Feeding of waste milk to Holstein calves affects antimicrobial resistance of Escherichia coli and Pasteurella multocida isolated from fecal and nasal swabs

The use of milk containing antimicrobial residues in calf feeding programs has been shown to select for resistant fecal Escherichia coli in dairy calves. However, information is scarce about the effects of feeding calves waste milk (WM) on the prevalence of multidrug-resistant bacteria. The objective of this study was to determine the antimicrobial resistance patterns of fecal E. coli and nasal Pasteurella multocida isolates from calves fed either milk replacer (MR) or WM in 8 commercial dairy farms (4 farms per feeding program). Fecal and nasal swabs were collected from 20 ± 5 dairy calves at 42 ± 3.2 d of age, and from 10 of these at approximately 1 yr of age in each study farm to isolate the targeted bacteria. Furthermore, resistance of E. coli isolates from calf-environment and from 5 calves at birth and their dams was also evaluated in each study farm. Resistances were tested against the following antimicrobial agents: amoxicillin-clavulanic acid, ceftiofur, colistin, doxycycline (DO), enrofloxacin (ENR), erythromycin, florfenicol, imipenem, and streptomycin. A greater number of fecal E. coli resistant to ENR, florfenicol, and streptomycin and more multidrug-resistant E. coli phenotypes were isolated in feces of calves fed WM than in those fed MR. However, the prevalence of fecal-resistant E. coli was also influenced by calf age, as it increased from birth to 6 wk of age for ENR and DO and decreased from 6 wk to 1 yr of age for DO regardless of the feeding program. From nasal samples, an increase in the prevalence of colistin-resistant P. multocida was observed in calves fed WM compared with those fed MR. The resistance patterns of E. coli isolates from calves and their dams tended to differ, whereas similar resistance profiles among E. coli isolates from farm environment and calves were observed. The findings of this study suggest that feeding calves WM fosters the presence of resistant bacteria in the lower gut and respiratory tracts of dairy calves.     

Exposure to antimicrobials through the milk diet or systemic therapy is associated with a transient increase in antimicrobial resistance in fecal Escherichia coli of dairy calves

The objective of this prospective cohort study was to describe the relationship between exposure to antimicrobials, through both the milk diet and systemic therapy, and to describe antimicrobial resistance of fecal Escherichia coli in dairy calves pre- and postweaning. A convenience sample of 15 Minnesota dairy farms was chosen, representing 3 equal cohorts of milk diet fed to preweaned calves: medicated milk replacer (MMR), nonmedicated milk replacer (NMR), or pasteurized nonsaleable milk (PNM). Five newborn calves were enrolled on each farm, with fecal samples collected from each calf at 1, 3, 5, and 16 wk of age. After isolation, 3 colonies of E. coli were randomly selected from each sample to determine antimicrobial susceptibility by minimum inhibitory concentration (Sensititer, Thermo Scientific, Waltham, MA) to 8 antimicrobials in 8 classes. The isolate was given an antimicrobial resistance score (ARS) according to the number of antimicrobial classes to which it was resistant. Any isolate resistant to 3 or more antimicrobials was defined as being multidrug resistant (MDR). Relationships between ARS and MDR (dependent variables) and possible explanatory variables were analyzed using mixed multivariable linear and logistic regression models, respectively, with critical P-values adjusted for multiple contrasts. Seventy percent of isolates were resistant to sulfadimethoxine. For wk 1 and 3, the mean ARS values were greatest for fecal E. coli from calves fed MMR or PNM compared with NMR, with no difference in ARS values between the MMR and PNM groups at either time point. At wk 5, the mean ARS value was greatest for fecal E. coli from calves fed MMR (3.56 ± 0.45; mean ± SE), intermediate for calves fed PNM (2.64 ± 0.45), and lowest for calves fed NMR (1.54 ± 0.45). However, by wk 16, the mean ARS values were ≤1.0 and did not differ among milk diets. Evaluation of the proportion of isolates with MDR mirrored the results of the ARS analysis (MDR more prevalent in MMR and PNM groups preweaning; no difference among milk diets at 16 wk). There was a tendency for an increase in ARS at wk 5 (1.28 ± 0.70), and the odds for MDR in fecal E. coli were estimated to be 5.2 (95% confidence interval = 0.67, 35.7) and 101.1 (95% confidence interval = 1.15, >999.9) higher at wk 3 and 5 if the calf was treated with a systemic antimicrobial within the 14-d period before sampling. These findings suggest that exposure to antimicrobials through the milk diet or systemic therapy may result in a transient increase in resistance in fecal E. coli, but once the antimicrobial pressure is removed, susceptible E. coli are able to flourish again, resulting in an overall decrease in resistance.     

