Specific DNA fragments coding for ST1 and LT1 toxins, and K88 (F4) adhesin in enterotoxigenic Escherichia coli

Ten porcine strains of enterotoxigenic Escherichia coli possessing the K88 (F4) adhesion fimbriae, were selected for study of enterotoxin- and fimbriae-encoding plasmids. Plasmid DNA, separated according to size by gel electrophoresis was transferred to nylon membranes by Southern blotting, and hybridized with enzyme-labelled oligonucleotide probes for ST1 and LT1 enterotoxins, and a 32P-labelled probe for the F4 fimbriae. Plasmids possessing the enterotoxin genes ranged from 50 MDa to 78 MDa in size. The ST1 genes were located on a common 8-MDa EcoR1 restriction endonuclease fragment, while the LT1 genes were located on a 4.5-MDa EcoR1 fragment from the different plasmids. Plasmids with the F4 genes ranged from 50 MDa to 118 MDa in size, but the F4 encoding genes were located on a common 3-MDa HindIII restriction endonuclease fragment. ST1 and LT1 genes were found on the same plasmid in only one strain, LT1 and F4 genes on the same plasmids in 5 strains, while no plasmid contained genes for both ST1 and F4.     

Spread of extended-spectrum beta-lactamase producing Escherichia coli isolates in Swedish broilers mediated by an incI plasmid carrying blaCTX-M-1

Background

The already high and increasing occurrence of extended-spectrum beta-lactamases (ESBL) producing Escherichia coli in European broiler populations is of concern due to the fact that third and fourth generation cephalosporins are deemed critically important in human medicine. In Sweden 34% of the broilers carry ESBL/pAmpC producing E. coli in their gut, despite the absence of a known selection pressure such as antimicrobial usages. The aim of the current study was to characterise a selection of E. coli strains carrying the bla_CTX-M-1, to determine if the spread was due to a specific clone.

Findings

Ten isolates carrying bla_CTX-M-1 from Swedish broilers belonged to eight different multi-locus sequence types with three isolates belonging to ST155. The ST155 isolates were identical as assessed by PFGE. The bla_CTX-M-1 was in all isolates carried on a plasmid of replicon type incI, which also transferred resistance to tetracycline and sulfamethoxazole.

Conclusion

The occurrence of ESBL-producing E. coli in the Swedish broilers is not due to the emergence of a single clone, but rather the spread of a specific incI plasmid carrying bla_CTX-M-1.     

Characterisation of Escherichia fergusonii isolates from farm animals using an Escherichia coli virulence gene array and tissue culture adherence assays

Escherichia fergusonii has been associated with a wide variety of intestinal and extra-intestinal infections in both humans and animals but, despite strong circumstantial evidence, the degree to which the organism is responsible for the pathologies identified remains uncertain. Thirty isolates of E. fergusonii collected between 2003 and 2004 were screened using an Escherichia coli virulence gene array to test for the presence of homologous virulence genes in E. fergusonii. The iss (increased serum survival) gene was present in 13/30 (43%) of the test strains and the prfB (P-related fimbriae regulatory) and ireA (siderophore receptor IreA) genes were also detected jointly in 3/30 (10%) strains. No known virulence genes were detected in 14/30 (47%) of strains. Following confirmatory PCR and sequence analysis, the E. fergusoniiprfB, iss and ireA genes shared a high degree of sequence similarity to their counterparts in E. coli, and a particular resemblance was noted with the E. coli strain APEC O1 pathogenicity island. In tissue culture adherence assays, nine E. fergusonii isolates associated with HEp-2 cells with a ‘localised adherence’ or ‘diffuse adherence’ phenotype, and they proved to be moderately invasive. The E. fergusonii isolates in this study possess both some phenotypic and genotypic features linked to known pathotypes of E. coli, and support existing evidence that strains of E. fergusonii may act as an opportunistic pathogens, although their specific virulence factors may need to be explored.     

