Changes in various metabolic parameters in blood and milk during experimental Escherichia coli mastitis for primiparous Holstein dairy cows during early lactation

Background:The objective of this study was to characterize the changes in various metabolic parameters in blood and milk during IMI challenge with Escherichia coli(E.coli) for dairy cows during early lactation.Thirty,healthy primiparous Holstein cows were infused(h = 0) with-20-40 cfu of live ? coli into one front mammary quarter at ~4-6wk in lactation.Daily feed intake and milk yield were recorded.At-12,0,3,6,12,18,24,36,48,60,72,96,108,120,132,144,156,168,180 and 192 h relative to challenge rectal temperatures were recorded and quarter foremilk was collected for analysis of shedding of E.coli.Composite milk samples were collected at-180,-132,-84,-36,-12,12,24,36,48,60,72,84,96,132 and 180 h relative to challenge(h = 0) and analyzed for lactate dehydrogenase(LDH),somatic cell count,fat,protein,lactose,citrate,beta-hydroxybutyrate(BHBA),free glucose(fglu),and glucose-6-phosphate(G6P).Blood was collected at-12,0,3,6,12,18,24,36,60,72,84,132 and 180 h relative to challenge and analyzed for plasma non-esterified fatty acids(NEFA),BHBA and glucose concentration.A generalized linear mixed model was used to determine the effect of IMI challenge on metabolic responses of cows during early lactation.Results:By 12 h,E.coli was recovered from challenged quarters and shedding continued through 72 h.Rectal temperature peaked by 12 h post-challenge and returned to pre-challenge values by 36 h post-IMI challenge.Daily feed intake and milk yield decreased(P 0.05) by 1 and 2 d,respectively,after mastitis challenge.Plasma BHBA decreased(12 h;P 0.05) from 0.96 ± 1.1 at 0 h to 0.57 ±0.64 mmol/L by 18 h whereas concentration of plasma NEFA(18 h) and glucose(24 h) were significantly greater,11 and 27%,respectively,after challenge.In milk,fglu,lactose,citrate,fat and protein yield were lower whereas yield of BHBA and G6 P were higher after challenge when compared to pre-challenge values.Conclusions:Changes in metabolites in blood and milk were most likely associated with drops in feed intake and milk yield.However,the early rise in plasma NEFA may also signify enhanced adipose tissue lipolysis.Lower concentrations of plasma BHBA may be attributed to an increase transfer into milk after IMI.Decreases in both milk lactose yield and%after challenge may be partly attributed to reduced conversion of fglu to lactose.Rises in G6 P yield and concentration in milk after challenge(24 h) may signify increased conversion of fglu to G6 P.Results identify changes in various metabolic parameters in blood and milk after IMI challenge with E.coli in dairy cows that may partly explain the partitioning of nutrients and changes in milk components after IMI for cows during early lactation.     

Genome-wide association for milk production and lactation curve parameters in Holstein dairy cows

The aim of this study was to identify genomic regions associated with 305-day milk yield and lactation curve parameters on primiparous (n = 9,910) and multiparous (n = 11,158) Holstein cows. The SNP solutions were estimated using a weighted single-step genomic BLUP approach and imputed high-density panel (777k) genotypes. The proportion of genetic variance explained by windows of 50 consecutive SNP (with an average of 165 Kb) was calculated, and regions that accounted for more than 0.50% of the variance were used to search for candidate genes. Estimated heritabilities were 0.37, 0.34, 0.17, 0.12, 0.30 and 0.19, respectively, for 305-day milk yield, peak yield, peak time, ramp, scale and decay for primiparous cows. Genetic correlations of 305-day milk yield with peak yield, peak time, ramp, scale and decay in primiparous cows were 0.99, 0.63, 0.20, 0.97 and −0.52, respectively. The results identified three windows on BTA14 associated with 305-day milk yield and the parameters of lactation curve in primi- and multiparous cows. Previously proposed candidate genes for milk yield supported by this work include GRINA, CYHR1, FOXH1, TONSL, PPP1R16A, ARHGAP39, MAF1, OPLAH and MROH1, whereas newly identified candidate genes are MIR2308, ZNF7, ZNF34, SLURP1, MAFA and KIFC2 (BTA14). The protein lipidation biological process term, which plays a key role in controlling protein localization and function, was identified as the most important term enriched by the identified genes.     