Escherichia coli of poultry food origin as reservoir of sulphonamide resistance genes and integrons

The antimicrobial resistance phenotype and genotype, the flanking regions of sulphonamide resistance genes and the integrons were analyzed in 166 Escherichia coli isolates recovered from poultry meat in Tunisia. High percentages of resistance were detected to ampicillin, streptomycin, nalidixic acid, sulphonamide and tetracycline (66-95%), and lower percentages to gentamicin, amoxicillin-clavulanic acid and cefoxitin (1-4%). The bla_TEM, tet(A)/tet(B), aph(3′)-Ia, aac(6′)-Ib-cr, aac(3)-II and cmlA genes were identified in 92, 82, 29, 2, 2 and 7 isolates, respectively. Class 1 and/or class 2 integrons were detected in 52% of E. coli isolates and five different gene cassette arrangements were identified in the variable regions of class 1 integrons, which included antimicrobial resistance determinants. Sixty-eight isolates contained the sul1 gene and 37 of them presented this gene into a class 1 integron structure. The sul3 gene was detected associated with non-classic class 1 integrons in 4 out of 46 sul3-positive isolates. The sul2 gene was detected in 66 isolates, 51 of them were linked to strA/B genes in seven different genetic structures. Seventy-three-per-cent of integron-positive isolates presented resistance to at least five different antimicrobial families versus 38.7% of integron-negative isolates. Our study highlights the role of commensal E. coli isolates from poultry meat as an important reservoir for sulphonamide resistance genes and integrons carrying antimicrobial resistance genes.     

Escherichia coli strain Nissle 1917: significant reduction of neonatal calf diarrhea

Inappropriate daily use of antimicrobial drugs for the treatment of intestinal diseases is associated with an increased risk of antibiotic resistance. Thus, the establishment of new forms of therapy is still needed. Our objective was to examine the effect of the nonpathogenic Escherichia coli strain Nissle 1917 on the prophylaxis and treatment of neonatal calf diarrhea in a hypothesis-generating study (study I) and a subsequent confirmatory clinical study (study II) under field conditions. Both trials were designed as consecutive, placebo-controlled, single-blind comparisons of 2 groups of animals. Immediately after birth, healthy calves were assigned to either the E. coli Nissle 1917 or the placebo group. The study medication was administered orally 1/d before the first feeding. The treatment was continued for the first 10 to 12 d of life. For each animal, the studies ended on d 20 to 22 of life. In both trials, the number of calves developing diarrhea was defined as the primary target criterion. A total of 335 newborn calves were included in the studies (study I: n = 172; study II: n = 163). Study I showed that the incidence of diarrhea was 65.2% under placebo and 26.5% under E. coli Nissle 1917. In study II, the corresponding figures were 63.0% under placebo and 12.2% under E. coli Nissle 1917. It can be concluded that the administration of viable E. coli bacteria, strain Nissle 1917, has a clear beneficial effect on the prophylaxis and treatment of neonatal calf diarrhea.     

Proteolysis in milk during experimental Escherichia coli mastitis

This work consisted of the intramammary infections (IMI) of 8 heifers by high doses of Escherichia coli to study both the proteolytic activity in milk and the resulting peptides. Therefore, a milking kinetic has been followed, and several parameters have been studied, such as proteose peptones (PP) fraction (quantitative and qualitative changes), plasmin activity (PA), milk somatic cell count (SCC), and bacterial count. A qualitative study of milk proteins and PP was performed by sodium dodecyl sulfate-PAGE, and the peptides recovered in PP during the acute phase of inflammation were amino-terminal micro-sequenced. A BSA increase in milk over time supported the hypothesis of an increase in the permeability of the epithelial barrier. A significant increase in PP content, considered to be an indicator of proteolysis, was observed from postinfusion hours (PIH) 12 to 48. Both the E. coli bacterial count and the SCC increased from PIH 3 to 216. Plasmin activity was increased noticeably from PIH 15 to 24. The respective increases in SCC, bacterial count, and PA suggest their involvement in a global mechanism responsible for the increase in proteolysis in milk after E. coli challenge. Somatic cell count and E. coli may be involved from PIH 3 to 216, and PA involvement might be highlighted during the maximum proteolysis, from PIH 15 to 24. A qualitative study of PP fraction by electrophoresis revealed the apparition of 5 peptide bands: P1 and P2 previously recovered during the lipopolysaccharide challenge, and E1 (27.0 kDa), E2 (15.5 kDa), and E3 (9.0 kDa) were specific to E. coli challenge; E1, E2, and E3 contained casein fragments. The roles played by leukocytes and E. coli are discussed.     