Characterization of Erysipelothrix rhusiopathiae strains isolated from acute swine erysipelas outbreaks in Eastern China

Recently, a series of acute swine erysipelas outbreaks occurred in Eastern China. Eight strains isolated from cases of septicemia were determined as serotype 1a, and 4 of the isolates were resistant to acriflavine. One isolate strain named {"type":"entrez-nucleotide","attrs":{"text":"HX130709","term_id":"383559396","term_text":"HX130709"}}HX130709 was attenuated on agar media containing acriflavine dye. The 432-bp hypervariable region in spaA gene of the field and attenuated strains were amplified and sequenced. It was further compared with the vaccine strain G₄T₁₀, and thus, the eight field strains can be divided into four spaA-types. The partial spaA gene analysis also showed that no point mutations occurred among different archived passages of {"type":"entrez-nucleotide","attrs":{"text":"HX130709","term_id":"383559396","term_text":"HX130709"}}HX130709 during the attenuation. Results of pulsed-field gel electrophoresis showed that eight distinct patterns with 22 to 30 DNA fragment bands were produced from field strains, and twelve distinct patterns with 23 to 27 DNA fragment bands were produced from different passages of the attenuated strains. Mouse pathogenicity test showed that the mortality of the mice infected with 10⁴ CFU field strains was 100% and the attenuation of strain {"type":"entrez-nucleotide","attrs":{"text":"HX130709","term_id":"383559396","term_text":"HX130709"}}HX130709 occurred between 46 and 50 passages. All the field and attenuated strains were highly sensitive to β-lactam antibiotics, tetracyclines and macrolides. So, we can make conclusions that the acute swine erysipelas outbreaks in Eastern China were caused by serotype 1a E. rhusiopathiae strains with different biochemical characteristics, and the virulence of serotype 1a E. rhusiopathiae strains is unrelated with some point mutations in 432-bp hypervariable region of the spaA gene.     

Cultivation, LD(50) determination and experimental model of Streptococcus suis serotype 2 strain HA9801

The effects of nutritional components and submerged culture conditions on colony-forming unit (CFU) counts by Streptococcus suis serotype 2 strain HA9801 in flask culture was investigated, and the optimal medium and cultivation conditions was confirmed by using a 50 l bioreactor. The LD₅₀ values of HA9801 in pigs before and after fermentation were 1.8 × 10⁷ CFU, which indicated that the virulence of HA9801 was very stable in the fermentation process. In addition, an experimental model that closely mimics naturally occurring disease in conventional pigs was established.     

Colonization antigens,antibiotic resistance and plasmid content of enterotoxigenic Escherichia coli isolated from piglets with diarrhoea in galicia (North-Western Spain)

Escherichia coli colonies isolated from 50 diarrhoeic and 29 healthy piglets were investigated for several properties related to pathogenicity, such as production of heat-labile (LT) and heat-stable (STa) enterotoxins, presence of K88 and K99 colonization antigens, mannose-resistant haemagglutinating activity (MRHA), β-haemolysis and antibiotic resistance. The objective was to establish the toxic and adhesive abilities of E. coli strains that cause porcine diarrhoea in Galician farms. Fifty-seven colonies from 14 diarrhoeic piglets formed STa, while no STa⁺ colony was detected from healty piglets. Thirty-four of the 57 STa⁺ colonies were resistant to gentamicin. Sixteen representative STa⁺ strains isolated from the 14 infected piglets were serotyped, investigated for plasmid content and examined by electron microscopy. Of these STa⁺ strains, 15 belonged to serotype 0141:K85ab and carried on their surface the fimbrial antigen P987. The remaining representative STa⁺ strain belonged to serotype 0101:K30 and was K99⁺, being the only STa⁺ strain with MRHA activity. All 15 STa⁺ P987⁺ strains possessed a similar plasmid pattern, with three plasmids ranging in molecular weight from 33 × 10⁶ to 74 × 10⁶; nine of the gentamicin-resistant strains possessed an additional plasmid of molecular weight 16 × 10⁶, which was absent in the six gentamicin-sensitive strains. Strains producing LT or K88 antigen were not detected. Forty-one MRHA⁺ colonies were isolated at similar rates from both diarrhoeic and healthy piglets. Twelve of the 19 non-enterotoxigenic MRHA⁺ strains of which the O-group was established, belonged to serogroups (01, 02, 07, 08, 09 and 075) typical of the human E. coli strains that cause extraintestinal infections. Finally, a statistically significant association between haemolytic and MRHA activities in porcine E. coli was found. In conclusion, it was found that STa⁺E. coli strains belonging to serotype 0141:K85ab:P987 are associated with porcine diarrhoea in Galicia. Additionally, no correlation between the isolation of non-ETEC MRHA⁺ strains and diarrhoea was observed.     

