ABSTRACT
This paper presents the analysis and design of recurrent neural network (RNN) and proportional and integral(PI) controller based hybrid control(HC) of doubly fed induction generator (DFIG). The proposed HC shows the quick dynamic and good transient response during the sudden changes in wind speed as well as generator speed. Further performance of proposed hybrid controller has been analyzed independently with RNN and PI control as its components. The HC along with RNN gives effective performance compared to conventional DTC (CDTC) and PI DTC in terms of flux ripples,torque ripples and settling time. The proposed technique is implemented in real-time simulator (RTS) based OPAL-RT and MATLAB/SIMULINK environment at a rating of 5.5 KW system under steeply and randomly change in wind velocity.
ABSTRACT
Seed hardness trait has a profound impact on cooking time and canning quality in dry beans. This study aims to identify the unknown genetic factors and associated molecular markers to better understand and tag this trait. An F2:7 recombinant inbred line (RIL) population was derived from a cross between the hard and soft seeded black bean parents (H68-4 and BK04-001). Eighty-five RILs and the parental lines were grown at two locations in southern Manitoba during years 2014-2016. Seed samples were harvested manually at maturity to test for seed hardness traits. The hydration capacity and stone seed count were estimated by soaking the seeds overnight at room temperature following AACC method 56-35.01. Seed samples from 2016 tests were also cooked to determine effect of seed hardness on cooking quality. For mapping of genomic regions contributing to the traits, the RIL population was genotyped using the genotype by sequencing (GBS) approach. The QTL mapping revealed that in addition to the major QTL on chromosome 7 at a genomic location previously reported to affect seed-hydration, two novel QTL with significant effects were also detected on chromosomes 1 and 2. In addition, a major QTL affecting the visual appeal of cooked bean was mapped on chromosome 4. This multi-year-site study shows that despite large environmental effects, seed hardness is an oligo-genic and highly heritable trait, which is inherited independently of the cooking quality scored as visual appeal of cooked beans. The identification of the QTLs and development of SNP markers associated with seed hardness can be applied for common bean variety improvement and genetic exploitation of these traits.
ABSTRACT
OBJECTIVES: We recently showed that diminished motor cortical excitability is associated with high levels of post-stroke fatigue. Motor cortex excitability impacts movement parameters such as reaction and movement times. We predicted that one or both would be influenced by the presence of post-stroke fatigue. METHODS: 41 first-time stroke survivors (high fatigue n=21, Fatigue Severity Scale 7 (FSS-7) score >5; low fatigue n=20, FSS-7 score <3) participated in the study. Movement times, choice and simple reaction times were measured in all participants. RESULTS: A three way ANOVA with fatigue (high and low), task (movement time, simple reaction time and choice reaction time) and hand (affected and unaffected) as the three factors, revealed a significant difference between affected (but not unaffected) hand movement times in the high compared to low fatigue groups. Reaction times, however, were not different between the high-fatigue and low-fatigue groups in either the affected or unaffected hand. CONCLUSIONS: Previously, we showed that motor cortex excitability is lower in patients with high post-stroke fatigue. Our current findings suggest that post-stroke fatigue (1) is a problem of movement speed (possibly a consequence of diminished motor cortex excitability) and not movement preparation, and (2) may have a focal origin confined to the lesioned hemisphere. We suggest that low motor cortex excitability in the lesioned hemisphere is a viable therapeutic target in post-stroke fatigue.