Incidence of Escherichia coli O157:H7 and E. coli virulence factors in US bulk tank milk as determined by polymerase chain reaction

Samples of bulk tank milk from dairies across the United States, taken as part of the National Animal Health Monitoring Dairy 2002 survey, were analyzed for the presence of several genes encoding virulence factors associated with enterohemorrhagic forms of Escherichia coli (EHEC) using real-time and conventional PCR assays. Samples from 859 farms in 21 states were collected and enriched in EC medium at 42.5°C to amplify any E. coli present, and DNA was isolated from the resulting biomass. The eaeA gene encoding intimin, a virulence factor associated with enteropathogenic forms of E. coli and EHEC, was detected in 199 (23%) of the samples. Thirty-six samples (4.2%) were positive for eaeA, the gamma allele of the translocated intimin receptor (γ-tir), found in EHEC strains of O157:H7, and one or both shiga-like toxin genes (stx1 and stx2), a combination that may be indicative of the presence of O157:H7 EHEC. Testing these 36 samples with a commercially available real-time PCR kit for detection of O157:H7 indicated that 5 samples could be contaminated with O157:H7. A multiplex PCR to detect the presence of fliC, rfbE, and hlyA genes found in O157:H7 reduced to 2 (0.2% of all samples) the number of samples likely to be contaminated with this organism. A strain of O157:H7 (eaeA⁺, γ-tir⁺, stx2⁺, rfbE⁺, fliC⁺, hlyA⁺) was subsequently isolated from one sample. Thirty-four eaeA-positive samples did not contain detectable γ-tir but did contain one or both of the stx genes suggesting the presence of EHEC strains other than O157:H7. These results indicate a low incidence of O157:H7 in bulk tank milk but suggest that a risk from other enteropathogenic and EHEC forms of E. coli may exist and that PCR targeting virulence factors associated with highly pathogenic forms of E. coli may be an effective means of detecting potential dangers in raw milk.     

Role of dietary copper in enhancing resistance to Escherichia coli mastitis

The role of dietary copper in enhancing resistance to Escherichia coli mastitis was investigated in first-lactation heifers. Twenty-three primigravid Holstein heifers were maintained on a basal (6.5 ppm copper; -Cu) diet or a diet supplemented (20 ppm) with copper sulfate (+Cu) beginning 60 d prepartum through 42 d of lactation. Liver biopsies and blood samples were taken for liver and blood minerals and plasma ceruloplasmin. Milk samples were taken weekly postpartum for bacteriology. The overall mean liver Cu concentration was about threefold higher, and the overall mean plasma Cu concentration was greater in the +Cu group than the -Cu group. At 34 d of lactation, one pathogen-free quarter per animal was infused with 22 cfu of Escherichia coli strain 727. Plasma Cu was greater at -24, 0, 18, 24, 36, 96, 192, and 240 h relative to infusion for +Cu animals. Plasma Zn concentration was higher at 24 h for the +Cu group. Milk bacterial count (log10 cfu/ml) was lower at 12, 18, and 48 h for the +Cu group. Somatic cell count (log10/ml) was lower at 18 h in +Cu animals. Clinical score at 24 h was lower for +Cu cows, while at 144 h, clinical score was lower for -Cu cows. Rectal temperature was lower at 18 h for the +Cu group. Plasma ceruloplasmin and Fe, dry matter intake and milk production did not differ. Copper supplementation reduced the clinical response during experimental E. coli mastitis, but duration was unchanged.     