Metabolic changes in red cells in response to adhesion of porcine K88 fimbriated Escherichia coli

Red cells glycolytic enzymes attached and nonattached to K88⁺ Escherichia coli were assayed. Hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase and glutathione reductase activities, were measured. E. coli with K88ab fimbriae, E. coli with K88ac fimbriae, and isolated K88ab fimbriae were investigated for their effect on the above enzymes. Different changes were obtained with K88ab + bacteria compared with K88ac + bacteria. Purified fimbriae gave a third set of responses.     

Characterization of a plasmid-encoded adhesin of an avian pathogenic Escherichia coli (APEC) strain isolated from a case of swollen head syndrome (SHS)

An avian pathogenic Escherichia coli (APEC) strain designated SHS4, isolated from a chicken with clinical signs of swollen head syndrome (SHS), adhered to but did not invade Hep-2 and tracheal epithelial cells. The PCR amplified fimA, csgA and tsh gene sequences. It produced Ia, Ib, E1, E3, K, and B colicins, but not colicin V and aerobactin. It harboured two plasmids of 60 and 98MDa and was resistant to streptomycin and tetracycline. Conjugation with a nalidixic acid (Na) resistant K-12 recipient strain (MS101) showed that the 98MDa plasmid did not transfer, whereas transfer of the 60MDa plasmid resulted in concomitant transfer of adhesion to Hep-2 and tracheal epithelial cells, production of the colicins Ia, E1, E3, and K, and the tsh-related DNA sequence. Transposon (TnphoA) mutagenesis of strain TR4 gave rise to strain Mut23, which lost its adhesive capacities, but was still able to express the same colicins as did strain TR4. PCR was able to amplify the tsh-related DNA sequence in this strain and a molecular probe based on transposon TnphoA indicated that the transposon was inserted in the 60MDa plasmid. Based on these results, we suggest that the 60MDa plasmid have adhesion genes, which may be responsible for the initial colonization of the upper respiratory tract of chickens.     

Pseudomonads from rabbits and their sensitivity to antibiotics and natural antimicrobials

The sensitivity/resistance of Pseudomonas spp. isolated from rabbits gastrointestinal tract and faeces to antibiotics, enterocins and herbal extracts was tested in this study. The counts of Pseudomonas-like bacteria were higher in faeces (3.23-6.16 log₁₀ CFU/mL/g) than in caecum (1.36-4.08 log₁₀ CFU/mL/g). Nineteen isolates (16 faecal, 3 caecal) were oxidase positive. The strains were allotted by phenotypization to Pseudomonas spp., Brevundimonas diminuta and Brevundimonasvesicularis. High percentage of resistant strains was observed to all antibiotics. The tested strains were more susceptible to natural substances, mainly to plant extracts oregano (95%) and sage extracts (58%). Comparing the antibacterial effect of antibiotics and enterocins against rabbits pseudomonads, enterocins were more effective; the strongest inhibitory activity was determined in the case of partially purified enterocins PPBs EF2019, EK13 and EF55.     

