Serum antibody coupled with the construction of gentamicin sulfate for the Escherichia coli targeted drug

Conjugates of the Escherichia coli serum antibody (Ab) with the gentamicin sulfate (GM sulfate) have been evaluated for the assessment of their ability to eradicate E. coli in vitro. The combination of every component in the targeted drug is the amino of serum antibody and gentamicin sulfate combined with the hydroxyl of PEG6000 by hydrogen bond, which forms stable complexes. The half-life of this complex is 4.83 h, and it is non-toxic side effects and low immunogenic. After the combination of antibody and gentamicin sulfate, the targeting of antibody is quite good. In vitro anti-bacterial activity of gentamicin sulfate increased 1000 times, and the clinical curative effect enhanced 100 times, this means higher efficacy and safety.     

Synthesis, cloning, and expression of Mycoplasma suis inorganic pyrophosphatase gene using PCR-based accurate synthesis and overlap-extension PCR, and its immunogenicity analysis

Mycoplasma suis (M. suis), a hemotrophic pathogen of pigs, causes economic losses in swine production throughout the world. Inorganic pyrophosphatase (ppa) is a very important gene in M. suis. The ppa gene of M. suis was synthesized by PCR-based accurate synthesis (PAS) and overlapextension PCR, inserted into vector pMD18-T, and then subcloned to the prokaryotic expression vector pET28c.The recombinant plasmid pET28c_ppa was transformed to E. coli BL21 for expression under induction of isopropyl thiogalactoside. The expressed product was identified by SDS–PAGE and Western blot, which suggested that the recombinant protein has good antigenicity. Piglets were immunised with purified recombinant protein, and specific antibodies to the recombinant protein were detected in piglet serum. The results show that the ppa gene can be efficiently expressed in E. coli and that the expressed recombinant protein can elicit a specific serum antibody response in piglets. PAS and overlap-extension PCR were first used to synthesize the ppa of M. suis. They provide simple, rapid, reliable and relatively inexpensive methods to synthesize, clone, and express genes. The experiment conducted in this paper will enable future research into the role and function of the ppa gene.     

Field trial evaluating changes in prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers medicated with enrofloxacin, apramycin and amoxicillin

The present study investigates, under field conditions, the influence of antimicrobial administration on prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers. For this purpose, a group of 16,000 commercial broiler chickens was treated with enrofloxacin from day 1 to day 3, gentamicin from day 19 to day 21, and ampicillin from day 26 to day 28. A control group of 16,000 broilers was placed in the same controlled environment poultry house. Fecal (from both groups) and feed samples were collected at regular intervals. Few E. coli isolates were obtained from either farm environment or poultry feed samples, while enterococci were found to be ubiquitous among these samples. The frequency of resistance against most antimicrobials tested was significantly higher (P < 0.05) in E. coli isolated from broilers receiving intermittent antimicrobial pressure than that from non-medicated broilers, whereas in enterococci these differences were only observed among structurally related antimicrobial drugs and over a short period of time. By the time the broilers reached market age (33 days), several multi-resistant E. coli and enterococci were detected in the feces of the medicated group. Results suggest that antimicrobial resistance in E. coli was mainly medication-dependent, whereas among enterococci, changes observed over time were apparently influenced by factors apart from antimicrobial exposure, namely the resistance organisms previously present in farm environment and those present in feedstuffs.     

Prediction of pharmacokinetic profiles of ampicillin sodium, gentamicin sulfate, and combination ampicillin sodium-gentamicin sulfate in serum and synovia of healthy horses

