Prevalence of virulence genes in Escherichia coli strains recently isolated from young pigs with diarrhea in the US

Enterotoxigenic Escherichia coli (ETEC)-associated post-weaning diarrhea (PWD) is economically one of the most important diseases for the swine industry. Porcine ETEC strains typically express K88 or F18 fimbria and heat-labile (LT) and/or heat-stable (STa, STb) enterotoxins. However, recent studies indicate that EAST1 toxin, adhesin involved in diffuse adherence (AIDA-I) and porcine attaching and effacing-associated factor (paa) may also be expressed by ETEC strains associated with diarrhea. To better understand the virulence factors of E. coli strains that cause PWD, we applied PCR to screen for K88, F18, F41, 987P and K99 fimbrial genes; LT, STa, STb, Stx2e and EAST1 toxic genes; and AIDA-I, paa and EAE adhesin genes in E. coli strains recently isolated from young pigs with PWD in the US. Of 304 E. coli isolates from diarrheic pigs submitted for testing, 175 (57.6%) strains possessed fimbrial genes: K88 (64.6%), F18 (34.3%), F41 (0.57%), K99 (0.57%), 987P (0); toxin genes: LT (57.7%), STb (72.6%), STa (27.4%), STx2e (17.4%), EAST1 (35%); and adhesin genes: AIDA-I (26.9%), paa (60%), EAE (1.1%). All toxin genes except the EAST1 toxin gene, were almost exclusively associated with K88+ or F18+ isolates, and most of these isolates carried multiple toxin genes. The non-fimbrial adhesin paa was found present in over half of the K88+ isolates. A total of 129 (42%) isolates carried no fimbrial genes, including 66 (21.7%) isolates that did not have any of the above virulence genes. These results suggest a broad array of virulence genes associated with PWD in pigs.     

Isolation and association of Escherichia coli AIDA-I/STb, rather than EAST1 pathotype, with diarrhea in piglets and antibiotic sensitivity of isolates.

To identify emerging Escherichia coli that have the potential to cause diarrhea in pigs, the prevalence of E. coli pathotypes was determined among 170 and 120 isolates from diarrheic and nondiarrheic piglets, respectively. The isolates were tested for F4, F5, F6, F18, and F41 fimbriae, for E. coli attaching and effacing (EAE), porcine attaching and effacing-associated (Paa), and adhesin involved in diffuse adherence (AIDA-I) factors, for LT, STa, STb, and enteroaggregative heat-stable (EAST1) enterotoxins, and for Shiga toxins (Stxl, Stx2, and Stx2e), using DNA hybridization and polymerase chain reaction. All isolates were O-serotyped and tested for antibiotic resistance against 10 drugs. Seventeen different pathotypes, accounting for 40.0% of the isolates, were recovered from diarrheic piglets. The main pathotypes included EAST1 (13.5%), F4/LT/STb/EAST1 (6.5%), AIDA-I/STb/EAST1 (4.1%), F5/STa (2.9%), EAE/EAST1 (2.9%), and AIDA-I/F18 (2.3%). Only 3 pathotypes, EAE (11.7%), EAST1 (10.8%), and EAE/EAST1 (3.3%), were recovered from nondiarrheic piglets. Paa factor was detected in 8.8% and 7.5% of isolates from diarrheic and nondiarrheic piglets, respectively, and always was associated with other virulence determinants. Overall, 22.9% of isolates from diarrheic piglets appeared to be enteropathogens: enterotoxigenic E. coli (11.7%), enteropathogenic E. coli (3.5%), and E. coli isolates (3.0%) for which none of the above adherence factors was detected. Pathotypes AIDA-I/STb/EAST1 and AIDA-I/STb were isolated only from diarrheic piglets and accounted for 4.7% of isolates. Strains of these pathotypes induced diarrhea when inoculated into newborn colostrum-deprived pigs, in contrast to an isolate positive only for EAST1, which did not induce diarrhea. Antibiotic sensitivity test showed that isolates of the AIDA-I/STb/EAST1 and AIDA-I/STb pathotypes were the only strains sensitive to enrofloxacin, gentamicin, neomycin, and trimethoprim-sulfamethoxazole. This study showed that at least 20.5% of isolates from diarrheic piglets appeared to be associated with AIDA-I/STb pathotype and that EAST1 pathotype is probably not an important marker for diarrhea in piglets.     

