Polymyxin B: pharmacokinetics of single doses given intravenously and intramuscularly to turkeys, and minimal inhibitory concentrations for Escherichia coli and Pasteurella multocida.

The 50% and 90% minimal inhibitory concentrations (MIC50 and MIC90) of polymyxin B for avian Escherichia coli and Pasteurella multocida isolates were determined by the agar plate dilution method. Polymyxin B at approximate MIC level in serum was bactericidal for E. coli in 2 to 4 hours. Aqueous polymyxin B sulfate was administered by a single bolus intravenous injection into turkeys at 10,000 IU/kg, and by a single bolus intramuscular injection at 5,000, 10,000 or 20,000 IU/kg. Effective serum drug concentrations after intramuscular injection (MIC50 levels or greater) were maintained for E. coli for 7.0 hr (10,000 IU/kg) and 11.5 hr (20,000 IU/kg), and for P. multocida for 3.0 hr (10,000 IU/kg) and 4.1 hr (20,000 IU/kg). Pharmacokinetic parameters were calculated by non-compartmental methods. Elimination time half-lives, mean residence time, clearance, and apparent volume of distribution at steady state (Vdss) were all much higher for i.m. injection of 20,000 IU/kg than for i.m. injection of 10,000 IU/kg. We postulate that there exists a minimal tissue-interaction threshold concentration (MTC) at which polymyxin B can enter previously unavailable compartments or bind to previously refractory tissue components. Bioavailability of polymyxin B injected i.m. was 0.904 for the 10,000 IU/kg dose and 0.675 for the 20,000 IU/kg dose. Dosage intervals necessary to produce minimal steady state concentrations (Cssmin) equal to the MIC were calculated. Certain aspects of the use of the parameter Vdss, and limitations on the use of dosage interval calculations for polymyxin B, are discussed. One week after i.m. injection of polymyxin B at 10,000 IU/kg, high tissue drug levels were present, especially in bound form in liver. Following single injections, no toxic effects on turkeys were observed.     

Efficacy of avian pneumovirus vaccines against an avian pneumovirus/Escherichia coli O2:K1 dual infection in turkeys

The clinical, pathological and microbiological outcome of a challenge with avian pneumovirus (APV) and Escherichia coli O2:K1 was evaluated in turkeys vaccinated with an attenuated APV vaccine and with or without maternally derived antibodies. Two groups of two-week-old poults, one with and one without maternally derived antibodies against APV, were vaccinated oculonasally with attenuated APV subtype A or B. A third group remained unvaccinated. Eleven weeks later, the turkeys were inoculated intranasally with either virulent APV subtype A, or E. coli O2:K1, or with both agents three days apart. After the dual infection, birds vaccinated with attenuated subtype A or B, and with or without maternally derived antibodies, had lower mean clinical scores than the unvaccinated birds. In the vaccinated birds, virus replication was significantly reduced and no bacteria were isolated, except from the birds vaccinated with attenuated subtype B. In the unvaccinated turkeys, large numbers of E. coli O2:K1 were isolated from the turbinates of the dually infected birds between one-and-a-half and seven days after they were inoculated.     

Experimental infection of turkeys with avian pneumovirus and either Newcastle disease virus or Escherichia coli

Avian pneumoviruses (APVs) are RNA viruses responsible for upper respiratory disease in poultry. Experimental infections are typically less severe than those observed in field cases. Previous studies with APV and Escherichia coli suggest this discrepancy is due to secondary agents. Field observations indicate APV infections are more severe with concurrent infection by Newcastle disease virus (NDV). In the current study, we examined the role of lentogenic NDV in the APV disease process. Two-week-old commercial turkey poults were infected with the Colorado strain of APV. Three days later, these poults received an additional inoculation of either NDV or E. coli. Dual infection of APV with either NDV or E. coli resulted in increased morbidity rates, with poults receiving APV/NDV having the highest morbidity rates and displaying lesions of swollen infraorbital sinuses. These lesions were not present in the single APV, NDV, or E coli groups. These results demonstrate that coinfection with APV and NDV can result in clinical signs and lesions similar to those in field outbreaks of APV.     