Subject(s)
Fatigue/etiology , Motor Cortex/physiopathology , Stroke/complications , Cross-Sectional Studies , Fatigue/physiopathology , Humans , Psychomotor Performance/physiology , Reaction Time/physiology , Severity of Illness Index , Stroke/physiopathologyABSTRACT
The present investigation was undertaken to study the effect of treatments and packaging on the quality of dried carrot slices during storage. Carrot cultivar 'Nantes' was sliced into 4.5 mm thick slices which were blanched in water at 95 °C for 4 min followed by dipping in 6% potassium metabisulphite (KMS) solution for 40 min and 350 ppm potassium sorbate solution for 10 min prior to two stage phase drying i.e. at 90 ± 5 °C for 2 h and further drying at 60 ± 5 °C for 7 h in a cross-flow hot air cabinet dryer. The dried carrot slices were packed in 50 g packages of aluminium foil laminate (AFL) (polyethylene, aluminium foil and polyester) and high density polyethylene (HDPE) pouches having 32.5 µm and 56.0 µm thickness respectively and stored under ambient conditions i.e.18.5-29.1 °C temperature and 44.4-60.4% relative humidity for 6 months. Significant (p ≤ 0.05) increase was observed in the moisture content, water activity, reducing sugars and non-enzymatic browning while total solids, total soluble solids, titratable acidity, ascorbic acid, total sugars, pectin, rehydration ratio, sulphur dioxide, sorbic acid and carotenoids decreased significantly (p ≤ 0.05) during storage. Carrot slices pre-treated with 6% KMS and packed in AFL pouches were found to retain best physico-chemical quality. The curried product and soup prepared from dried slices from the same had highly acceptable sensory quality with initial overall acceptability scores 8.2 and 8.5 for curried slices and soup respectively on 9-point hedonic scale. The overall acceptability scores decreased from 8.2 to 7.9 and 8.5 to 7.7 in curried product and soup respectively after 6 months storage. All the samples were microbially safe during 6 months of storage.
ABSTRACT
Genetic studies were undertaken to determine the inheritance and genomic location of uncharacterised seedling resistance to leaf rust, caused by Puccinia hordei, in the barley cultivar Ricardo. The resistance was shown to be conferred by a single dominant gene, which was tentatively designated RphRic. Bulk segregant analysis (BSA) and genetic mapping of an F(3) mapping population using multiplex-ready SSR genotyping and Illumina GoldenGate SNP assay located RphRic in chromosome 4H. Given that this is the first gene for leaf rust resistance mapped on chromosome 4H, it was designated Rph21. The presence of an additional gene, Rph2, in Ricardo, was confirmed by the test of allelism. The seedling gene Rph21 has shown effectiveness against all Australian pathotypes of P. hordei tested since at least 1992 and hence represents a new and useful source of resistance to this pathogen.
Subject(s)
Basidiomycota/physiology , Chromosome Mapping/methods , Disease Resistance/genetics , Genes, Plant/genetics , Hordeum/genetics , Inheritance Patterns/genetics , Seedlings/microbiology , Chromosome Segregation/genetics , Chromosomes, Plant/genetics , Crosses, Genetic , Genotype , Hordeum/immunology , Hordeum/microbiology , Microsatellite Repeats/genetics , Plant Diseases/genetics , Plant Diseases/microbiology , Polymorphism, Single Nucleotide/genetics , Seedlings/geneticsABSTRACT
A study was conducted to estimate nitrate content in commonly used fodder crops, viz., berseem (Trifolium alexandrinum), bajra (Pennisetum glaucum), maize (Zea mays), oats (Avena sativa), sorghum (Sorghum vulgare) and toriya (Brassica napus), collected from the fields of different villages of Punjab and farms of the university, and to evaluate the factors associated with nitrate accumulation in these crops. The nitrate level was highest in sorghum on dry matter basis, followed by oats and toriya, berseem, maize and bajra. The nitrate content was also determined in fodder samples harvested from young and mature stages and in different parts of plants. The stem part of forages had higher content than leaves; however, concentrations were low in mature crops as compared to immature ones. The environmental and soil factors associated with it are discussed and correlated with the experimental findings.