Pharmacokinetics of ampicillin sodium (11 mg/kg), gentamicin sulfate (2.2 mg/kg), and combination ampicillin sodium-gentamicin sulfate were determined for serum and synovia of healthy horses given single-dose IV injection and were not found to be different from those from other reports; however, a prolonged terminal gamma-phase for gentamicin (8,498 +/- 1,842 minutes) in serum of horses was found to exist. Pharmacokinetic interaction between combination ampicillin sodium-gentamicin sulfate was not observed int he serum or synovia. Prediction of ampicillin sodium or gentamicin sulfate concentrations in synovia, based on serum-based pharmacokinetics, cannot be accomplished solely upon analysis of peripheral-compartment pharmacokinetics. However, once equilibrium is achieved between synovia and extracellular fluid in the peripheral compartment, the decrease in drug concentrations in synovia parallels that in serum. Therefore, after 6 hours, synovial concentrations of gentamicin sulfate can be predicted based on peripheral-compartment pharmacokinetics, using an appropriate correction factor. The significance of these findings need to be correlated with clinical conditions so that a pharmacostatistical model for the prediction of synovial concentrations of drug(s) during treatment of horses with septic arthritis can be developed.     

救必应中药血清与抗菌药联合对产ESBLs细菌抑菌效果的研究

本试验主要探讨救必应中药血清与抗菌药联合诱导细菌传代的体外抑菌活性。制备救必应中药血清,采用二倍微量稀释法体外检测抗菌药的最小抑菌浓度(MIC),然后救必应中药血清与抗菌药联合诱导产超广谱β-内酰胺酶(extended spectrumβ-lactamases,ESBLs)细菌传代。救必应中药血清与抗菌药联合诱导细菌传代对耐药大肠杆菌有不同程度的抑菌活性。救必应中药血清可明显地增强β-内酰胺类抗菌药、氨基糖苷类抗菌药、喹诺酮类抗菌药和卡巴氧类抗菌药对耐药菌的抑制作用。     

Resistance to Serum Complement,iss, and Virulence of Avian Escherichia coli

Control of avian colibacillosis is hampered by lack of easily identifiable markers for virulent Escherichia coli. Resistance to serum complement appears to be a widespread trait of virulent avian E. coli, suggesting that bacterial factors promoting survival in serum may be useful in discriminating between virulent and avirulent isolates. Such distinguishing factors may prove useful in diagnostic protocols or as targets in future colibacillosis control protocols. Interestingly, the factors responsible for resistance to complement differ in the E. coli isolated from mammalian and avian hosts, which may reflect differences in the nature of avian and mammalian colibacillosis. In some cases, genetic determinants for serum complement resistance in avian E. coli are found on aerobactin- or Colicin V-encoding plasmids. One such gene, iss, first described for its role in the serum resistance associated with a ColV plasmid from a human E. coli isolate, occurs much more frequently in isolates from birds with colibacillosis than in faecal isolates from healthy birds. Efforts to identify the genomic location of iss in a single, virulent avian E. coli isolate have revealed that it occurs in association with several purported virulence genes, all linked to a large conjugative R plasmid. At this time, it is not known whether iss merely marks the presence of a larger pathogenicity unit or is itself a contributor to virulence. Nevertheless, the presence of the complement-resistance determinant, iss, may be a marker of virulent avian E. coli exploitable in controlling avian colibacillosis.     

Plasmid encoded β-lactamases resistant to inhibition by clavulanic acid produced by calf faecal coliforms

Two new plasmid encoded β-lactamase enzymes produced by a strain of Escherichia coli and a strain of Citrobacter freundii isolated from calf faeces have been characterised. Both enzymes were similar to TEM-1 in terms of substrate and inhibition profiles and physical properties but differed from TEM-1 in being far less susceptible to the β-lactamase inhibitors clavulanic acid or tazobactam. In each case transfer of the the plasmid E coli K12 rendered it clinically resistant to the combination of amoxycillin and clavulanic acid. The β-lactamase from the E coli had an iso-electric point (pI) of 5·4 and was encoded on a plasmid of 95 Kbp which also mediated resistance to tetracycline, sulphonamides, apramycin, streptomycin and gentamicin. The β-lactamase from the C freundii had a pI of 5·2 and was encoded on a 75 Kbp plasmid which also mediated resistance to trimethoprim, chloramphenicol, apramycin, gentamicin and tobramycin.     

Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens

Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with immune sera from commercial meat-type chickens with clinical outbreak of Clostridium infections. In addition to the genes encoding EF-Tu and PFO, C. perfringens alpha-toxin and necrotic enteritis B-like (NetB) toxin were also expressed in Escherichia coli and their corresponding recombinant proteins were purified. Using the four recombinant proteins as target antigens in ELISA immunoassays, high serum antibody titers were observed not only in chickens with clinical signs of Clostridium infections, but also in apparently healthy animals from the same disease-endemic farm. By contrast, no antibodies against any of the proteins were present in the serum of a specific pathogen-free bird. In ELISA using recombinant proteins of C. perfringens, the levels of anti-bacterial protein antibodies were also higher in chickens which were experimentally induced to show NE clinical signs after co-infection with C. perfringens and Eimeria maxima compared with uninfected controls. These results show that two antigenic C. perfringens proteins, EF-Tu and PFO can be useful detection antigens for C. perfringens-afflicted infections in commercial poultry.     

Antibody Response in Sheep Following Immunization with Streptococcus bovis in Different Adjuvants

Recent studies have shown that immunization with Streptococcus bovis using Freund's complete adjuvant (FCA) may confer protection against lactic acidosis in sheep. The major objective of this study was to compare the antibody responses to S. bovis in a practically acceptable adjuvant (Freund's incomplete adjuvant (FIA); QuilA; dextran sulphate (Dex); Imject Alum; or Gerbu) and in FCA. Thirty-five sheep were randomly allocated to 7 treatment groups. Six groups were immunized with S. bovis in an adjuvant; the other group served as the non-immunization control. The primary immunization was administered intramuscularly on day 0, followed by a booster injection on day 28. Immunization with FCA induced the highest saliva and serum antibody responses. The saliva antibody concentrations in the FIA and QuilA groups were significantly higher than those in the Alum, Dex and Gerbu groups (p<0.01). The serum antibody concentration in the FIA group was significantly higher than those in the QuilA, Alum, Dex and Gerbu groups (p<0.01). Immunization enhanced the antibody level in faeces (p<0.05), but there was no significant difference between the different adjuvant groups (p>0.05). Seven and 14 days following booster immunization, the saliva antibody levels induced by QuilA and/or FIA were comparable with the level stimulated by FCA (p>0.05). There was a strongly positive correlation (R ² = 0.770, p<0.01) between the antibody concentrations in saliva and serum. Compared with the controls, a higher faecal dry matter content was observed in the animals immunized with either FCA or QuilA. The change in faecal dry matter content was positively associated with the faecal antibody concentration (R ² = 0.441, p<0.05). These results indicate that FIA and QuilA were effective at inducing high levels of antibody responses to S. bovis, and suggest that either Freund's incomplete adjuvant or QuilA may be useful for preparing a practically acceptable vaccine against lactic acidosis.     

Prevalences of resistance to seven antimicrobials among fecal Escherichia coli of swine on thirty-four farrow-to-finish farms in Ontario, Canada

Fecal specimens were composited and a hydrophobic-grid membrane-filter method was used to measure antimicrobial resistance to ampicillin 16 μg/ml, carbadox 30 μg/ml, gentamicin 4 μ/ml, nitrofurantoin 32 μg/ml, spectinomycin 16 μg/ml, sulfisoxazole 32 μg/ml and tetracycline 8 μg/ml among 8119 Escherichia coli isolates from 68 fecal samples collected on 34 farrow-to-finish swine farms marketing over 500 hogs/yr. The overall prevalences of resistance to antimicrobials among these isolates were: ampicillin 29%, carbadox 3.5%, gentamicin 0.6%, nitrofurantoin 27%, spectinomycin 28%, sulfasoxizole 38% and tetracycline 71%. Thirty to seventy-six per cent of the variations in prevalences were explained by between-farm differences.     