Inverse relationship between heat stable enterotoxin-b induced fluid accumulation and adherence of F4ac-positive enterotoxigenic Escherichia coli in ligated jejunal loops of F4ab/ac fimbria receptor-positive swine

Heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) increases bacterial adherence to porcine enterocytes in vitro and enhances small intestinal colonization in swine. Heat-stable enterotoxin-b (STb) is not known to affect colonization; however, through an induction of net fluid accumulation it might reduce bacterial adherence. The relationship between fluid accumulation and bacterial adherence in jejunal loops inoculated with ETEC strains that produce LT, STb, both, or neither toxin was studied. Ligated jejunal loops were constructed in weaned Yorkshire pigs in two independent experiments (Exp. 1, n = 5, 8-week-old; Exp. 2, n = 6, 6–8-week-old). Each pig was inoculated with six F4ac⁺ E. coli strains: (1) LT⁺, STb⁺ parent (WAM2317); (2) STb^? (ΔestB) mutant (MUN297); (3) MUN297 complemented with STb (MUN298); (4) LT^? STb^? (ΔeltAB ΔestB) mutant (MUN300); (5) MUN300 complemented with LT (MUN301); and (6) 1836-2 (non-enterotoxigenic, wild-type). Pigs were confirmed to be K88 (F4)ab/ac receptor-positive in Exp. 2 by testing for intestinal mucin-type glycoproteins and inferred to be receptor-positive in both Exp. 1 and 2 based on histopathologic evidence of bacterial adherence. Strains that produced STb induced marked fluid accumulation with the response (ml/cm) to WAM2317 and MUN298 significantly greater than that to the other strains (P < 0.0001). Conversely, bacterial adherence scores based on immunohistochemistry and CFU/g of washed mucosa were both lowest in the strains that expressed STb and highest in those that did not. For the two experiments combined, the Pearson correlation coefficient (R) between fluid volume (ml/cm) and log CFU per gram was ?0.57021 (P < 0.0001); R² = 0.3521 (n = 197). These results support the hypothesis that enterotoxin-induced fluid accumulation flushes progeny organisms into the lumen of the bowel, thereby increasing the likelihood of fecal shedding and transmission of the pathogen to new hosts.     

Distribution of virulence genes in Escherichia coli strains isolated from diarrhoeic piglets in the Slovak Republic

Ninety-two Escherichia coli isolates from 14 to 28-day-old piglets that died because of diarrhoea were examined for genes for fimbriae (F4, F5, F6, F18 and F41), enterotoxins (STa, STb and LT), verotoxin (VT2e or Stx2e) and enteroaggregative heat-stable enterotoxin 1 (EAST1) by polymerase chain reaction. Twenty-two strains (24%) carried a gene for F4, whereas genes for F18, F6 and F5 + F41 were detected in 10.8, 3.3 and 1.1% of strains respectively. Genes for STb, LT, STa and Stx2e were detected in 40.2, 26.1, 14.1 and 1.1% of strains respectively. The astA gene was detected in 49 (53.3%) isolates, 35 of which also carried genes for enterotoxins and/or fimbriae. The major genotypes reached at (in decreasing order of prevalence) were F4/STb/LT/EAST1, F18/STa/STb/EAST1, STb/EAST1, F6/STa/STb/EAST1 and F18/STb/EAST1.     

Antimicrobial resistant Escherichia coli isolates in cattle and house sparrows on two Czech dairy farms