ABSTRACT
Johne's disease is chronic granulomatous infectious enteritis of animals caused by Mycobacterium avium subspecies paratuberculosis. A total of 153 animals from 19 dairy farms, 2 gaushalas (unproductive-animal rehabilitation centers), 2 goat and 2 sheep farms from different districts of the Punjab region were selected on the basis of clinical signs of disease. All samples from cattle (n=86), buffalo (n=34), goat (n=25) and sheep (n=26) were subjected to Ziehl-Neelsen staining and DNA extraction by a freeze and thaw method. Ziehl-Neelsen staining detected 71% samples positive for acid-fast bacilli whereas IS900 PCR detected 55% positive for Map DNA. IS1311 PCR-REA analysis of IS900 positive samples revealed 'Bison' type as the most prevalent (82%) genotype of Map, infecting all domestic ruminants. 'Cattle' type was present in a minority of cases (15%) from cattle, buffaloes and goats. This is the first report of 'Cattle' type Map from buffalo and goat species in India.
Subject(s)
Mycobacterium avium subsp. paratuberculosis/genetics , Paratuberculosis/diagnosis , Paratuberculosis/epidemiology , Polymerase Chain Reaction , Polymorphism, Genetic , Ruminants/microbiology , Animals , Bacterial Typing Techniques , Cattle , India/epidemiology , Molecular Epidemiology , Paratuberculosis/microbiology , Restriction MappingABSTRACT
Genotyping of Mycobacterium avium subspecies paratuberculosis (MAP) is important for precise classification of bacterium and for understanding the molecular epidemiology. The present study reports detection and typing of the MAP from milk. On the basis of clinical signs of diarrhea and/or weakness, the dairy animals suspected for Johne's disease were screened by Ziehl-Neelsen staining of fecal samples. The milk samples from 13 selected animals were processed for DNA extraction and direct IS900 polymerase chain reaction (PCR). MAP identified by IS900 PCR was genotyped using IS1311 PCR-restriction endonuclease analysis (REA). IS900 milk PCR revealed 30.8% animals positive for MAP, including 40% of the moderate and 50% of the heavy fecal shedders. All infected animals showed Bison type MAP in IS1311 PCR-REA. IS900 PCR can be used for screening of milk for MAP; however, the method needs to be evaluated for subclinical cases. IS1311 PCR-REA results indicated the predominance of Bison type MAP in the dairy animals of this region.
Subject(s)
Bacterial Typing Techniques , Cattle Diseases/microbiology , Milk/microbiology , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Paratuberculosis/microbiology , Sheep Diseases/microbiology , Animals , Buffaloes , Cattle , Dairying , Feces/microbiology , Female , Mycobacterium avium subsp. paratuberculosis/classification , SheepABSTRACT
Sera from 186 sheep, 83 cattle, and 103 water buffaloes from Punjab, India, were evaluated for antibodies to Toxoplasma gondii using a commercial ELISA kit. This study was planned using a 2-stage random sampling procedure and sampling software 'survey toolbox.' In the first step, villages were selected randomly from a sampling frame of all the villages of Punjab, followed by selection of owners and animals in the second step. Antibodies to T. gondii were found in 7 of 186 sheep, 2 of 83 cattle, and 3 of 103 buffaloes. Results indicate a low prevalence of T. gondii in ruminants tested.
Subject(s)
Antibodies, Protozoan/blood , Buffaloes/parasitology , Cattle Diseases/epidemiology , Sheep Diseases/epidemiology , Toxoplasma/immunology , Toxoplasmosis, Animal/epidemiology , Animals , Cattle , Cattle Diseases/immunology , Cattle Diseases/parasitology , Enzyme-Linked Immunosorbent Assay/veterinary , Female , India/epidemiology , Male , Sheep , Sheep Diseases/immunology , Sheep Diseases/parasitology , Toxoplasmosis, Animal/immunologyABSTRACT
Neospora caninum is one of most important causes of abortion in cattle worldwide, and dogs are an important risk factor for N. caninum infection in cattle. Antibodies to N. caninum were determined in 184 (126 rural, 58 urban) dogs from the Punjab State, India, using commercial monoclonal antibody-based competitive ELISA and found in 16.8% of the animals. The prevalence of N. caninum antibodies was significantly higher in rural dogs (21.4%, 27 of 126) than city dogs (6.9%, 4 of 58). To our knowledge this is the first report of N. caninum infection in canines from India.