Elevated milk soluble CD14 in bovine mammary glands challenged with Escherichia coli lipopolysaccharide

The purpose of this study was to determine whether soluble CD14 (sCD14) in milk was affected by stage of lactation, milk somatic cell count (SCC), presence of bacteria, or lipopolysaccharide (LPS)-induced inflammation. Milk samples from 100 lactating cows (396 functional quarters) were assayed for sCD14 in milk to determine effects of stage of lactation, SCC, and intramammary infection. The concentration of sCD14 was highest in transitional milk (0 to 4 d postpartum) and in milk with high SCC (> 750,000 cells/ml). Most of the infected quarters (> 80%) were infected by coagulase-negative staphylococci and yeast. No difference was found between noninfected and infected quarters. One quarter of six healthy lactating cows was challenged with 100 microg LPS in order to study the kinetics of sCD14 during an LPS-induced inflammation. Milk samples were collected at various intervals until 72 h after injection. Rectal temperature, milk tumor necrosis factor-alpha, and interleukin-8 increased immediately after challenge. The increase in sCD14 paralleled the increase in SCC, peaked at 12 h, and started to decline after 24 h. Serum leakage, as characterized by the level of bovine serum albumin in milk, peaked at 4 h and then gradually decreased. All parameters remained at basal levels in control quarters throughout the study. In vitro experiments indicated that neutrophils released sCD14 in response to LPS in a dose-dependent manner. The results indicate that the concentration of sCD14 was significantly increased in milk after LPS challenge. The increase was not likely due to serum leakage. Instead, infiltrated neutrophils might be the main source of increased sCD14 in milk during inflammation.     

Supplementation of lysolecithin in milk replacer for Holstein dairy calves: Effects on growth performance,health, and metabolites

Lysolecithin is an antiinflammatory emulsifier associated with improved apparent digestibility of total dietary fat and improved feed efficiency in dairy cattle. However, it is unknown if lysolecithin (LYSO) improves performance in calves. Moreover, since many conventional milk replacers use vegetable-sourced fat (e.g., palm oil), nutrient absorption and fecal score may be affected in neonatal calves. Thus, the objective of this study was to evaluate the effects of LYSO supplemented in milk replacer on performance, metabolites, and gut health of preweaned dairy calves. Holstein calves (n = 32) with adequate passive transfer were assigned in pairs (16 blocks) balanced by birth weight, date of birth, and sex at 1 d of age to randomly receive either LYSO (mixed in 2 milk replacer feedings at a rate of 4 g/d Lysoforte, Kemin Industries Inc., Des Moines, IA) or a milk replacer control (nothing added). Both treatments were fed 6 L/d milk replacer [22.5% crude protein, 16.2% crude fat (vegetable oil fat source) on a dry matter basis with 14% solids] by bucket in 2 daily feedings for 56 d. Calves were individually housed in wooden hutches and offered a commercial calf starter (24.6% crude protein and 13.9% neutral detergent fiber) and water by bucket ad libitum. Feed refusals and calf health was assessed daily. Weights and blood metabolites (glucose, total serum protein, albumin, creatinine, triglycerides, and cholesterol) were sampled weekly, and calves completed the study before weaning at 56 d of age. The effects of LYSO on calf average daily gain, feed efficiency, and blood metabolites were evaluated using a linear mixed model with time as a repeated measure, calf as the subject, and block as a random effect in SAS (SAS Institute Inc., Cary, NC). The effect of LYSO to improve the odds of abnormal fecal score was evaluated using a logistic model. Supplementation of LYSO increased average daily gain (control 0.28 ± 0.03 kg; LYSO 0.37 ± 0.03 kg; least squares means ± standard error of the mean) and increased feed efficiency (gain-to-feed; control 0.25 ± 0.03; LYSO 0.32 ± 0.03). Similarly, LYSO calves had a higher final body weight at d 56 (control 52.11 ± 2.33 kg; LYSO 56.73 ± 2.33 kg). Interestingly, total dry matter intake was not associated with LYSO despite improved average daily gain (total dry matter intake control 1,088.7 ± 27.62 g; total dry matter intake LYSO 1,124.8 ± 27.62 g). Blood glucose, albumin, creatinine, triglycerides, and cholesterol were not associated with LYSO. Indeed, only total serum protein had a significant interaction with LYSO and age at wk 5 and 6. Moreover, control calves had a 13.57 (95% confidence interval: 9.25–19.90) times greater odds of having an abnormal fecal score on any given day during the diarrhea risk period from d 1 to 28. The inclusion of LYSO as an additive in milk replacer in a dose of 4 g/d may improve performance, and calf fecal score, preweaning. Further research should investigate the mechanisms behind the effects of LYSO on fat digestibility in calves fed 6 L/d of milk replacer with vegetable-sourced fat.     