Mutant prevention concentration of orbifloxacin: comparison between Escherichia coli,Pseudomonas aeruginosa,and Staphylococcus pseudintermedius of canine origin

Background

The mutant prevention concentration (MPC) is an important parameter to evaluate the likelihood of growth of fluoroquinolone-resistant mutants for antimicrobial-pathogen combinations. The MPCs of fluoroquinolones for different canine pathogens have not been compared. In this study, we compared for the first time orbifloxacin MPCs between susceptible strains of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus pseudintermedius of canine origin.

Methods

More than 10¹⁰ CFU/ml of 10 strains of each bacterial species were inoculated onto Muller-Hinton agar supplemented with different concentrations of orbifloxacin from 1× to 64× minimum inhibitory concentration (MIC) and the MPCs were recorded. MICs of original strains and of mutants arising after exposure to sub-MPC concentrations (one per original strain) were determined in the presence or absence of efflux pump inhibitors (EPIs). The effects of quinolone resistance-determining region (QRDR) mutations were also examined.

Results

MPCs were significantly higher for P. aeruginosa (16–128 μg/ml) than for E. coli (0.5–32 μg/ml). MPCs for S. pseudintermedius varied between the low-susceptible (16–128 μg/ml) and the high-susceptible strains (4–16 μg/ml) and were the most broadly distributed among the three species. Regarding resistance mechanisms, only one QRDR mutation in gyrA was found in all of the 10 mutants of E. coli and in 4 of the 10 mutants of P. aeruginosa, whereas mutations in both grlA and gyrA were found in 3 mutants and one mutation in grlA was found in 2 mutants among the 10 mutants of S. pseudintermedius. In the presence of an EPI, the MICs of P. aeruginosa mutants decreased markedly, those of E. coli mutants decreased moderately, and those of S. pseudintermedius mutants were unaffected.

Conclusions

MPCs of orbifloxacin vary between bacterial species of canine pathogens, possibly due to the diversity of the main fluoroquinolone resistance mechanism among these species. Therefore, the type of bacterial species should be taken into consideration when using fluoroquinolone drugs such as orbifloxacin in canines.     

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.     

Preslaughter diet management in sheep and goats: effects on physiological responses and microbial loads on skin and carcass

Sixteen crossbred buck goats (Kiko x Spanish; BW = 32.8 kg) and wether sheep (Dorset x Suffolk; BW = 39.9 kg) were used to determine the effect of preslaughter diet and feed deprivation time (FDT) on physiological responses and microbial loads on skin and carcasses. Experimental animals were fed either a concentrate (CD) or a hay diet (HD) for 4 d and then deprived of feed for either 12-h or 24-h before slaughter. Blood samples were collected for plasma cortisol and blood metabolite analyses. Longisimus muscle (LM) pH was measured. Skin and carcass swabs were obtained to assess microbial loads. Plasma creatine kinase activity (863.9 and 571.7 ± 95.21 IU) and non-esterified fatty acid concentrations (1,056.1 and 589.8 ± 105.01 mEq/L) were different (P < 0.05) between sheep and goats. Species and diet treatments had significant effects on the ultimate pH of LM. Pre-holding total coliform (TCC) and aerobic plate counts (APC) of skin were significantly different between species. Goats had lower (P < 0.05) TCC (2.1 vs. 3.0 log₁₀ CFU/cm²) and APC (8.2 vs. 8.5 log₁₀ CFU/cm²) counts in the skin compared to sheep. Preslaughter skin E. coli counts and TCC were different (P < 0.05) between species. Goats had lower (P < 0.05) counts of E. coli (2.2 vs. 2.9 log₁₀ CFU/cm²) and TCC (2.3 vs. 3.0 log₁₀ CFU/cm²) in the skin compared with those in sheep. Diet, species, and FDT had no effect (P > 0.05) on E. coli and TCC in carcass swab samples. The APC of carcass swab samples were only affected (P < 0.05) by the FDT. The results indicated that preslaughter dietary management had no significant changes on hormone and blood metabolite concentrations and sheep might be more prone for fecal contamination than goats in the holding pens at abattoir.     

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.     