Subject(s)
Antibodies, Protozoan/blood , Coccidiosis/veterinary , Dog Diseases/epidemiology , Neospora/immunology , Age Factors , Animals , Coccidiosis/epidemiology , Coccidiosis/immunology , Dog Diseases/immunology , Dogs , Enzyme-Linked Immunosorbent Assay/veterinary , Female , India/epidemiology , Male , Risk Factors , Rural Health , Seroepidemiologic Studies , Sex FactorsABSTRACT
Present pilot study is the first attempt in the country to estimate sero-prevalence of Bovine Johne's disease (BJD) by screening cattle and buffaloes representing large population belonging to farmer's and farm herds in the home tracts (Uttar Pradesh (UP) and Punjab) of Hariana cattle and Murrah buffaloes in North India. Indigenous and in-house plate ELISA kit (using protoplasmic antigen from native Mycobacterium avium subsp. paratuberculosis 'Bison type' strain of goat origin), originally developed for goats and sheep was standardized in bovines and used for screening. For this study, 33 villages of south and west UP were randomly selected and surveyed from 2001 to 2003. There were 7943 farmer's families having 38,251 livestock, including cattle, buffaloes, goats and sheep (per family 4.8% livestock). Numerically, buffaloes and cattle were 54.7% and 22.1%, respectively. Serum samples were collected from 726 animals (4.2% of 16, 981 livestock with 4375 farmer's families) located in 33 randomly surveyed villages. Serum samples (699), submitted to Epidemiology Department of Veterinary College (Punjab Agricultural University, Ludhiana), in the year 2004 by farmer's and organized farm herds (Buffaloes, 372, Cattle, 327), were screened by this ELISA kit. Soluble protoplasmic antigen was prepared from Map (S 5) 'Bison type' strain isolated from a terminally sick goat with Johne's disease. Of the total 1425 bovine (Buffaloes and cattle) serum samples screened using indigenous ELISA kit, sero-prevalence of Johne's disease was 29.0% (28.6% in buffalo and 29.8% in cattle) in Northern India. State-wise sero-prevalence was 31.9% and 23.3% in UP and Punjab, respectively. In UP, of the 601 randomly sampled buffaloes, sero-prevalence was 40.3% (16.6% in young and 40.9% adults) and 25.5% (10.5% in young and 26.3% adults) in south and west UP, respectively. Of the 125 cattle screened, sero-prevalence was 42.6% (nil in young and 44.4% adults) and 30.0% (nil in young and 30.6% adults) in south and west UP, respectively. Of the 699 serum samples screened from Ludhiana, Punjab, sero-prevalence of BJD was 23.0%. Sero-prevalence was 23.3% (12.1% in young and 24.4% in adults) and 26.9% (27.2% in young and 26.8% in adults) in buffaloes and cattle, respectively. High prevalence of BJD in buffaloes in native tract of Murrah breed, and Hariana breed of cattle correlated with poor per-animal productivity and BJD may be the major cause. Indigenous ELISA kit was rapid, economic and sensitive test for large-scale screening of buffaloes and cattle population against incurable BJD.