Rectal smears of calves, cows and young bulls, as well as cloacal smears of house sparrows (Passer domesticus), from farms at the villages of Šumice and Troskotovice, Czech Republic, were examined for E. coli resistant to 12 antimicrobials. The resistant isolates were tested for antimicrobial-resistance genes and integrons. Totals of 40% (n = 183), 3% (n = 95), 0% (n = 33), and 9% (n = 54) of Escherichia coli isolates from calves, cows, young bulls and house sparrows, respectively, were antimicrobial resistant. The following genes were identified in cattle E. coli isolates: tetA, tetB (isolates resistant to tetracycline), bla_TEM (beta-lactams), strA, aadA (streptomycin), sul1, sul2 (sulphonamides), and cat, floR (chloramphenicol). Seven of 16 antimicrobial-resistant calf isolates from the Šumice farm possessed class 1 integrons with the aadA1 gene cassette integrated, 1 kb in size. On the Troskotovice farm, eight of 57 antimicrobial-resistant calf isolates possessed class 1 integrons. Integrons of 1.5 kb with the dhfr1- aadA1 gene cassette were found in four isolates, followed by a 1 kb integron with the aadA1 gene found in three isolates, and a 1.7 kb integron with the dhfr17-aadA5 gene cassette and the phenotype ASSuTSxtNaCipCCfG. The prevalence of resistant E. coli in calves compared to adult cattle was much higher and probably was influenced by oral antimicrobial usage in calves, feeding with milk and colostrum from treated cows, as well as mechanisms unrelated to antimicrobial drug selection. Although house sparrows lived together with the cattle and came into contact with cattle waste on the farm, they were not infected by resistant E. coli isolates with the same characteristics as those found in cattle.     

Computed tomographic morphology and clinical features of extrahepatic portosystemic shunts in 172 dogs in Japan

Canine extrahepatic congenital portosystemic shunts (EH-cPSS) are classified into several anatomical types, depending on the origin and termination of the shunt vessel. The aim of this retrospective study was to determine the proportion and clinical features of each anatomical shunt type in a population of dogs presented to a veterinary teaching hospital in Japan. Dogs diagnosed with EH-cPSS using computed tomographic (CT) portography were included (n = 172) and shunts were classified based on previous reports. Clinical data were collected from case records and analysed statistically. The most common anatomical type was the spleno-phrenic shunt (n = 64), followed by the spleno-azygos (n = 38), right gastric-caval (n = 29), spleno-caval (n = 21), right gastric-caval with caudal loop (n = 9), right gastric-phrenic (n = 6), colono-caval (n = 3), spleno-phrenic and azygos (n = 1), and porto-caval (n = 1) shunts. Spleno-phrenic and spleno-azygos shunts were diagnosed more frequently in older dogs than right gastric-caval and spleno-caval shunts (P < 0.05). The portal vein/aortic (PV/Ao) ratio was significantly larger in dogs with spleno-phrenic shunts than in dogs with spleno-azygos, right gastric-caval or spleno-caval shunts (P < 0.05). The PV/Ao ratio was significantly larger in dogs with spleno-azygos shunts than in dogs with right gastric-caval shunts. Dogs with spleno-phrenic shunts had significantly lower serum alkaline phosphatase activities than those with right gastric-caval or spleno-caval shunts. Dogs with spleno-phrenic shunts had significantly lower fasting ammonia concentrations than those with spleno-caval shunts.     

Western European epidemiological survey for parvovirus and coronavirus infections in dogs

An epidemiological survey for canine parvovirus (CPV) and canine coronavirus (CCoV) infections was conducted in Western Europe. A total of 156 faecal samples were collected from dogs with diarrhoea in Spain (n = 47), Italy (n = 39), France (n = 26), Germany (n = 21), the United Kingdom (n = 8), Belgium (n = 10), and the Netherlands (n = 5). Using molecular assays for virus detection and characterisation, CPV and CCoV were found to be widespread in European dog populations, either alone or in mixed infections. In agreement with previous reports, the original type CPV-2 was shown not to circulate in European dogs. The recently identified virus variant CPV-2c was predominant in Italy and Germany and present at high rates in Spain and France but was not detected in the UK or Belgium. Except for the UK, CCoV genotype I was identified in all European countries involved in the survey, albeit at a lower prevalence rates than CCoV genotype II.     