Effects of crossbreed pregnancies on the abortion risk of Neospora caninum-infected dairy cows

Previous studies have shown that the use of beef bull semen significantly reduces the rate of abortions due to Neospora caninum in artificially inseminated (AI) seropositive dairy cows. In addition, certain beef breeds could be more resistant to N. caninum infection and abortion than others. The aim of the present study was to determine whether different crossbreed pregnancies, those derived from Limousin, Charolais, Piedmontese or Belgian Blue semen, carry different risks of abortion in Neospora-infected dairy cows. The effects of possible interactions between maternal levels of N. caninum antibodies and the different breed crosses were also evaluated. The study was performed on five commercial Holstein–Friesian dairy herds in Northeast Spain with previously confirmed diagnoses of N. caninum infection in aborted foetuses. The study population was comprised of 1115 pregnancies: 633 pregnancies recorded after AI using Holstein–Friesian semen from 18 bulls and 482 after AI using beef semen from 27 bulls (304 inseminations using semen from Limousin bulls, 191 from Belgian Blue bulls, 89 from Piedmontese bulls and 49 from Charolais bulls). Abortion rates were 32.2% (155/482) and 15.2% (96/633) for seropositive cows inseminated with Holstein–Friesian and beef breed semen, respectively. Logistic regression analysis revealed the herd and the interaction between maternal N. caninum antibody titre and the different crossbreeds as significant factors affecting the abortion rate. Lowest abortion rates, similar to that shown by seronegative animals in the analysed herds (3.2%, 239/7432), were observed in dams AI using Limousin semen that had low (<30 relative index (RI) units) N. caninum antibody titres (2.1% abortion, 3/145) and these cows were used as reference. Compared to the cows used as reference, cows with low N. caninum antibody titres (<30 RI units) showed a similar risk of abortion when inseminated with Piedmontese or Charolais bull semen, but higher risk of abortion when inseminated with Holstein (17.9 times) or Belgian Blue (7.2 times) bull semen. All cows with high N. caninum antibody titres (≥30 RI units) had a higher risk of abortion, ranging from 8.9 times (cows inseminated with Limousine semen) to 37.8 times (cows inseminated with Piedmontese semen), compared to the cows used as reference. In conclusion, different crossbreed pregnancies carried different abortion risks in Neospora-infected dairy cows. The use of beef bull semen dramatically reduced the risk of abortion in dairy cows, especially if Limousin breed semen was used. Moreover, this reduction was found to be dependent on the N. caninum antibody titre such that the lowest incidence of abortions was recorded in Limousin semen inseminated cows with low antibody titres. Insemination of Neospora-seropositive cows with beef bull semen could both reduce the risk of abortion and avoid breeding replacements for infected cattle.     

Evaluation of a serum-based PCR assay for the diagnosis of canine monocytic ehrlichiosis

The aim of this study was to estimate the relative diagnostic sensitivity and specificity of a polymerase chain reaction (PCR) assay in the serum of dogs with naturally occurring non-myelosuppressive canine monocytic ehrlichiosis (CME), and to investigate the association between PCR positivity and immunofluorescence antibody (IFA) titres for Ehrlichia canis. Serum samples obtained from 38 dogs with non-myelosuppressive CME and 12 healthy dogs were analyzed retrospectively. Each serum sample was analyzed in triplicate using an E. canis-specific nested PCR assay targeting a 389 bp sequence of the 16S rRNA gene. E. canis DNA was amplified in 24 of 38 (63.1%) affected dogs; all samples from healthy dogs were negative. A high level of agreement was found among the PCR replicates (P < 0.0001). Median IFA titre of the 24 PCR-positive dogs was significantly lower than that of the PCR-negative infected dogs (P = 0.0029), indicating that E. canis DNA may circulate prior to the development of a high antibody titre. Serum-based PCR analysis is suggested for the early diagnosis of CME when whole blood samples are not available.     