丙酸镁对泌乳早期奶牛体况、泌乳性能和代谢参数的影响

选用36头经产奶牛,根据泌乳期、上一泌乳期305 d产奶量和预产期,采用随机区组设计分为4组,对照组饲喂基础日粮,处理1,2和3组分别在基础日粮基础上添加丙酸镁50,100和150 g/d,研究丙酸镁对泌乳早期奶牛采食量、泌乳性能、血液代谢参数和尿酮浓度的影响。结果表明,添加丙酸镁对奶牛的采食量、乳脂率、乳蛋白率、乳糖率和乳干物质率无显著影响,添加丙酸镁100和150 g/d对产乳量、饲料转化效率、体况及代谢参数有改善,该二处理组产奶量、饲料转化效率、体况评分、能量平衡、血浆葡萄糖和胰岛素浓度均显著高于对照组(P<0.05),而血浆游离脂肪酸和β-羟丁酸浓度显著低于对照组(P<0.05),尿酮浓度(除100 g/d组产后7 d测定值与50 g/d组无显著差异外)显著低于对照组和50 g/d组(P<0.05)。根据试验结果,丙酸镁适宜添加量为100 g/d。     

Comparative proteomic analysis of proteins expression changes in the mammary tissue of cows infected with Escherichia coli mastitis

Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and α-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis.     

Effects of malic acid on feed intake, milk yield, milk components and metabolites in early lactation Holstein dairy cows

The objective of this study was to evaluate the effects of malic acid (MA) on feed intake, milk yield and composition, blood metabolites and energy balance in early lactation Holstein dairy cows from 1 to 63 day in milk (DIM). Twenty-eight multiparous Holstein dairy cows, blocked by lactation number, previous 305-d mature equivalent milk production, and expected calving date, were arranged into four groups in a randomized block design. Treatments were: control (without MA), LMA, MMA and HMA with 70, 140 and 210 g malic acid per cow per day, respectively. The supplement of food grade MA (99.8% of MA) was hand-mixed into the top one-third of the daily ration. Cows were fed ad libitum a total mixed ration consisting of equal proportion of forage and concentrate. Milk yield increased (P = 0.04), but feed intake and milk components were not affected (P > 0.05) by MA supplementation. The energy balance, expressed as the difference between energy input and output, tended to be higher (P = 0.08) for MA supplemented cows during the 63-DIM period MA and supplemented cows showed a trend (P = 0.07) toward less loss of BW during the 63-day period, especially during the first 21-day of lactation. Concentrations of plasma glucose and serum insulin were higher for cows fed LMA, MMA, and HMA relative to control and linearly (P < 0.01) increased with increasing MA supplementation. Concentrations of non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA) and urine ketones were lower for MA-supplemented cows at 7, 14 and 21 DIM of lactation and linearly (P < 0.01) decreased with increasing MA supplementation. Although feed intake was not affected, milk yield increased, MA-supplemented cows experienced higher concentrations of plasma glucose and serum insulin, lower concentrations of plasma BHBA and NEFA, and lower concentrations of urine ketones, suggesting that nutrient digestibilities and energy availability may have been improved.     

traT and CNF2 genes of Escherichia coli isolated from milk of healthy cows and sheep

The objectives of the present study were to isolate Escherichia coli from milk of apparently healthy cows and sheep and to investigate the presence of traT and cytotoxic necrotising factor-2 (CNF2) virulence genes by multiplex polymerase chain reaction (PCR). Milk samples collected from a total of 1028 apparently healthy ruminants (737 cows and 291 sheep) in eastern Turkey were streaked onto 5% sheep-blood agar. E. coli was isolated and identified by biochemical tests in 5.9% (61/1028) of milk samples. Correct amplification with the molecular length of 232 bp was obtained from all E. coli isolates by the species-specific PCR. The isolation rates of the agent were calculated to be 7.6% (56/737) in cows and 1.7% (5/291) in sheep. The difference between these proportions was statistically significant (p < 0.001). Multiplex PCR showed that traT and CNF2 genes were present in 62.3% and 6.6% of all isolates, respectively. Both genes were present in 16.4% of the isolates, with only 14.7% having neither gene.     