Genotyping of Streptococcus agalactiae strains isolated from fish,human and cattle and their virulence potential in Nile tilapia

Streptococcus agalactiae (Lancefield group B; GBS) is a pathogen that causes meningoencephalitis in fish, mastitis in cows, and neonatal sepsis in humans. The objective of this study was to characterize S. agalactiae isolated from fish (n = 27), cows (n = 9), and humans (n = 10) using pulsed-field gel electrophoresis (PFGE) and to investigate the virulence of the identified strains in Nile tilapia (Oreochromis niloticus). The PFGE types were determined by dendogram analyses and the in vivo virulence was evaluated by experimental infection (using i.p. and immersion routes) of Nile tilapia. Among the fish strains, 5 different PFGE patterns were observed and 21 strains showed the same genetic pattern. In some farms two or three profiles occurred simultaneously. The bovine and human strains exhibited high genetic diversity and few relationships were established among S. agalactiae strains from the three host origins analyzed. Eight S. agalactiae strains from fish caused high mortality of Nile tilapia. Three bovine strains infected Nile tilapia (by i.p. route) and two of those strains caused clinical signs of meningoencephalitis. All human strains (n = 5) infected Nile tilapia (by i.p. route) and meningoencephalitis was induced by one strain (by both i.p. and immersion routes). In conclusion, the analyzed strains from the three natural hosts did not show genetic relatedness, yet some of the bovine and human strains were able to infect fish and cause meningoencephalitis. We suggest that genetic linkage is not a prerequisite for S. agalactiae to cross the host-specific barrier.     

An unmatched case controlled study of clinicopathologic abnormalities in dogs with Bartonella infection

We compared clinicopathologic findings in dogs with Bartonella infection to Bartonella spp. negative dogs suspected of a vector-borne disease. Cases (n = 47) and controls (n = 93) were selected on the basis of positive or negative enrichment culture PCR results, respectively. Signalment, clinicopathologic findings and treatments were extracted from medical records. DNA sequencing identified Bartonella henselae (n = 28, 59.6%), Bartonella vinsonii subsp. berkhoffii (n = 20, 42.6%), Bartonella koehlerae (n = 3, 6.4%), Bartonella volans-like (n = 3, 6.4%) and Bartonella bovis (n = 1, 2.1%). There were no significant differences in age, breed, size, sex or neuter status between cases and controls. Dogs infected with Bartonella sp. often had a history of weight loss [OR = 2.82; 95% CI: 1.08–7.56] and were hypoglobulinemic [OR = 4.26; 95% CI: 1.31–14.41]. With the exception of weight loss and hypoglobulinemia, clinicopathologic abnormalities in Bartonella-infected dogs in this study were similar to dogs suspected of other vector-borne infections.     

Isolation of Escherichia coli from piglets in South Korea with diarrhea and characteristics of the virulence genes

Escherichia coli was isolated from the feces of 122 piglets with diarrhea on 55 farms in Korea. The virulence genes of each isolate were characterized by polymerase chain reaction (PCR). Of the 562 isolates, 191 carried 1 or more of the virulence genes tested for in this study. Of the 191 isolates, 114 (60%) carried 1 or more of the genes for enterotoxigenic E. coli (ETEC) fimbriae F4, F5, F6, F18, and F41 and ETEC toxins LT, STa, and STb, 57 (30%) carried 1 or more of the genes for the Shiga-toxin-producing E. coli (STEC) toxins Stx1, Stx2, and Stx2e, and 21% and 37% carried the gene for enteropathogenic E. coli intimin and for enteroaggregative E. coli toxin, respectively. Collectively, our results indicate that other pathotypes of E. coli as well as ETEC can be strongly associated with diarrhea in piglets. In addition, detection of the genes for Stx1 and Stx2 indicates that pigs are reservoirs of human pathogenic STEC.     

Genotypic Prevalence of the Adhesin Involved in Diffuse Adherence in Escherichia coli Isolates in Pre‐weaned Pigs with Diarrhoea in Korea

A total of 1002 Escherichia coli strains isolated from pre‐weaned pigs with diarrhoea on 1114 swine farms were screened for the presence of the adhesin involved in diffuse adherence (AIDA) gene by polymerase chain reaction (PCR). Escherichia coli isolates that carried AIDA genes were also tested by PCR for the detection of five fimbriae (F4, F5, F6, F18 and F41), heat‐stable (STa, STb) and heat‐labile (LT) enterotoxin, enteroaggregative E. coli heat‐stable enterotoxin 1 (EAST1), and Shiga toxin 2 oedema disease (Stx2e) genes. Twenty‐three (2.3%) of the 1002 E. coli isolates carried the gene for AIDA. Among 23 isolates shown to carry genes for AIDA, three carried the AIDA gene as the only shown virulence factor. Other isolates carried other virulence factor genes in addition to AIDA. Four isolates carried genes for at least one of the fimbrial adhesins and enterotoxins. Sixteen isolates carried genes for enterotoxins only. The AIDA may represent an additional virulence determinant in pre‐weaned pigs with diarrhoea.     