<Emphasis Type="Italic">Theileria</Emphasis> (<Emphasis Type="Italic">Babesia</Emphasis>) <Emphasis Type="Italic">equi</Emphasis> and <Emphasis Type="Italic">Babesia caballi</Emphasis> Infections in Horses in Galicia,Spain

The control of equine piroplasmosis is becoming increasingly important to maintain the international market open to the horse industry. The purpose of this study was to demonstrate the occurrence of equine piroplasmosis (Theileria equi and Babesia caballi) in Galicia, north-west Spain, and to compare haematological and serum biochemistry parameters between non-parasitaemic horses and horses parasitaemic with T. equi and B. caballi. Sixty serum samples (control group) were taken from healthy horses pastured on two farms, and examined for evidence of equine T. equi and B. caballi infection by indirect fluorescent antibody test (IFAT). Of the 60 samples, 24 (40%) and 17 (28.3%) samples were positive for T. equi and B. caballi, respectively. Twelve (20%) samples were positive for both parasites. Haematology and serum biochemistry were compared between controls and a series of 36 horses clinically affected by T. equi (25) or B. caballi (11). Compared with the healthy group, there was a 43% and 37% decrease in the haematocrit for T. equi and B. caballi infection, respectively. Parasitaemic horses presented an intense anaemia and serum biochemistry signs of liver damage. The anaemia was more severe in T. equi-infected than in B. caballi-infected horses. Our results suggest that equine piroplasmosis is widespread in the region and is a cause for concern.     

Studies on Cellulitis and Other Disease Syndromes Caused by Escherichia coli in Broilers in Sri Lanka

Cellulitis caused by Escherichia coli in broilers results in substantial losses to the broiler industry in North America and Europe due to condemnations at slaughter. The objective of this study was to identify cellulitis in broilers in Sri Lanka and to characterize the E. coli from cellulitis and other colibacillosis lesions. Twenty-four farms from the low- and mid-country were selected and bacterial isolations were obtained from 241 birds. Two hundred and ninety-one gross lesions were observed in these 241 birds and 162 E. coli isolates were obtained. Cellulitis was observed in 21% of the birds. Twenty-one per cent of the birds had multiple lesions due to E. coli. The frequency of detection of other disease syndromes was 162 (67%) birds with pericarditis, 26 (11%) airsacculitis, 24 (10%) hepatitis, 12 (5%) perihepatitis, and 16 (7%) polyserositis (a combination of pericarditis, perihepatitis and airsacculitis). Serogroups O78, O2, O85 and O88 were distributed among the 32% of typable E. coli and 81% of isolates were assigned to three biotypes. Forty-four per cent of the E. coli isolates produced aerobactin and 88% demonstrated resistance to the bactericidal effect of normal chicken serum. The majority of the E. coli isolates were resistant to the antibiotics commonly used in poultry. All the E. coli isolates were non-haemolytic and 25% of the isolates produced K1 capsule. This study demonstrated the presence of cellulitis in Sri Lanka and this report describes some of the phenotypic characteristics of the E. coli isolates.     

Low levels of antibacterial drug resistance expressed by Gram-negative bacteria isolated from poultry carcasses in New Zealand

Abstract

AIM: To provide baseline data on the levels and patterns of antibacterial drug resistance expressed by Gram-negative bacteria isolated from poultry carcasses in New Zealand.

METHODS: Between July and December 2006, isolates of Escherichia coli (n=407) and Salmonella spp. (n=3) originating from carcass-rinse samples were submitted by testing laboratories affiliated to five major poultry processing plants. Isolates of Campylobacter jejuni (n=193) originating from retail poultry carcasses in 2005–2006 were retrieved from the Massey University archives. All isolates underwent disc diffusion susceptibility testing against panels of 12 (Enterobacteriaceae) and six (Campylobacter spp.) antibacterial drugs. Cephalothin-resistance in isolates of E. coli was confirmed using ETest strips, and confirmation of the resistance phenotypes for a subset of C. jejuni isolates used microbroth dilution assays. Patterns within the resistance phenotypes of the isolates were investigated using hierarchical clustering, and logistic regression modelling.