Occurrence and characterization of methicillin-resistant staphylococci from bovine mastitis milk samples in Finland

Background

Methicillin-resistant staphylococci (MRS) are increasingly being isolated in bovine mastitis. The aim of our study was to evaluate the occurrence of MRS in Finnish mastitis milk samples and characterize the MRS isolates using molecular methods.

Results

Methicillin-resistant S. aureus (MRSA) was a rare finding in bovine mastitis in Finland. Only two out of 135 (1.5%) S. aureus isolates were positive for mec genes. One of these carried mecA and was of spa type t172, SCCmec type IV and ST375, and the other harboured mecC, being spa type t3256, and ST130. MRSA ST375 is common among human MRSA isolates in Finland, but this is the first report in the country of bovine mecC MRSA. In coagulase-negative staphylococci (CoNS) originating from bovine mastitis, methicillin resistance was more common. In the two CoNS collections studied, 5.2% (17/324) and 1.8% (2/110) of the isolates were mecA positive. Eighteen of these were methicillin-resistant S. epidermidis (MRSE), which were divided into 6 separate PFGE clusters. One pulsotype was detected in different parts of the country, indicating clonal spread. Most MRSE (13/18) were of SCCmec type IV, one was of type V and four were non-typeable. Comparison with a human staphylococcal database indicated that bovine MRSE strains were not closely related to human MRSE isolates.

Conclusions

The occurrence of MRS, especially MRSA, in bovine mastitis in Finland was low. Most methicillin-resistant bovine CoNS are MRSE, and we found evidence of a bovine MRSE strain that may spread clonally. This is the first report of a Finnish bovine isolate of MRSA_mecC ST130. The study provides a baseline for further MRS monitoring.     

Role of fimbriae F18 for actively acquired immunity against porcine enterotoxigenic Escherichia coli

Enterotoxigenic (ETEC) and enterotoxaemic (ETEEC) Escherichia (E.) coli that express F18 (F107) fimbriae colonize the small intestine and cause diarrhoea and/or oedema disease in weaned pigs. So far, two antigenic variants of F18 can be distinguished with a common antigenic factor designated ‘a’ and two specific factors called ‘b’ and ‘c’. In this study the existence of crosswise anti-colonization immunity between E. coli strains that express F18ab or F18ac fimbrial variants, respectively, was demonstrated. Weaned pigs of susceptible genotype with respect to susceptibility to adhesion of E. coli with fimbriae F18 were inoculated with E. coli strains 3064^STM (O157:K-:H-:F18ab; resistant to streptomycin) and 8199^RIF (O141ab:K-:H4:F18ac; resistant to rifampicin). The faecal shedding was compared subsequent to immunization and homologous or heterologous challenge. An enzyme-linked immunosorbent assay (ELISA) was applied to measure IgA, IgM and IgG antibodies against the F18ab and F18ac antigens in saliva, faeces, serum and intestinal wash samples. About 8 log CFU/g of the inoculated strains were found in faeces of all pigs following immunization as well as in non-immunized controls after challenge. Bacterial counts of the inoculated strains after challenge were between 2 and 5 log lower, without any difference between homologous and heterologous challenge. Intestinal colonization with fimbriated E. coli resulted in production of significantly increased levels of anti-fimbrial antibodies, especially IgA, in serum and intestinal wash samples. There were higher levels of homologous than of heterologous anti-fimbrial antibodies. Production of antibodies against F18a or against another common fimbrial antigen is probably responsible for crosswise anti-colonization immunity between E. coli strains with F18ab and F18ac fimbrial variants. Serum F18-specific IgA may be a useful indicator of a mucosal immune response directed against F18 fimbriae.     