Genetic analysis of multi-drug resistance and the clonal dissemination of β-lactam resistance in Salmonella Infantis isolated from broilers

An epidemiologic study was conducted to investigate the incidence and characterize the antimicrobial resistance determinants, analyzing plasmid profiles, and establishing the genetic relationship among β-lactam-resistant isolates of Salmonella Infantis from broilers in Southern Japan. A total of 120 isolates were recovered from 56 flocks belonging to 44 holdings during 2004–2006. The percentages of resistance were as follows: ampicillin (24%), cephalothin (23%), cefoxitin (0%), ceftazidime (11%), cefotaxime (11%), chloramphenicol (0%), kanamycin (7.5%), ofloxacin (20%), oxytetracycline, streptomycin and sulfamethoxazole (100%) and trimethoprim (75%). The incidence of bla_TEM-encoded β-lactam resistance in 2004–2006 was significantly higher than in 1998–2003 (P < 0.001). BlnI-digested PFGE patterns generated two related clusters implicated in the dissemination of β-lactam resistance. Two types of plasmid profiles were observed and two plasmids of ca. 50 and 180-kb size were carried by β-lactam-resistant isolates. Streptomycin resistance was conferred by aadA1 (n = 116), aadA1-aadA2 (n = 1), and aadA1-strA-strB (n = 3). Resistances to kanamycin, oxytetracycline, sulfamethoxazole and trimethoprim were conferred by aphA1 (n = 9, 100%), tetA (n = 120, 100%) sul1 (n = 120, 100%) and dfrA5 (n = 90, 100%), respectively. Two types of class 1 integrons were detected: 1.0 kb (n = 120) and, 1.0/1.5 kb (n = 3). Integrons of 1.0/1.5 kb were found in isolates with the aadA1-strA-strB gene combination. For the first time, all S. Infantis isolates showed resistance to at least three classes of antimicrobial agents; and the intestinal tract of healthy poultry was a reservoir of the extended-spectrum cephalosporin-resistant isolates of serovar Infantis.     

Analysis of the AIDA-I gene sequence and prevalence in Escherichia coli isolates from pigs with post-weaning diarrhoea and oedema disease

Three hundred and twenty-four strains of Escherichia coli isolated from weaned pigs with diarrhoea or oedema disease in Eastern China were screened by multiplex PCR for the presence of the gene encoding adhesin involved in diffuse adhesion I (AIDA-I). Two AIDA-I positive strains were subjected to analysis of the nucleotide sequence of the complete orfA and orfB of the AIDA gene. The AIDA-I positive E. coli isolates were also assessed for five fimbriae (F4, F5, F6, F18 and F41) by monoclonal antibodies and for toxin genes (STa, STb, LT, EAST1, Stx2e) by PCR. Twenty-one (6.5%) of the isolates possessed AIDA-I genes. Of these isolates, two carried AIDA-I genes as the only demonstrated virulence factors, and the remaining isolates carried other virulence factor genes. Comparing the AIDA-I sequence from porcine and human sources, a high homology of orfA both in porcine E. coli and human E. coli was observed. However, each orfB of the two porcine E. coli isolates was 3864 nucleotides long compared with 3861 for the E. coli 2787 orfB, and showed 96.5% homology to E. coli 2787. The data indicated (1) that AIDA-I may be an occasional virulence factor in post-weaning diarrhoea and oedema disease in pigs, (2) that it has the potential to transfer between porcine and human E. coli, and (3) that there is a genetic diversity in orfB between human and porcine E. coli.     