RESULTS: The majority of isolates (71.5% E. coli, 99% C. jejuni, and all three Salmonella spp. isolates) were fully susceptible to the drugs that were tested. Four (1%) E. coli isolates showed resistance to three or more drugs. The proportions of susceptible E. coli differed between the five processing plants. Resistances were detected in E. coli isolates, using disc diffusion to cephalothin (18.2%), ampicillin (4.4%), tetracycline (4.4%) and gentamicin (1.5%). There was an association between cephalothin-resistant isolates of E. coli and decreased susceptibility to gentamicin. Using ETests to ascertain the minimum inhibitory concentrations (MIC) of E. coli for cephalothin gave inconsistent results. One of 193 C. jejuni isolates was resistant to erythromycin, and microbroth dilution assays confirmed that this panel of C. jejuni was generally susceptible to antibacterial drugs.

CONCLUSIONS: The levels of resistance shown by Gram-negative bacteria isolated from chicken carcasses in New Zealand are among the lowest reported around the world. No resistance to extended-spectrum cephalosporin drugs was detected in E. coli, suggesting that CTX-M and AmpC beta-lactamases are rare or absent. Salmonella spp. are rarely isolated from poultry carcasses during routine testing in New Zealand, and the isolates identified during this study were fully susceptible to the drugs tested. A panel of C. jejuni isolates originating from retail poultry carcasses were susceptible to first-line and second-line antibacterial drugs. The use of cephalothin as a marker of resistance to first-generation cephalosporins may not be appropriate for non-type-specific E. coli of animal origin.     

Elution of metronidazole and gentamicin from polymethylmethacrylate beads

OBJECTIVE: To characterize the elution and bioactivity of metronidazole and gentamicin sulfate polymerized, individually and in combination, with polymethylmethacrylate (PMMA). STUDY DESIGN: In vitro experimental study. METHODS: PMMA beads containing metronidazole (3 concentrations), gentamicin sulfate, or metronidazole and gentamicin sulfate were immersed in 5 mL of phosphate-buffered saline in triplicate. Eluent was replaced at specified time intervals for 1 or 21 days, and antibiotic concentrations were measured by high-performance liquid chromatography. Changes in antibiotic bioactivity attributable to polymerization or copolymerization of the antibiotics with PMMA, ethylene oxide sterilization, and storage of AIPMMA beads containing metronidazole were evaluated. RESULTS: Antibiotic elution patterns were similar for all groups. Day 1 elution for groups containing metronidazole or gentamicin individually represented a mean 63%-66% and 79%, respectively, of the 21-day total. Approximately 50% of the day 1 elution occurred during the first hour. The elution of metronidazole was dose dependent. The elution of metronidazole (day 3-21) and gentamicin (all days) was significantly greater when metronidazole and gentamicin were combined (P <.05). The addition of metronidazole delayed polymerization of PMMA. Neither polymerization nor copolymerization of metronidazole and gentamicin with PMMA, gas sterilization, or 2-month storage of beads containing metronidazole significantly affected antimicrobial bioactivity. CONCLUSIONS: Metronidazole elution from PMMA was dose dependent. Copolymerization of metronidazole and gentamicin sulfate in PMMA resulted in increased rates of elution. Intraoperative preparation of metronidazole-impregnated PMMA beads is not practical, but sterilization and storage for 2 months should not affect efficacy. CLINICAL RELEVANCE: The local delivery of biologically active metronidazole and gentamicin by elution from PMMA is feasible.     

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from Mossoró, Rio Grande do Norte, Brazil

Toxoplasma gondii is an obligate intracellular parasite with a variety of hosts, responsible for reproductive problems and economic losses in sheep flocks. Neospora caninum was recently identified and its clinical presentation in sheep is similar to that of toxoplasmosis, which can cause repeated abortions, though less frequently in this species. In order to confirm the prevalence of these agents in the city of Mossoró, Rio Grande do Norte, Brazil, 409 serum samples from adult sheep (364 females and 45 males) were tested by the indirect immunofluorescence antibody test, using cut-off point at a dilution of 1:64 and 1:50 for T. gondii and N. caninum, respectively. From the 35 properties examined, 23 (65.7%) had at least one seropositive animal for T. gondii and six (17.1%) for N. caninum. The prevalence of seropositive animals for T. gondii was 20.7% and for N. caninum 1.8%. There was no association between the presence of the agent’s antibody and gender, reports of reproductive problems and presence of dogs and/or cats in the properties. T. gondii is well distributed and N. caninum has low prevalence in sheep and in the properties of the studied region.     