丙酸钙对泌乳早期奶牛泌乳性能和代谢产物的影响

选用32头经产奶牛,根据泌乳期、上个泌乳期305 d产奶量和预产期,采用随机区组设计分为4组,对照组饲喂基础日粮,处理1、2、3组分别在基础日粮基础上添加丙酸钙100、200、300 g/d.结果显示,日粮添加丙酸钙对奶牛的采食量、乳脂率、乳蛋白率、乳糖率和乳干物质率无显著影响,200、300 g/d组产奶量和饲料转化效率显著高于对照组(P<0.05).200、300 g/d组血浆葡萄糖和胰岛素浓度显著高于对照组(P<0.05),而血浆游离脂肪酸和β-羟丁酸浓度显著低于对照组(P<0.05);200、300 g/d组尿酮浓度显著低于对照组和100 g/d组(P<0.05).试验结果表明,丙酸钙适宜添加量为200 g/d.     

Essential role of neutrophils but not mammary alveolar macrophages in a murine model of acute Escherichia coli mastitis

Mastitis, the inflammation of the mammary gland, is an important disease affecting dairy animals worldwide. The disease is caused by mammary pathogenic bacteria and Escherichia coli are frequently implicated. Virulence factors of mammary pathogenic E. coli are only partially known and intramammary challenge with LPS elicits neutrophil recruitment in experimental bovine and murine mastitis models. We have previously shown that neutrophil recruitment in LPS-induced murine mastitis is strictly dependent on mammary alveolar macrophages. However, the relative role of alveolar macrophages and blood neutrophils in E. coli mastitis is not well defined. To this end, we selectively depleted mammary alveolar macrophages or blood neutrophils before intramammary challenge with E. coli strain P4 (ECP4). Mice depleted of alveolar macrophages prior to intramammary challenge recruited neutrophils normally and restricted bacterial growth and interstitial invasion. Importantly however, upon depletion of alveolar macrophages, ECP4 invaded the mammary alveolar epithelial cells and formed intracellular bacterial communities. In contrast, neutrophil depletion prior to intramammary infection with ECP4 was associated with unrestricted bacterial growth, tissue damage, severe sepsis and mortality. This study suggests that neutrophils but not alveolar macrophages provide essential antimicrobial defense against mammary pathogenic E. coli. Furthermore, we show here similar invasion after depletion of alveolar macrophages as in our previous studies showing that LPS/TLR4 signaling on alveolar macrophages abrogates ECP4 invasion of the mammary epithelium. Interestingly, similar ECP4 invasion and formation of intracellular communities were also observed following intramammary infection of either iNOS gene-deficient or IL-1 receptor type 1 gene-deficient mice.     

Modelling the dynamics of intramammary E. coli infections in dairy cows: understanding mechanisms that distinguish transient from persistent infections

The majority of intramammary infections with Escherichia coli in dairy cows result in transient infections with duration of about 10 days or less, although more persistent infections (2 months or longer) have been identified. We apply a mathematical model to explore the role of an intracellular mammary epithelial cell reservoir in the dynamics of infection. We included biological knowledge of the bovine immune response and known characteristics of the bacterial population in both transient and persistent infections. The results indicate that varying the survival duration of the intracellular reservoir reproduces the data for both transient and persistent infections. Survival in an intracellular reservoir is the most likely mechanism that ensures persistence of E. coli infections in mammary glands. Knowledge of the pathogenesis of persistent infections is essential to develop preventive and treatment programmes for these important infections in dairy cows.     