Bovine-associated MRSA ST398 in The Netherlands

During routinely screening (50.000 milk samples on an annual basis) 14 MRSA ST398 strains were identified in the period of January 2008 to September 2008 in 14 different dairy herds located in the provinces Overijssel and Gelderland, The Netherlands. Molecular analysis was performed by Cfr9I PFGE, ST398-specific diagnostic PCR, spa typing, SCCmec typing and Panton-Valentine Leukocidin (PVL) gene PCR. The molecular analyses of 14 MRSA (one MRSA strain per herd) strains revealed that all strains belong to ST398 with 3 closely related spa types (t011, t108 and t889, all commonly found in pigs) and carry 2 different SCCmec types, IVa and V. All MRSA strains were resistant to two or more classes of antibiotics and also PVL negative. The majority of farms (n = 9, 64%) harboured combined livestock with both cows and pigs present. Our study contributes to the growing evidence that MRSA ST398 is transmitted among various animal species and can be considered as an etiological agent of mastitis in dairy cows.     

Isolation,in vitro propagation,genetic analysis,and immunogenic characterization of an Ehrlichia canis strain from southeastern Brazil

Amplification of the 16S rRNA gene from a blood sample obtained from a dog in southeastern Brazil was used to confirm a naturally acquired Ehrlichia (E.) canis infection. Following isolation and culturing of the new bacterial strain called Uberlândia, partial sequences of the dsb and p28 genes were obtained. The dsb partial sequence of the novel strain was 100% similar to dsb gene sequences of E. canis obtained from different geographic areas around the world. Conversely, the p28 partial sequence for the E. canis Uberlândia strain differed at several nucleotides from other sequences available in GenBank. To confirm the antigenic profile of the Uberlândia strain, an indirect immunofluorescence assay against E. canis antigens was performed using dog sera collected from two different areas in Brazil (Uberlândia and São Paulo). The results suggest that both antigens were able to identify animals seropositive for E. canis in Brazil since these Brazilian strains appear to be highly conserved.     

Molecular characterization of tetracycline- and quinolone-resistant Aeromonas salmonicida isolated in Korea

The antibiotic resistance of 16 Aeromonas (A.) salmonicida strains isolated from diseased fish and environmental samples in Korea from 2006 to 2009 were investigated in this study. Tetracycline or quinolone resistance was observed in eight and 16 of the isolates, respectively, based on the measured minimal inhibitory concentrations. Among the tetracycline-resistant strains, seven of the isolates harbored tetA gene and one isolate harbored tetE gene. Additionally, quinolone-resistance determining regions (QRDRs) consisting of the gyrA and parC genes were amplified and sequenced. Among the quinolone-resistant A. salmonicida strains, 15 harbored point mutations in the gyrA codon 83 which were responsible for the corresponding amino acid substitutions of Ser⁸³→Arg⁸³ or Ser⁸³→Asn⁸³. We detected no point mutations in other QRDRs, such as gyrA codons 87 and 92, and parC codons 80 and 84. Genetic similarity was assessed via pulsed-field gel electrophoresis, and the results indicated high clonality among the Korean antibiotic-resistant strains of A. salmonicida.     

Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine

In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 10⁹ CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID₅₀ of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 × 10⁹ CFU of p3D-NT56/S. cho were protected in 9 days.     

Fimbriae in Escherichia coli isolated from the small intestine of piglets

Ninety E. coli strains, isolated from piglets which had died from neonatal diarrhea, were tested for the presence of K88, K99, 987P and type 1 fimbriae. Two or more types of fimbriae were demonstrated in 14 of the strains, a single fimbria! type in 44 strains while in 32 strains no fimbriae were detected. Of the 14 E. coli strains with more than 1 type of fimbriae, 12;, 10, 8 and 4 strains showed K88, K99, 987P and type 1, respectively.Twelve E. coli strains were isolated from piglets which had died in the neonatal period without showing signs of neonatal diarrhea at necropsy. One strain showed 987P and 3 strains showed type 1 fimbriae, while the remaining 8 strains were unfimbriated.Sixteen fimbriated E. coli strains were subcultured in order to examine the stability of fimbrial expression in the strains. The K88 and the type 1 fimbriae were regularly expressed, while the K99 and 987P were inconsistently demonstrated.