Prevalence of shiga toxin-producing Escherichia coli in dairy cattle and their products

The main objective of this review was to assess the role of dairy cattle and their products in human infections with Shiga toxin-producing Escherichia coli (STEC). A large number of STEC strains (e.g., members of the serogroups O26, O91, O103, O111, O118, O145, and O166) have caused major outbreaks and sporadic cases of human illnesses that have ranged from mild diarrhea to the life-threatening hemolytic uremic syndrome. These illnesses were traced to O157 and non-O157 STEC. In most cases, STEC infection was attributed to consumption of ground beef or dairy products that were contaminated with cattle feces. Thus, dairy cattle are considered reservoirs of STEC and can impose a significant health risk to humans. The global nature of food supply suggests that safety concerns with beef and dairy foods will continue and the challenges facing the dairy industry will increase at the production and processing levels. In this review, published reports on STEC in dairy cattle and their products were evaluated to achieve the following specific objectives: 1) to assemble a database on human infections with STEC from dairy cattle, 2) to assess prevalence of STEC in dairy cattle, and 3) to determine the health risks associated with STEC strains from dairy cattle. The latter objective is critically important, as many dairy STEC isolates are known to be of high virulence. Fecal testing of dairy cattle worldwide showed wide ranges of prevalence rates for O157 (0.2 to 48.8%) and non-O157 STEC (0.4 to 74.0%). Of the 193 STEC serotypes of dairy cattle origin, 24 have been isolated from patients with hemolytic uremic syndrome. Such risks emphasize the importance and the need to develop long-term strategies to assure safety of foods from dairy cattle.     

Antimicrobial resistance in Campylobacter coli isolated from pigs in two provinces of China

The aim of this study was to determine the prevalence, antimicrobial resistance and molecular epidemiology of Campylobacter coli isolated from swine in China. A total of 190 C. coli isolates obtained from two slaughter houses and ten conventional pig farms in Shandong (SD, n = 95) and Ningxia (NX, n = 95) provinces were tested for their susceptibility to 14 antimicrobials. A high prevalence (> 95%) of ciprofloxacin and tetracycline-resistant strains was observed in both SD and NX. The erythromycin and clindamycin resistance rates of C. coli from NX (ERY: 54.7% CLI: 43.2%) were higher than those from SD (ERY: 37.9%, CLI: 35.8%). A significant difference (P < 0.05) was observed in erythromycin resistance rate, but not (P > 0.05) in clindamycin resistance rate. while the resistance rates of ampicillin and kanamycin in NX (AMP: 34.7%, KAN: 43.2%) were significantly lower (P < 0.05) than those in SD (AMP: 51.6%, KAN: 71.6%). None of the tested isolates were resistant to phenicols. The majority of the isolates from both provinces (SD: 80% and NX: 73.7%) showed multi-drug resistance profiles. The point mutations of A2075G in the 23S rRNA and C257T in the gyrA gene were detected in 98% (87/89) of macrolide resistant isolates and all ciprofloxacin resistant isolates, respectively. In addition, all tetracycline-resistant isolates harbored the tet(O) gene. The high prevalence of antimicrobial resistance in C. coli strains derived from pigs in China was observed and was likely due to the extensive use of various antimicrobials. Prudent use of antimicrobial agents on farms should be further emphasized to control the dissemination of antimicrobial resistant C. coli.     

Effects of feeding pasteurized waste milk to dairy calves on phenotypes and genotypes of antimicrobial resistance in fecal Escherichia coli isolates before and after weaning

The aim of this study was to evaluate the effects of feeding pasteurized waste milk (pWM) to calves on antimicrobial resistance of fecal Escherichia coli at both phenotypic and genotypic levels. Fifty-two Holstein female calves (3 ± 1.3 d of age) were fed 1 of the 2 different types of milk: milk replacer (MR) without antimicrobials or pWM with β-lactam residues until weaning at 49 d of age. Fecal swabs of all calves were obtained on d 0, 35, and 56 of the study and 3 E. coli isolates per sample were studied. Phenotypic resistance was tested by the disk diffusion method against a panel of 12 antimicrobials. A total of 13 resistance genes consisting of β-lactam, sulfonamide, tetracycline, and aminoglycoside families were examined by PCR. Feeding pWM to calves increased the presence of phenotypic resistance to ampicillin, cephalotin, ceftiofur, and florfenicol in fecal E. coli compared with MR-fed calves. However, the presence of resistance to sulfonamides, tetracyclines, and aminoglycosides was common in dairy calves independent of their milk-feeding source, suggesting other factors apart from the feeding source are involved in the emergence of antimicrobial resistance.     