The change of dry matter intake, milk yield and bone turnover in primiparous cows compared to multiparous cows during early lactation

Data from six primiparous and nine multiparous Holstein cows were used to clarify the difference of Ca and P mobilization between primiparous and multiparous cows during early lactation. The dry matter intake (DMI) of primiparous cows was lower (P < 0.01) than those of multiparous cows. Milk yield was lower in primiparous cows at 7, 14, 21 (P < 0.01), and 28 days (P < 0.05) after parturition. There was no significant difference in milk Ca and P concentrations between primiparous and multiparous cows. There were no significant differences in plasma Ca and P concentrations between primiparous and multiparous cows. The plasma P level at 7 days postpartum in primiparous cows was lower (P < 0.05) than 28 days postpartum. The concentration of plasma osteocalcin (OC) measured as bone formation marker of primiparous cows was significantly higher than multiparous cows (P < 0.01) at 21 and 28 days postpartum. The urinary deoxypyridinoline (DPD) as bone resorption marker of primiparous cows tended to be higher (P < 0.10) than multiparous cows at 21 days after parturition and decreased to the same level as that of multiparae toward the peak lactation. These results show that Ca and P mobilization of primiparous cows are more active than multiparous cows.     

All blood, No stool: enterohemorrhagic Escherichia coli O157:H7 infection

Enterohemorrhagic Escherichia coli serotype O157:H7 is a pathotype of diarrheagenic E. coli that produces one or more Shiga toxins, forms a characteristic histopathology described as attaching and effacing lesions, and possesses the large virulence plasmid pO157. The bacterium is recognized worldwide, especially in developed countries, as an emerging food-borne bacterial pathogen, which causes disease in humans and in some animals. Healthy cattle are the principal and natural reservoir of E. coli O157:H7, and most disease outbreaks are, therefore, due to consumption of fecally contaminated bovine foods or dairy products. In this review, we provide a general overview of E. coli O157:H7 infection, especially focusing on the bacterial characteristics rather than on the host responses during infection.     

Pathotyping avian pathogenic Escherichia coli strains in Korea

To examine the genetic background of avian pathogenic Escherichia coli (APEC) that affects virulence of this microorganism, we characterized the virulence genes of 101 APEC strains isolated from infected chickens between 1985~2005. Serotypes were determined with available anti-sera and median lethal doses were determined in subcutaneously inoculated chicks. The virulence genes we tested included ones encoding type 1 fimbriae (fimC), iron uptake-related (iroN, irp2, iucD, and fyuA), toxins (lt, st, stx1, stx2, and vat), and other factors (tsh, hlyF, ompT, and iss). Twenty-eight strains were found to be O1 (2.0%), O18 (3.0%), O20 (1.0%), O78 (19.8%), and O115 (2.0%) serotypes. The iroN (100%) gene was observed most frequently followed by ompT (94.1%), fimC (90.1%), hlyF (87.1%), iss (78.2%), iucD (73.3%), tsh (61.4%), fyuA (44.6%), and irp2 (43.6%). The strains were negative for all toxin genes except for vat (10.9%). All the strains were classified into 27 molecular pathotypes (MPs). The MP25, MP19, and MP10 pathotypes possessing iroN-fimC-ompT-hlyF-iucD-tsh-iss-irp2-fyuA (22.8%), iroN-fimC-ompT-hlyF-iucD-tsh-iss (21.8%), and iroN-fimC-ompT-hlyF-iss (11.9%) genotypes, respectively, were predominant. Redundancy of iron uptake-related genes was clearly observed and some strains were associated with higher mortality than others. Therefore, strains with the predominant genotypes can be used for diagnosis and vaccine.     

The expression of plasmid mediated afimbrial adhesin genes in an avian septicemic Escherichia coli strain

An Escherichia coli strain (SEPT13) isolated from the liver of a hen presenting clinical signs of septicaemia had a LD₅₀ of 4.0 × 10⁵ CFU/ml in one-day-old chickens, expressed Ia, Ib, E1, E3, K and B colicins and aerobactin. The strain was ampicillin and streptomycin resistant, and found to have fimA, csgA and tsh DNA related sequences; it could adhere to and invade HEp-2 and tracheal epithelial cells, expressed fimbriae (observed by electron microscopy), and had five plasmids of 2.7, 4.7, 43, 56, and 88 MDa. Transposon mutagenesis of strain SEPT13, with transposon TnphoA, resulted in a mutant strain named ST16 that had a LD₅₀ of 1.2 × 10¹² CFU/ml. All other biological characteristics of strain ST16 were the same as those detected for strain SEPT13 except for the migration of an 88 MDa plasmid to the 93 MDa position indicating the insertion of the transposon into the 88 MDa plasmid. The 93 MDa plasmid of strain ST16 was transferred, by electroporation assay, to non-pathogenic receptor strains (E. coli strains K12 MS101 and HB101), resulting in transformant strains A and B, respectively. These strains exhibited adhesion properties to in vitro cultivated HEp-2 cells but did not have the capacity for invasion. The adherence occurred despite the absence of fimbriae; this finding suggests that the 88 MDa plasmid has afimbrial adhesin genes.     