Subject(s)
Buffaloes , Cattle Diseases/epidemiology , Enzyme-Linked Immunosorbent Assay/veterinary , Mycobacterium avium subsp. paratuberculosis/genetics , Paratuberculosis/epidemiology , Reagent Kits, Diagnostic/veterinary , Animals , Antibodies, Bacterial/blood , Cattle , Cattle Diseases/blood , Genotype , Goat Diseases/microbiology , Goats , India/epidemiology , Mycobacterium avium subsp. paratuberculosis/classification , Mycobacterium avium subsp. paratuberculosis/immunology , Paratuberculosis/blood , Pilot Projects , Reproducibility of Results , Seroepidemiologic StudiesABSTRACT
Country lacks indigenous diagnostic kits against Johne's disease in animals. Indigenous ELISA and IS 900 PCR kits, originally developed for goats and sheep, have been adapted for screening of lactating cows. Multiple diagnostic tests were used to screen 26 lactating dairy cows against Johne's disease. Milk ELISA was evaluated with fecal culture, milk culture and fecal PCR. Of the 26 samples from lactating cows, 84.6, 96.1, 88.4 and 23.0% were positive in fecal culture, milk culture, m-ELISA and m-PCR, respectively. Comparatively milk sediment and milk fat culture detected 84.6 and 76.9% cows positive, respectively. Comparatively fecal culture and milk culture detected 84.6 and 96.1% cows positive, respectively. M-ELISA detected 11.5, 0.0, 11.5, 61.0 and 15.3%, cows as negative, suspected, low positive, positive and strong positive, respectively. There was good correlation between milk and fecal culture with m-ELISA. Three negative cows in m-ELISA were also detected in milk and fecal culture. Of the 26 decontaminated fecal samples, 23.0% cows were positive using specific IS 900 f-PCR. Comparative evaluation of m-ELISA with fecal and milk culture showed agreement in 80.7 and 84.6% cows, respectively. Sensitivity of m-ELISA with respect to fecal and culture was 90.9 and 95.6%, respectively. Comparative evaluation of four tests (milk culture, fecal culture, m-ELISA and f-PCR) showed that only 15.3% cows were detected in all the four tests. In three tests (fecal and milk culture and m-ELISA), 57.6% cows were detected positive. None of the cow was exclusively detected in f-PCR. Of the four diagnostic tests used milk culture was most sensitive (96.15%), followed by fecal culture (86.6%), m-ELISA (76.9%) and IS 900 PCR (23.0%) for the diagnosis of bovine Johne's disease (BJD). Milk ELISA detected only one cow extra, which was negative in milk culture. In view of the simplicity, rapidity and efficacy present milk ELISA kit employing soluble protoplasmic antigen from native Map 'Bison type' genotype of goat origin can be reliable for screening of bovine population against Johne's disease in India.
Subject(s)
Cattle Diseases/diagnosis , Enzyme-Linked Immunosorbent Assay/veterinary , Feces/microbiology , Milk/microbiology , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Paratuberculosis/diagnosis , Polymerase Chain Reaction , Animals , Cattle , Female , Goats , India , Sensitivity and SpecificityABSTRACT
Neospora caninum is now recognized as a major cause of abortion in cattle worldwide, but there is no report of N. caninum infection in cattle in India. Serum samples from 427 dairy cattle and 32 dairy water buffaloes from 7 organized dairy farms located in Punjab, India, were tested for N. caninum antibodies using a commercial monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Antibodies to N. caninum were found in 35 of 427 cattle from 6 of the 7 farms; 9.6% of cows, 5.1% of heifers, and 5.0% of calves were seropositive, suggesting postnatal transmission of N. caninum on the farm. Antibodies to N. caninum were found in 16 of 32 buffaloes tested from 2 dairy farms. In total, 64 cattle and 16 buffalo sera already tested by ELISA were also evaluated by an indirect fluorescent antibody test (IFAT) to verify ELISA results. Of the 64 cattle samples, 29 sera were negative by both tests and of the 35 ELISA-positive sera, 12 had IFAT titers of 1:100 or higher (1 had IFAT titer of 100, 2 had IFAT titer of 200, and 9 had IFAT titers of 400 or higher). Of the 16 buffalo sera positive by ELISA, 1 had an IFAT titer of 1:400. Thus, antibodies to N. caninum were demonstrated in cattle sera by 2 serologic methods. To our knowledge this is the first report of N. caninum infection in cattle and buffaloes in India.