Agglutination of Brucella abortus cells by sera from cattle experimentally infected with Escherichia coli

Infection of normal adult cattle and sporadic serological Brucella abortus reactors, which no longer had diagnostic titers to B. abortus, with a culture of Escherichia coli isolated from a cow (or its environment) resulted in the production of a primary IgM serum antibody response to both B. abortus and E. coli. Cattle injected with heat killed E. coli and guinea pigs or young calves lacking natural agglutinins to B. abortus injected with live E. coli, produced serum antibody only to E. coli. The subsequent reinjections of live E. coli into the former two groups of cattle resulted in all but one animal in each group producing IgM ‘secondary’ responses to B. abortus of decreasing magnitude, while the anti-E. coli responses increased and eventually switched to synthesis of IgG class antibody. The remaining two animals produced a series of ‘secondary’ responses of IgM antibody to B. abortus similar in amplitude and duration to the primary immune response. The anti-E. coli agglutinins of these two animals increased in titer and IgG class antibody to E. coli was evident after several injections of E. coli. These results indicate that infection of cattle by E. coli can cause a problem in the serological fiagnosis of B. abortus infection. Speculations on the cause of this cross-reaction and ways of minimizing misdiagnosis are discussed.     

A Single-chain Fragment Variable Recombinant Antibody against F5 Fimbria of Enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> Inhibits Agglutination of Horse Red Blood Cells Induced by F5 Protein

Bovine colibacillosis caused by enterotoxigenic Escherichia coli (ETEC) is a worldwide problem. Adhesion of ETEC to intestinal cell receptors mediated by the surface protein F5 fimbriae is the initial step in the establishment of colibacillosis. Prevention of ETEC F5⁺ adhesion to enterocytes protects newborn calves against collibacillosis. On the enterocytes, the F5 fimbriae bind to a ganglioside that is also found on horse red blood cells. Thus, the presence of F5 fimbriae induces haemagglutination, which is useful as an indicator in a functional assay system. In this study, recombinant anti-F5 scFv antibody fragment produced in E. coli HB2151 reacted with F5 fimbriae in ELISA and Western immunoblot, and prevented haemagglutination induced by the binding of the F5 fimbriae to its natural host receptors on horse red blood cells. Given the ease with which recombinant antibodies can be mass-produced, the presently described scFv may hold promise as a prophylactic agent for colibacillosis.     

Development and evaluation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against Campylobacter fetus in cattle

Campylobacteriosis is a zoonosis that occurs worldwide. Infection with Campylobacter fetus (C. fetus) causes infertility and abortion in sheep and cattle. The current study focuses on the SapA gene of C. fetus that encodes surface array proteins and plays an important role in the virulence of C. fetus. The SapA-N (1398 bp) and SapA-C (1422 bp) fragments were amplified from the C. fetusSapA gene using polymerase chain reaction (PCR), and the corresponding recombinant proteins rSapA-N and rSapA-C were expressed in Escherichia. coli BL21 cells. Results of Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the immunological activity of rSapA-N was higher than that of rSapA-C (P < 0.05). Therefore, rSapA-N was selected to establish an indirect ELISA for detecting antibodies against C. fetus. The diagnostic criteria were as follows: S/P ? 0.45: positive; S/P < 0.4: negative; 0.45 > S/P ? 0.4: suspected. The specificity and sensitivity of our method were 94.3% and 88.6%, respectively. Moreover, no cross-reactions were observed between rSapA-N and serum samples that were positive for other bovine bacterial pathogens diseases such as Mycobacterium avium subspecies paratuberculosis. One hundred and two serum samples from cows that had experienced abortion were tested. Four and 2 C. fetus-positive serum samples were found among the 70 bovine brucellosis-positive samples and the 32 infectious bovine rhinotracheitis (IBR)-positive samples, respectively. The findings suggest that the rSapA-N-based ELISA method has immense potential in future applications.