奶源大肠杆菌的分离鉴定及耐药性分析

目的了解原料乳和乳房炎奶样中大肠杆菌的污染情况及菌株耐药性。方法通过选择培养和聚合酶链式反应方法对2个奶牛场采集到的206份奶样(129份乳房炎牛奶样品和77份原料乳样品)进行大肠杆菌的分离鉴定,采用药敏纸片法对分离株进行25种常用抗生素耐药特征检测。结果 206份奶样中大肠杆菌的污染率为8.3%(17/206),其中乳房炎和原料乳样品的污染率分别为7.0%(9/129)和10.4%(8/77)。从17份污染的样本中共分离到34株大肠杆菌,其中乳房炎奶样分离到18株,原料乳分离到16株。药敏结果显示,奶样分离株对氨苄西林耐药最为普遍(44.1%,15/34),对头孢类抗生素也有较强耐药性[如头孢唑啉和头孢噻吩(20.6%,7/34)]。最常见的耐药谱为AMP(11.8%,4/34),AMP-CXM-CFZ-KF-F和AMP-CXM-CFZ-CTX-PRLCRO-KF(5.9%,2/34)。此外,A,B奶牛场分离株的耐药率(P=0.007)和耐药谱总体差异显著(P=0.043)。结论奶样中存在大肠杆菌的污染情况,菌株普遍对氨苄西林和头孢类抗生素耐药且部分对非兽用抗生素也有一定的耐药性。因此,为避免耐药大肠杆菌对人类,尤其是抵抗力较弱的老年人和婴幼儿的感染和中毒,除应加强对奶源地的管理外,还需防止抗生素的滥用。     

Increase of Escherichia coli inoculum doses induces faster innate immune response in primiparous cows

The objective of the current study was to evaluate the dynamics of infection and the immunological response to varying numbers of Escherichia coli injected into the mammary glands of primiparous cows during the periparturient period. Primiparous cows have been shown to be more resistant to intramammary E. coli challenge, and an increase of the inoculum dose by 2 log10 units induced a more rapid clinical response and clearance of the organisms. Recognition of lipopolysaccharide (LPS) is a key event in the innate immunity response to gram-negative infection and is mediated by the accessory molecules CD14 and LPS-binding protein (LBP). Primiparous cows were inoculated with 1 x 10(4) (Group A; n=8) or 1 x 10(6) (Group B; n=8) cfu E. coli P4:O32 in their 2 left quarters during the periparturient period. Clinical examination and analysis of blood and milk parameters, including IL-8, complement fragment 5a (C5a), LBP, and soluble CD14 (sCD14), were performed from d -4 to d +3 relative to infection. Primiparous cows in Group B initiated a more rapid clinical response following intramammary infection (IMI), resulting in typical clinical signs and changes in blood and milk parameters approximately 3 h earlier compared with primiparous cows in Group A. Based on average milk production in the noninfected quarters on d +2 postinoculation, all heifers reacted as moderate responders. Distinct differences in the kinetic patterns of rectal temperature, somatic cell count (SCC), IL-8, C5a, LBP, and sCD14 were observed between both groups during the early phase of inflammation. Both C5a and IL-8 increased before cellular influx into the infected glands, followed by increases in sCD14 and LBP. In conclusion, primiparous cows are able to clear an intramammary E. coli infection efficiently. Moreover, increasing the inoculum dose induces a more rapid inflammatory reaction, mainly because of early activation of the innate host immune response.     