Subject(s)
Antibodies, Protozoan/blood , Buffaloes/parasitology , Cattle Diseases/epidemiology , Coccidiosis/veterinary , Neospora/immunology , Abortion, Veterinary/epidemiology , Abortion, Veterinary/parasitology , Animals , Cattle , Coccidiosis/epidemiology , Enzyme-Linked Immunosorbent Assay/veterinary , Female , Fluorescent Antibody Technique, Indirect/veterinary , India/epidemiology , Pregnancy , Sensitivity and Specificity , Seroepidemiologic StudiesSubject(s)
Buffaloes , Gangrene/veterinary , Tail/pathology , Amputation, Surgical/methods , Amputation, Surgical/veterinary , Animals , Antidiarrheals/administration & dosage , Boric Acids/administration & dosage , Dermatologic Agents/administration & dosage , Gangrene/drug therapy , Insecticides/administration & dosage , Kaolin/administration & dosage , Necrosis , Powders , Zinc Oxide/administration & dosageABSTRACT
A random survey was conducted to study the epidemiology of brucellosis in Punjab (India), using the 'Survey Toolbox' sampling software. A two-stage sampling procedure was adopted: in the first stage, villages were selected, and in the second the selection of animals was made. In all, 52 villages were selected randomly from a sampling frame of all the villages of Punjab. The total number of animals in these villages was 18,644, out of which 973 animals (approximately 5%) belonging to various owners were randomly selected. Serum samples collected from the animals were screened for Brucella antibodies by an avidinbiotin enzyme-linked immunosorbent assay, which showed the apparent overall prevalence of brucellosis to be 12.09% (true prevalence, 11.23%). The prevalence varied from a low of 0% to a high of 24.3% in various districts. Higher variance (0.08) was noted within villages than between different villages (0.03). The prevalence rates among buffaloes and cattle were 13.4% and 9.9%, respectively. The seroprevalence of brucellosis was found to be significantly higher (chi square = 24.50, p < 0.001) in animals with a history of abortion (33.87%) than in those without such a history (11.63%).
Subject(s)
Abortion, Veterinary/microbiology , Antibodies, Bacterial/blood , Brucellosis, Bovine/epidemiology , Software , Animals , Cattle , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/veterinary , Female , India/epidemiology , Male , Pregnancy , Seroepidemiologic StudiesABSTRACT
The efficacy of two formulations of abamectin, i.e. oral and injectable was determined against ivermectin-resistant strain of T. colubriformis in sheep. Twenty-four lambs were infected with 10,000 third stage larvae of ivermectin-resistant strain of T. colubriformis. Twenty-four days post-infection, the lambs were divided randomly into four groups of six animals each according to egg counts. The first group was left untreated and kept as a control. The second group was treated with ivermectin (oral) at 0.2mg kg(-1) body weight. The third group was treated with oral formulation of abamectin at 0.2mg kg(-1) body weight. The fourth group was treated with injectable formulation of abamectin at 0.2mg kg(-1) body weight. Fecal egg count and controlled slaughter tests were employed to determine the efficacy of abamectin (oral and injection) against ivermectin-resistant strain of T. colubriformis in sheep. Reduction in arithmetic mean fecal egg counts achieved by ivermectin (oral), abamectin (oral) and abamectin (injection) was 66, 98 and 76%, respectively 10 days after treatment. Ivermectin (oral), abamectin (oral) and abamectin (injection) reduced arithmetic mean worm burden by 63, 97 and 74%, respectively. The findings demonstrated that abamectin oral formulation was more effective than abamectin injection against ivermectin-resistant strain of T. colubriformis in sheep.