Differential expression of pro-inflammatory and anti-inflammatory cytokines during experimental infection with low or high virulence bovine viral diarrhea virus in beef calves

The objective was to compare the mRNA expression of pro-inflammatory (TNF-α, IL-1β, IFN-γ, IL-2, IL-12, IL-15) and anti-inflammatory (IL-4, IL-10, TGF-β) cytokines, after experimental infection with low or high virulence noncytopathic (ncp) bovine viral diarrhea virus (BVDV). Thirty BVDV-naïve, beef calves were intranasally inoculated with low (LV; n = 10, SD-1) or high (HV; n = 10, 1373) virulence ncp BVDV or with BVDV-free cell culture medium (Control, n = 10). Calves were euthanized on day 5 post-inoculation, and tracheo-bronchial lymph node and spleen samples were collected for mRNA expression through quantitative-RT-PCR. mRNA levels of pro-inflammatory (TNF-α, IL-1β, IL-2, IFN-γ) and anti-inflammatory (IL-4 and IL-10) cytokines were up-regulated in tracheo-bronchial lymph nodes of HV, but not in LV, compared to the control group (P < 0.05). IL-12 mRNA level was up-regulated in tracheo-bronchial lymph nodes of both LV and HV groups (P ≤ 0.05). A significant up-regulation of IL-15 mRNA was observed in tracheo-bronchial lymph nodes for LV calves (P < 0.002), but not for HV calves. Experimental inoculation with BVDV-2 1373 stimulated significant mRNA expression of pro-inflammatory and anti-inflammatory cytokines. In contrast, inoculation with BVDV-1a SD-1 only resulted in up-regulation of IL-12 and IL-15 mRNA, which is associated with activation of macrophages and NK cells during innate immune response.     

A field trial of infrared thermography as a non-invasive diagnostic tool for early detection of digital dermatitis in dairy cows

Infrared thermography (IRT) was used to detect digital dermatitis (DD) prior to routine claw trimming. A total of 1192 IRT observations were collected from 149 cows on eight farms. All cows were housed in tie-stalls. The maximal surface temperatures of the coronary band (CB) region and skin (S) of the fore and rear feet (mean value of the maximal surface temperatures of both digits for each foot separately, CB_max and S_max) were assessed. Grouping was performed at the foot level (presence of DD, n = 99; absence, n = 304), or at the cow level (all four feet healthy, n = 24) or where there was at least one DD lesion on the rear feet, n = 37). For individual cows (n = 61), IRT temperature difference was determined by subtracting the mean sum of CB_max and S_max of the rear feet from that of the fore feet.Feet with DD had higher CB_max and S_max (P < 0.001) than healthy feet. S_max was significantly higher in feet with infectious DD lesions (M-stage: M2 + M4; n = 15) than in those with non-infectious M-lesions (M1 + M3; n = 84) (P = 0.03), but this was not the case for CB_max (P = 0.12). At the cow level, an optimal cut-off value for detecting DD of 0.99 °C (IRT temperature difference between rear and front feet) yielded a sensitivity of 89.1% and a specificity of 66.6%. The results indicate that IRT may be a useful non-invasive diagnostic tool to screen for the presence of DD in dairy cows by measuring CB_max and S_max.     

Quadricuspid aortic valve and associated abnormalities in the dog: Report of six cases

ObjectivesTo describe the clinical and echocardiographic findings in dogs with quadricuspid aortic valve (QAV).BackgroundQAV is a rare canine congenital heart disease which has been reported only three times in the young dog.Animals, materials and methodsSix dogs (0.3- to 13-year-old) with QAV diagnosed by two-dimensional echocardiography were retrospectively evaluated. Medical records, echocardiograms, and follow-ups were reviewed.ResultsAccording to aortic cusp morphology, QAV was classified as type A (n = 1), type B (n = 4) or type C (n = 1). QAV was associated with at least one other heart disease in all of the dogs including, ventricular septal defect (n = 1), enlarged left coronary ostium (n = 4), degenerative mitral valve disease (MVD, n = 1) and patent ductus arteriosus (PDA, n = 3). Mild to moderate aortic regurgitation was also detected in all dogs by continuous-wave and color-flow Doppler echocardiography. QAV was diagnosed in four asymptomatic dogs referred for evaluation of a heart murmur. The remaining two dogs had QAV and PDA with evidence of mild exercise intolerance and moderately retarded growth. The PDA was surgically corrected in both dogs and at the time of writing, 1–2.5 years after the initial diagnosis, none of the six animals shows evidence of clinical signs.ConclusionQAV is a cause of aortic insufficiency. It may incidentally be found by two-dimensional echocardiography in dogs of various ages in association with other congenital or acquired cardiac abnormalities.     