Changes in circulating adiponectin and metabolic hormone concentrations during periparturient and lactation periods in Holstein dairy cows

Although our previous report demonstrated that adiponectin and AdipoR1 gene expressions changed among different lactation stages in the bovine mammary gland, its in vivo kinetics remain unclear in ruminant animals. In this study, we investigated the changes in circulating concentrations of adiponectin, as well as other metabolic hormones and metabolites, (i) during the periparturient period and (ii) among different lactation stages, in Holstein dairy cows. In experiment 1, serum adiponectin concentrations increased after parturition. Serum insulin concentrations were lower in the postpartum than prepartum period, whereas serum growth hormone (GH) concentrations increased in the postpartum period. Serum nonesterified fatty acids (NEFA) levels were increased during the postpartum period and were dependent on the parity. In experiment 2, there was no significant difference in plasma adiponectin concentrations among lactational stages. Plasma insulin concentrations tended to be lower in early lactation while plasma GH levels tended to be higher. Plasma NEFA concentrations were significantly lower in mid‐ and late‐lactation stages than non‐lactation stages. These findings indicate that elevation of serum adiponectin might be involved in energy metabolism just around parturition, and might exert its action through regulation of receptor expression levels in target tissues in each lactational stage in Holstein dairy cows.     

Feed intake, lactation performance, blood metabolites and fertility in early lactation dairy cows grazing a timothy pasture

The present study was conducted to investigate feed intake, milk yield, milk composition, blood metabolites and fertility in early lactation dairy cows grazing a timothy pasture. Fourteen multiparous Holstein cows that calved between 20 May and 19 July were used over a 3‐year period. The stocking rate was 3.6–4.3 cow/ha. Concentrates were fed separately at 9.5–11.5 kg/day per cow (dry matter basis) from 1 to 13 weeks postparturition. Herbage intake was estimated using chromium oxide as an indigestible marker. The mean contents of crude protein, total digestible nutrients and neutral detergent fiber of pasture during the 3‐year study period were 22.3%, 71.8% and 51.7%, and those of total diet were 18.9%, 77.3% and 40.3%, respectively. The mean herbage dry matter intake was 13.0 kg/day from 2 to 13 weeks postparturition during the study, total dry matter intake was 23.7 kg/day, the total digestible nutrients sufficiency rate was 105%, milk yield was 39.7 kg/day, and milk fat percentage was 3.30%. The decrease in bodyweight postparturition was slight. Urea nitrogen concentrations in serum were below 18.3 mg/dL. The mean days to first estrus and days open were 36 and 104 days, respectively. These results indicate that energy deficiency, decrease in bodyweight and fertility in early lactation barely occur when high producing dairy cows are fed enough grazing grass and suitable concentrates.     

Virulence factors and genetic variability of uropathogenic Escherichia coli isolated from dogs and cats in Italy

In this study, the association between virulence genotypes and phylogenetic groups among Escherichia (E.) coli isolates obtained from pet dogs and cats with cystitis was detected, and fingerprinting methods were used to explore the relationship among strains. Forty uropathogenic E. coli (UPEC) isolated from dogs (n = 30) and cats (n = 10) in Italy were analysed by polymerase chain reaction (PCR) for the presence of virulence factors and their classification into phylogenetic groups. The same strains were characterized by repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR techniques. We found a high number of virulence factors such as fimbriae A, S fimbriae (sfa) and cytotoxic necrotizing factor 1 (cnf1) significantly associated with phylogenetic group B2. We demonstrated a high correlation between α-hemolysin A and pyelonephritis C, sfa, and cnf1 operons, confirming the presence of pathogenicity islands in these strains. In addition, UPEC belonging to group B2 harboured a greater number of virulence factors than strains from phylogenetic groups A, B1, and D. REP- and ERIC-PCR grouped the UPEC isolates into two major clusters, the former grouping E. coli strains belonging to phylogenetic group B2 and D, the latter grouping those belonging to groups A and B1. Given the significant genetic variability among the UPEC strains found in our study, it can be hypothesized that no specific genotype is responsible for cystitis in cats or dogs.     