Subject(s)
Anthelmintics/administration & dosage , Ivermectin/analogs & derivatives , Ivermectin/administration & dosage , Sheep Diseases/drug therapy , Sheep Diseases/parasitology , Trichostrongylosis/drug therapy , Trichostrongylosis/veterinary , Trichostrongylus/growth & development , Administration, Oral , Animals , Drug Resistance , Feces/parasitology , Injections, Subcutaneous/veterinary , Intestine, Small/parasitology , Ivermectin/pharmacology , Parasite Egg Count/veterinary , Random Allocation , Sheep , Trichostrongylosis/parasitologyABSTRACT
The purpose of this study was to compare the pathological effects of Shiga toxin-producing Escherichia coli (STEC) that vary in their association with bovine and human disease. Shiga toxin-producing E. coli of serotypes associated with both dysentery in calves and hemolytic uremic syndrome (HUS) in humans (O5:H-, O26:H11, O111:H-,O113:H21) were compared with O157:H7 STEC, which are associated with HUS in humans but not with disease in calves. The STEC were administered orally to 80 day-old chicks and into ligated loops in the ileum and colon of four 2- to 6-day-old calves. Examination of the ceca of the chickens 10 d postchallenge showed no adherence or tissue abnormality for any isolate. The calves were euthanized 8 to 10 h postinoculation, and sections of the intestinal loops were examined by light microscopy, transmission and scanning electron microscopy, and immunohistochemistry. All strains showed consistent focal adherence associated with mild lesions in the colon. Attaching and effacing lesions were observed with the eae-positive strains. Ileal lesions were similar to the colonic ones but were sometimes severe, with marked polymorphonuclear leukocyte proliferation in the lamina propria. It is concluded that chickens were unsuitable for studying interaction of STEC with the intestine and that there was no difference in the interaction of the ligated calf intestine with STEC of serotypes associated with disease in calves compared with O157:H7 STEC.
Subject(s)
Cattle Diseases/microbiology , Colon/microbiology , Escherichia coli Infections/veterinary , Escherichia coli O157/pathogenicity , Ileum/microbiology , Shiga Toxin/biosynthesis , Animals , Animals, Newborn , Bacterial Adhesion , Cattle , Cattle Diseases/pathology , Chickens , Colon/pathology , Escherichia coli Infections/microbiology , Escherichia coli Infections/pathology , Escherichia coli O157/physiology , Humans , Ileum/pathology , Immunohistochemistry/veterinary , Microscopy/veterinary , Microscopy, Electron/veterinary , Serotyping/veterinary , Species SpecificityABSTRACT
This study identified potential virulence markers in 93 eae-positive and 179 eae-negative Shiga toxin-producing Escherichia coli (STEC), isolated from a random sampling of healthy cattle in southwestern Ontario. PCR amplification was used to identify genes for enterohemorrhagic E. coli (EHEC)-hemolysin, the EAF plasmid, and bundle-forming pili (Bfp); adherence to HEp-2 cells and to bovine colonocytes, and the fluorescent actin staining (FAS) test were used to characterize interaction of the bacteria with epithelial cells. The EHEC-hemolysin sequences were detected in 98% of eae-positive isolates compared with 34% of eae-negative isolates. All isolates were negative for EAF and bfp sequences. There was 100% correlation between localized adherence (LA) to HEp-2 cells and the FAS test. Forty-eight (52%) of the eae-positive isolates were LA/FAS-positive, whereas none of the 179 eae-negative isolates was positive in either test. Among the eae-negative isolates, 20 (11%) showed diffuse adherence and 5 (2.8%) showed enteroaggregative adherence to HEp-2 cells. Seventy-three percent of the eae-positive isolates adhered to bovine colonocytes, whereas only 26% of 120 eae-negative isolates that were tested adhered. All 13 O157:H7 isolates were positive for eae and EHEC-hemolysin gene sequences, LA/FAS, and adherence to bovine colonocytes. It is concluded that possession of genes for eae and EHEC hemolysin is correlated with the serotype of STEC, that production of EHEC hemolysin was highly correlated with serotypes implicated in human disease, and that none of the potential markers that were examined can be used to predict the potential virulence of an isolate.