Development of multiplex polymerase chain reaction assays for detecting enterotoxigenic Escherichia coli and their application to field isolates from piglets with diarrhea.

Fimbriae and enterotoxins are major virulence factors associated with enterotoxigenic Escherichia coli (ETEC). In this study, 3 sets of multiplex polymerase chain reaction (mPCR) assays targeting fimbriae, enterotoxins, and other adherence factors were developed for detecting ETEC. A total number of 188 E. coli field isolates were examined, and percentages of E. coli strains carrying each virulence factors were as follows: F4 (7.45%), F5 (29.79%), F6 (6.38%), F18 (15.43%), F41 (3.72%), STa (10.11%), STb (20.74%), LT (9.57%), Stx2e (2.13%), EAST1 (42.02%), F1 (67.55%), AIDA-I (2.66%), and pAA (7.45%). Of the 188 E. coli field isolates examined, 25.53% were found to be pathogenic ETEC, having both fimbriae and enterotoxins. However, the ratio increased to 44.68% when the presence of other adhesins was considered as criteria for virulence. Among the adherence factors, F1 was found to be the most prevalent. AIDA-I and pAA were also found with similar ratio as compared with other virulence factors. In addition, virulence patterns carrying these alternate adhesive genes with enterotoxins were detected with significant ratio. Therefore, it is desirable that alternate adhesins be considered as markers for diagnosis of ETEC.     

Genotypic characterization of Malaysian human isolates of Streptococcus pneumoniae from carriage and clinical sources

This study characterized carriage and clinical pneumococcal isolates for serotypes, penicillin susceptibility, virulence genes and restriction fragment length polymorphism (RFLP) pattern of penicillin binding protein (PBP) genes. DNA fingerprint of isolates was generated by BOX-PCR. Majority of serotypes were 23F followed by 19F, 19A and 6A. Twenty-four percent of isolates were penicillin non-susceptible (PNSP). All of the targeted virulence genes were detected in all isolates with the exception of pili; 20.6% (n = 22) for PI-1 and 14.0% (n = 15) for PI-2. Of the 13 isolates which carried both PI-1 and PI-2, 10 were of clinical origin. Digested pbp-DNA produced three PBP-RFLP profiles for pbp1a (A1 to A3), six profiles for pbp2b (B1 to B6) and seven for pbp2x (X1 to X7) mostly in PNSPs. Based on BOX-PCR analysis, the majority of isolates were genetically diverse with a small number of potentially related isolates carrying pili genes. No obvious genotypic association was observed pertaining to carriage and clinical origin of isolates.     

Expression of trefoil factor genes in the duodenum and colon of dogs with inflammatory bowel disease and healthy dogs

Trefoil factors (TFF) are small peptides produced by goblet cells, which are crucial for epithelial restitution. In humans with inflammatory bowel disease (IBD), TFF expression is up-regulated as part of an unspecific repair mechanism. The goal of this study was to assess TFF gene expression in the gastrointestinal tract from dogs with IBD compared to healthy controls. Preliminary assessment by PCR revealed TFF1 and 3 expression in the small and large intestine, whereas TFF2 was amplified only in the stomach. Subsequent RT-qPCR (with relative quantification against 3 reference genes) on endoscopic duodenal (IBD n = 22, healthy controls n = 18) and colonic (IBD n = 12, controls n = 11) biopsies revealed that TFF1 expression was significantly up-regulated in the duodenum from IBD dogs (Mann–Whitney p = 0.001), whereas TFF3 expression was significantly lower in IBD colon compared to controls (t-test p = 0.018).This study demonstrates evidence for dysregulation of TFF gene expression in canine IBD. Up-regulation of TFF1 could signify ectopic expression as a compensatory repair-mechanism, whereas down-regulation of TFF3 could contribute to defective epithelial barrier function, respectively. Whether this is a cause or consequence of IBD could not be established.