Moderate inflammatory reaction during experimental Escherichia coli mastitis in primiparous cows

Nineteen primiparous cows were experimentally infected in 2 quarters with 1 x 10(4) (group A) or 1 x 10(6) (group B) cfu of Escherichia coli P4:O32 per quarter within 2 to 4 wk after parturition. Blood and milk samples were collected from all primiparous cows at regular time intervals from d -4 to d +3 relative to inoculation. Milk production rapidly decreased in both groups during E. coli mastitis, but recovery appeared to be faster in group B at d + 1 postinfusion (p.i.). The milk production losses in the noninfected quarters were substantial on the day of inoculation, which is probably due to pronounced systemic effects. However, on d + 2 p.i. milk production in the noninfected quarters nearly reached preinfection levels, indicating a moderate clinical severity following intramammary inoculation. None of the other severity criteria evolved towards a severe response pattern. Reticulorumen motility was inhibited in both groups during E. coli mastitis. The clinical episode was short lasting in both groups. Rectal temperature, heart rate, blood leukocyte count, number of colony-forming units, milk somatic cell count and several indicators for the disintegration of the blood-milk barrier returned to normal values within 24 to 72 h p.i. Primiparous cows reacted with a moderate inflammatory response following intramammary infusion with a relatively high dose of E. coli. Despite the use of a high inoculum dose, primiparous cows in both groups showed pronounced resistance against severe intramammary E. coli infection. A possible effect of the inoculum dose could be present, however, further research into the effect of the inoculum dose on the inflammatory response should be performed.     

Cloning of genes encoding colicin E2 in Lactococcus lactis subspecies lactis and evaluation of the colicin-producing transformants as inhibitors of Escherichia coli O157:H7 during milk fermentation

Colicin E2 (ColE2) is a proteinaceous bacterial toxin produced by some strains of Escherichia coli and other members of the Enterobacteriaceae that exhibits inhibitory activity against some strains of E. coli O157:H7. A 2.0-kb DNA fragment, containing the ColE2 structural gene ceaB and immunity gene ceiB from E. coli NCTC 50133 (pColE2-P9), was cloned into the lactococcal plasmid vector pNZ2103. The lysis gene, celB, was not cloned. The plasmid, pLR-E2, encoding the cloned genes was transformed into E. coli DH5α and Lactococcus lactis ssp. lactis LM0230 and PN-1 using electroporation. The bacteriocin ColE2 was expressed in transformants of both E. coli and L. lactis ssp. lactis. Secretion of ColE2 into media was verified by spot-on-lawn assays and measurement of ColE2 activity in the growth medium of transformants. The level of ColE2 produced by transformants containing pLR-E2 was similar to that produced by the parental strain, E. coli NCTC 50133 (pColE2-P9). Evaluation of a ColE2-producing transformant of L. lactis ssp. lactis as a starter culture revealed that, although ColE2 was produced by transformants and could be detected in milk during fermentation, the inhibitory activity of ColE2 against E. coli O157:H7 was significantly decreased in milk compared with buffered growth medium.     

Prevalence and characteristics of Shiga toxin-producing Escherichia coli in Swiss raw milk cheeses collected at producer level

The aim of this study was to describe the prevalence, serotypes, and virulence genes of Shiga toxin-producing Escherichia coli (STEC) isolated from raw milk cheese samples collected at the producer level with the purpose of determining whether raw milk cheeses in Switzer-land represent a potential source of STEC pathogenic for humans. Raw milk cheese samples (soft cheese, n = 52; semihard and hard cheese, n = 744; all produced from Swiss cows’, goats’, and sheep's milk) collected at the producer level throughout Switzerland within the national sampling plan during the period of March 2006 to December 2007 were analyzed. Of the 432 cheese samples obtained in the year 2006 and the 364 samples obtained in the year 2007, 16 (3.7%) and 23 (6.3%), respectively, were found to be stx positive. By colony dot-blot hybridization, non-O157 STEC strains were isolated from 16 samples. Of the 16 strains, 11 were typed into 7 E. coli O groups (O2, O15, O22, O91, O109, O113, O174), whereas 5 strains were nontypeable (ONT). Among the 16 STEC strains analyzed, stx₁ and stx₂ variants were detected in 1 and 15 strains, respectively. Out of the 15 strains with genes encoding for the Stx2 group, 4 strains were positive for stx₂, 6 strains for stx_2d2, 2 strains for stx_2-O118, 1 strain for stx_2-06, 1 strain for stx_2g, 1 strain for stx₂ and stx_2d2, and 1 strain for stx₂ and stx_2g. Furthermore, 3 STEC strains harbored E-hlyA as a further putative virulence factor. None of the strains tested positive for eae (intimin). Results obtained in this work reinforce the suggestion that semihard raw milk cheese may be a potential vehicle for transmission of pathogenic STEC to humans.     