Subject(s)
Acyltransferases , Bacterial Proteins/genetics , Bacterial Toxins/genetics , Cattle Diseases/microbiology , Escherichia coli Infections/veterinary , Escherichia coli Proteins , Escherichia coli/pathogenicity , Hemolysin Proteins/genetics , Amino Acid Sequence , Animals , Cattle , Escherichia coli/classification , Escherichia coli/genetics , Escherichia coli Infections/genetics , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Serotyping , Shiga Toxins , VirulenceABSTRACT
Intimin or EaeA protein has been implicated in the attaching/effacing lesion caused by entero-hemorrhagic Escherichia coli (EHEC) in the intestine but it is not produced by all EHEC and is therefore not adequate as a marker for EHEC. Hemolysins are produced by a high percentage of verotoxigenic E. coli (VTEC) and could be a marker for EHEC, but their distribution and relation to virulence are not known. We used PCR amplification to determine the presence or absence of eaeA sequences in 281 VTEC isolates from the feces of healthy cattle. There were 101 eaeA-positive isolates, which belonged to O groups 5, 26, 69, 80, 84, 98, 103, 111, 119, 145, 157 and 108 eaeA-negative isolates, which belonged to O groups 8, 22, 38, 113, 119, 116, 132, 153, 156 or untypable. All isolates were tested for hemolysis on horse blood agar and on washed sheep blood agar. PCR amplification was used to test for EHEC hemolysin, Ehly1, Ehly2 and alpha-hemolysin D sequences. Among eaeA-positive isolates 98% were positive for EHEC hemolysin sequences and were hemolytic on washed sheep red blood cell agar; the corresponding percentage for eaeA-negative isolates was 36%. Ehly1 and Ehly2 sequences were present in only 11 isolates (O groups 26, 84, 119 and 132). None of the eaeA-positive and 13 of the eaeA-negative isolates (including all 11 isolates of O group 132) were positive for alpha-hemolysin D gene sequences by PCR and alpha-hemolysin production on horse blood agar. We conclude that since Ehly1, Ehly2 and alpha-hemolysin occur at low frequency among bovine VTEC and serotypes implicated in human disease they are unlikely to be significant virulence factors. In contrast, EHEC hemolysin was present in almost all eaeA-positive VTEC isolates and in approximately one-third the eaeA-negative ones; it may be both a virulence marker and virulence factor but further testing is required. The study identified isolates with all combinations of eaeA and EHEC hly, which may be useful for further testing.
Subject(s)
Adhesins, Bacterial , Carrier Proteins , Escherichia coli Proteins , Escherichia coli/genetics , Hemolysin Proteins/genetics , Animals , Bacterial Outer Membrane Proteins/genetics , Bacterial Toxins/biosynthesis , Cattle , Escherichia coli/pathogenicity , Genotype , Horses , Humans , Phenotype , Polymerase Chain Reaction , Rabbits , Shiga Toxin 1ABSTRACT
Fecal samples from 335 dairy farm residents and 1458 cattle on 80 farms were tested for Vero cytotoxin (VT)-producing Escherichia coli (VTEC). Residents were also tested for antibodies to VT1 and O157 lipopolysaccharide (LPS). Residents and cattle on farms with VTEC-positive persons or E. coli O157:H7-positive cattle were retested. Twenty-one persons (6.3%) on 16 farms (20.8%) and 46% of cattle on 100% of the farms had VTEC in fecal samples. Human VTEC isolates included E. coli O157:H7 and 8 other serotypes, 4 of which were present in cattle on the same farms. More persons had antibodies to VT1 (41%) than to O157 LPS (12.5%). Seropositivity to O157 LPS was associated with isolation of E. coli O157:H7 on the farm (P = .022). Human VTEC infection was negatively associated with age (P < .05) and was not associated with clinical illness. Many dairy farm residents experience subclinical immunizing VTEC infections at a young age, which frequently involve non-O157 VTEC found in cattle.