Phylogenetic distribution of virulence genes in Escherichia coli isolated from bovine mastitis in Iran

One hundred and twenty seven Escherichia coli isolates from bovine mastitis were examined to detect the phylogenetic group/subgroups and a selection of virulence associated genes. Forty nine (38.58%) isolates belonged to group B1 the remaining isolates fell into four phylogenetic subgroups: A₀ (18.11%), A₁ (26.77%), D₁ (6.29%) and D₂ (10.23%). None of the isolates belonged to B2 group. Forty seven (37.00%) isolates were positive for at least one virulence gene, among them f17A was the most common gene, found in 20.47% of the isolates. Among the E. coli isolates, 11.81% had iucD, 9.44% f17c-A, 9.44% cnf2, 7.87% f17b-A, 6.29% afaD-8 and afaE-8, 3.14% f17d-A, 0.78% cnf1 and 0.78% clpG genes. All of the detected virulence genes were present alone or in combination with each other except clpG and f17d-A genes that were only found alone. None of the isolates contained the genes for F17a-A, intimin, P or S fimbriae.     

泌乳早期发情和乏情奶牛机体代谢和生殖机能的变化

在某一集约化牛场,随机选取奶牛30头,在奶牛产后发情期(50～90 d)内根据奶牛是否发情将奶牛分为发情组和乏情组。通过对试验奶牛血液生化指标(Glu、BHBA、NEFA)、内分泌指标(FSH、LH、LP、E2、P4)等进行调查和检测。结果显示:当奶牛发情时,机体血浆Glu、FSH、LH、E2和LP浓度高,血浆NEFA、P4浓度低,但乏情时正好相反。当奶牛发情时,血浆leptin与LH、E2呈显著正相关,与P4呈显著负相关,但乏情奶牛未呈现相关性。这些结果提示奶牛乏情与机体能量代谢状况和Ins、Gn、E2、P4、Lp分泌少有关。能量平衡能促进奶牛产后发情,与机体高水平血糖、Lp、FSH、LH、E2和低水平P4、NEFA有关。LP是奶牛泌乳早期发情的一个重要调控因子,在奶牛产后能量平衡、生产性能和繁殖性能之间起到一个平衡纽带的作用。     

Antimicrobial resistance of Escherichia coli isolates from canine urinary tract infections

This study determined the antimicrobial resistance profiles of Escherichia coli isolates from dogs with a presumptive diagnosis of urinary tract infection (UTI). Urine samples from 201 dogs with UTI diagnosed through clinical examination and urinalysis were processed for isolation of Escherichia coli. Colonies from pure cultures were identified by biochemical reactions (n=114) and were tested for susceptibility to 18 antimicrobials. The two most frequent antimicrobials showing resistance in Urinary E. coli isolates were oxytetracycline and ampicillin. Among the resistant isolates, 17 resistance patterns were observed, with 12 patterns involving multidrug resistance (MDR). Of the 69 tetracycline-resistant E. coli isolates, tet(B) was the predominant resistance determinant and was detected in 50.9% of the isolates, whereas the remaining 25.5% isolates carried the tet(A) determinant. Most ampicillin and/or amoxicillin-resistant E. coli isolates carried bla_TEM-1 genes. Class 1 integrons were prevalent (28.9%) and contained previously described gene cassettes that are implicated primarily in resistance to aminoglycosides and trimethoprim (dfrA1, dfrA17-aadA5). Of the 44 quinolone-resistant E. coli isolates, 38 were resistant to nalidixic acid, and 6 were resistant to nalidixic acid, ciprofloxacin and enrofloxacin. Chromosomal point mutations were found in the GyrA (Ser83Leu) and ParC (Ser80Ile) genes. Furthermore, the aminoglycoside resistance gene aacC2, the chloramphenicol resistant gene cmlA and the florfenicol resistant gene floR were also identified. This study revealed an alarming rate of antimicrobial resistance among E. coli isolates from dogs with UTIs.     

Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.