Occurrence, virulence genes and antibiotic resistance of Escherichia coli O157 isolated from raw bovine, caprine and ovine milk in Greece

The examination of 2005 raw bovine (n = 950), caprine (n = 460) and ovine (n = 595) bulk milk samples collected throughout several regions in Greece for the presence of Escherichia coli serogroup O157 resulted in the isolation of 29 strains (1.4%) of which 21 were isolated from bovine (2.2%), 3 from caprine (0.7%) and 5 from ovine (0.8%) milk. Out of the 29 E. coli O157 isolates, only 12 (41.4%) could be classified as Shiga-toxigenic based on immunoassay and PCR results. All 12 Shiga-toxigenic E. coli serogroup O157 isolates belonged to the E. coli O157:H7 serotype. All except one of the 12 Shiga-toxin positive isolates were stx₂-positive, five of which were also stx₁-positive. The remaining isolate was positive only for the stx₁ gene. All stx-positive isolates (whether positive for stx₁, stx₂ or stx₁ and stx₂) were also PCR-positive for the eae and ehxA genes. The remaining 17 E. coli O157 isolates (58.6%) were negative for the presence of the H7 flagellar gene by PCR, tested negative for Shiga-toxin production both by immunoassay and PCR, and among these, only four and three strains were PCR-positive for the eae and ehxA genes, respectively. All 29 E. coli O157 isolates displayed resistance to a wide range of antimicrobials, with the stx-positive isolates being, on average, resistant to a higher number of antibiotics than those which were stx-negative.     

Ecology of Escherichia coli O157:H7 in commercial dairies in southern Alberta

Shedding of Escherichia coli O157:H7 was monitored monthly over a 1-yr period by collecting pooled fecal pats (FECAL) and manila ropes orally accessed for 4 h (ROPE) from multiple pens of cattle in 5 commercial dairies in southern Alberta, Canada. Using immunomagnetic separation, E. coli O157:H7 was isolated from cows on 4 of the dairies and from 13.5% of FECAL and 1.1% of ROPE samples. Pulsed-field gel electrophoresis of XbaI- and SpeI-digested bacterial DNA of the 65 isolates produced 23 unique restriction endonuclease digestion patterns, although 92% of the isolates belonged to 3 restriction endonuclease digestion pattern clusters sharing a minimum 90% homology. Collection of positive isolates was 15 times more likely from June through September. Across dairies, peak somatic cell count occurred in July, August, September, and November. The likelihood of positive isolates was 2.6 times higher in calves and heifers compared with mature cows. This study indicates that ROPE would be of little value for the detection of E. coli O157:H7 in dairy herds unless oral contact with ROPE could be increased in mature animals. Additionally, mitigation strategies for E. coli O157:H7 should be targeted to the months of July, August, and September and toward immature animals for maximum impact. All farms displayed unique combinations of seasonality of shedding and diversity of E. coli O157:H7 subtypes. The fact that seasonal prevalence of E. coli O157:H7 largely coincided with peak somatic cell count within climatically controlled dairy barns suggests that similar environmental factors may be enhancing fecal shedding E. coli O157:H7 and the incidence of mastitis.     

Growth and health of Holstein calves fed milk replacers supplemented with antibiotics or Enteroguard

Forty-five Holstein calves were fed milk replacers containing either antibiotics [MRA (oxytetracycline at 138 mg/kg and neomycin at 276 mg/kg), n = 22)] or Enteroguard [MRE, a blend of fructooligosaccharides, allicin, and gut-active microbes at (129 mg/kg, n = 23)] from birth to 5 wk of age to compare effects on average daily gain and on incidence of scours. Performance was evaluated by measuring weight gain, feed efficiency, and fecal scores. The overall body weight gains and severity of scours were not different between treatments, nor were there differences in starter intake or mean body weight gain. During wk 2, the average gain of calves fed MRA was less than that of calves fed MRE (0.07 vs. 0.09 kg/d, P = 0.09), and greater during wk 5 (0.62 vs. 0.51 kg/d, P < 0.01); however, total gain for calves fed MRE was not different from calves fed MRA. Likewise, average feed efficiencies (gain/dry matter intake) were not different. Severity of scours, as measured by fecal scores, and concentrations of serum proteins, an indirect measure of immunoglobulins, were similar for calves fed MRA and MRE. The results suggest that antibiotics in milk replacers can be replaced with compounds such as fructooligosaccharides, probiotics, and allicin to obtain similar calf performance.