、设备、程序不当等。提出防治乳腺炎几点建议 :创造良好的饲养环境 ;补充维生素和微量元素 ;开发研制奶牛乳腺炎疫苗 ;奶牛乳腺炎的综合治疗     

The possible involvement of seagulls (Larus sp) in the transmission of salmonella in dairy cattle.

A series of infections in a herd of dairy cows by different salmonella serotypes over a seven year period is described. The source of infection appeared to be the private water supply contaminated by seagulls.     

The Pho regulon and the pathogenesis of Escherichia coli

During the course of infection, bacteria must coordinately regulate gene expression in response to environmental stimuli. The phosphate (Pho) regulon is controlled by the two component-regulatory system PhoBR. PhoBR is activated during starvation and regulates genes involved in phosphate homeostasis. Several studies have highlighted the importance of the Pho regulon in bacterial pathogenesis, showing how induction of PhoBR, in addition to regulating genes participating in phosphate metabolism, leads to modulation of many cellular processes. The pleiotropic effects of Pho regulon activation include attenuated virulence and alteration of many virulence traits, including adhesion to host cells and resistance to cationic antimicrobial peptides, acidity and oxidative stresses. This review provides an overview of the relationship between the Pho regulon and virulence in Escherichia coli and illustrates that, in addition to regulating phosphate homeostasis, the Pho regulon plays a key role in regulating stress responses and virulence.     

Binding of host glycosaminoglycans and milk proteins: possible role in the pathogenesis of Streptococcus uberis mastitis

The role of indirect binding of host proteins through glycosaminoglycans (GAGs) on adherence and internalization of Streptococcus uberis to bovine mammary epithelial cells was evaluated. Preincubation of S. uberis with GAGs followed by incubation with fetal bovine serum (FBS), bovine milk or milk proteins resulted in greater adherence to and internalization of S. uberis into mammary epithelial cells than observed in untreated controls. Highest values were detected, when final incubation was done with milk. Greater adherence to and internalization into mammary epithelial cells were observed when heparin sulfate (HEP) and milk were used compared with any other GAG and FBS. When individual milk proteins were used, greatest adherence and internalization were observed when S. uberis strains were pretreated with HEP followed by treatment with beta-casein. The findings of this study illustrate a pathogenic strategy of S. uberis that may occur during the very early stages of infection.     

Prevention of clinical coliform mastitis in dairy cows by a mutant Escherichia coli vaccine.

A prospective cohort study was undertaken in two commercial California dairies. The treatment group, 246 cows, received three doses of a whole cell bacterin of J5 Escherichia coli (mutant of E. coli O111:B4) plus Freund's incomplete adjuvant vaccine (two in the dry period and one after calving) while 240 unvaccinated cows served as controls. Thirty-five cases of clinical coliform mastitis were diagnosed, six in vaccinated cows and 29 in unvaccinated cows. Bacteria isolated from the clinical cases included 15 E. coli five Klebsiella pneumoniae, three K. oxytoca, three K. ozaenae, five Enterobacter aerogenes, three Serratia marcescens and one Serratia spp. Four control cows were culled, three of them because of chronic coliform mastitis and one because of postcoliform infection agalactia. Incidence rate of clinical gram-negative mastitis was 2.57% in vaccinated cows and 12.77% in unvaccinated cows. The estimated risk ratio, the measure of risk of having clinical gram-negative mastitis for vaccinated cows to unvaccinated cows, was 0.20 (p less than 0.005), indicating a strong relationship between vaccination and lack of clinical gram-negative mastitis. The results of this trial indicate that the administration of the E. coli J5 vaccine is protective against natural challenge to gram-negative bacteria, and reduces the incidence of clinical gram-negative mastitis in dairy cows during the first three months of lactation.     

Phenotypic and genotypic antimicrobial resistance patterns of Escherichia coli isolated from dairy cows with mastitis

Pulsed field gel electrophoresis (PFGE) patterns, susceptibility to 26 antimicrobial agents used in veterinary and human medicine, and prevalence of antimicrobial resistance genes of Escherichia coli isolated from cows with mastitis were evaluated. Among 135 E. coli isolates, PFGE analysis revealed 85 different genetic patterns. All E. coli were resistant to two or more antimicrobials in different combinations. Most E. coli were resistant to antimicrobials used in veterinary medicine including ampicillin (98.4%, >or=32 microg/ml) and many E. coli were resistant to streptomycin (40.3%, >or=64 microg/ml), sulfisoxazole (34.1%, >or=512 microg/ml), and tetracycline (24.8%, >or=16 microg/ml). Most E. coli were resistant to antimicrobials used in human medicine including aztreonam (97.7%, >or=32 microg/ml) and cefaclor (89.9%, >or=32 microg/ml). Some E. coli were resistant to nitrofurantoin (38%, >or=128 microg/ml), cefuroxime (22.5%, >or=32 microg/ml), fosfomycin (17.8%, >or=256 microg/ml). All E. coli were susceptible to ciprofloxacin and cinoxacin. Almost 97% (123 of 127) of ampicillin-resistant isolates carried ampC. Eleven of 52 (21.2%) streptomycin-resistant isolates carried strA, strB and aadA together and 29 streptomycin-resistant isolates (55.8%) carried aadA alone. Among 44 sulfisoxazole-resistant E. coli, 1 isolate (2.3%) carried both sulI and sulII, 12 (27.3%) carried sulI and 10 (22.7%) isolates carried sulII. Among 32 tetracycline-resistant isolates, 14 (43.8%) carried both tetA and tetC and 14 (43.8%) carried tetC. Results of this study demonstrated that E. coli from cows with mastitis were genotypically different, multidrug resistant and carried multiple resistance genes. These bacteria can be a reservoir for antimicrobial resistance genes and can play a role in the dissemination of antimicrobial resistance genes to other pathogenic and commensal bacteria in the dairy farm environment.     

The role of endotoxin in the pathogenesis of conform mastitis in sows

Summary

Sows were made tolerant to Escherichia coli endotoxin by daily intravenous (IV) injection of the pyrogen. A refractory state was induced, characterised by a markedly decreased fever. In contrast, intramammary (IMM) infusion of only a quarter of the endotoxin dose to which the animals were made tolerant by IV injection produced a markedly increased fever. This finding suggests that inflammatory endogenous mediators were released in the mammary glands and that their subsequent absorption into the blood circulation, and not the absorption of endotoxin caused fever.     

Prevalence of verotoxin-producing Escherichia coli (VTEC) in bovine coli mastitis and their antibiotic resistance patterns.

Between December 1996 and October 1997, milk samples from a total of 145 cows with coli mastitis were screened for the presence of verotoxin-producing E. coli (VTEC). VTEC were found in four (2.8%) out of the 145 samples. The four isolated strains proved to be verotoxin (VT) 1-, VT2- or VT1- and VT2-positive. However, no strain contained all three virulence factors tested. Further strain characterization was carried out by serotyping as well as by resistance pattern analysis.