猪肌注硫酸安普霉素的血清浓度测定

给６头健康猪单剂量肌肉注射国产硫酸安普霉素（２０ｍｇ／ｋｇ）,采取血样,用微生物法测定硫酸安普霉素的血清药物浓度。结果平均回收率为９９．０３％,血清是低检测浓度为０．１μｇ／ｍｌ,日内、日间变异系数为２．２％～５．１％,血清药物浓度在０．１～３．０μｇ／ｍｌ范围呈良好线性关系（ｒ＝０．９９６５）,用此法对６头猪肌注硫酸安普霉素的血清药物浓度测定,得出药时,曲线数据。     

Immune responses associated with homologous protection conferred by commercial vaccines for control of avian pathogenic Escherichia coli in turkeys

Avian pathogenic Escherichia coli (APEC) infections are a serious impediment to sustainable poultry production worldwide. Licensed vaccines are available, but the immunological basis of protection is ill-defined and a need exists to extend cross-serotype efficacy. Here, we analysed innate and adaptive responses induced by commercial vaccines in turkeys. Both a live-attenuated APEC O78 ΔaroA vaccine (Poulvac® E. coli) and a formalin-inactivated APEC O78 bacterin conferred significant protection against homologous intra-airsac challenge in a model of acute colibacillosis. Analysis of expression levels of signature cytokine mRNAs indicated that both vaccines induced a predominantly Th2 response in the spleen. Both vaccines resulted in increased levels of serum O78-specific IgY detected by ELISA and significant splenocyte recall responses to soluble APEC antigens at post-vaccination and post-challenge periods. Supplementing a non-adjuvanted inactivated vaccine with Th2-biasing (Titermax® Gold or aluminium hydroxide) or Th1-biasing (CASAC or CpG motifs) adjuvants, suggested that Th2-biasing adjuvants may give more protection. However, all adjuvants tested augmented humoral responses and protection relative to controls. Our data highlight the importance of both cell-mediated and antibody responses in APEC vaccine-mediated protection toward the control of a key avian endemic disease.     

Colonization of Escherichia coli in young turkeys and chickens.

In order to investigate the possibility of pathogenic Escherichia coli penetrating the bloodstream via the intestinal mucosa in normal and stressed turkeys and chickens, birds were inoculated orally with the bacteria or exposed environmentally to it. Immediately after hatch, intestines contained a substantial number of coliform bacteria that increased with time. In orally infected turkeys, the pathogenic bacteria (nalidixic-acid-resistant O78) replaced 10%-50% of the native coliform flora but could not be isolated from the trachea or blood. Environmentally exposed groups exhibited pathogenic bacteria in intestines but not in blood. Stressing of exposed turkeys resulted in isolation of the pathogenic bacteria from blood and even spleen. In orally infected broiler chickens, stress resulted in bacteremia and mortality. Chickens that were exposed to pathogenic bacteria at a young age and showed no mortality or morbidity demonstrated no detrimental effects due to challenge with the same pathogenic bacteria later in life. Stress seems to cause penetration of the pathogenic bacteria into the bloodstream, which in turn can cause severe disease and mortality.     

Studies on the use of a long-acting oxytetracycline in turkeys: serum levels and tissue residues following injection

Forty 6-week-old large white commercial turkeys were injected subcutaneously with a long-acting oxytetracycline formulation (69 mg/lb). The turkeys were divided into four groups of 10 birds each, and the birds in each group were bled twice at different times between 4 and 144 hours postinjection (PI) to determine serum levels of oxytetracycline. Two additional groups of turkeys were also given the long-acting oxytetracycline formulation mixed with either neomycin or a bacterin for Pasteurella multocida to determine if either of these compounds interfered with absorption of the oxytetracycline. Serum levels of oxytetracycline were 5.38 micrograms/ml, 1.59 microgram/ml, and 0.93 microgram/ml at 24, 48, and 72 hours PI, respectively, following an average dose of 69 mg/lb of body weight. These levels are all considered therapeutic. There appeared to be no interference with absorption of oxytetracycline when mixed with either neomycin or the bacterin. Tissue residues of oxytetracycline in the muscle, liver, and kidney were within tolerance levels by 3 weeks PI.     

Steroid levels after intramuscular injection of testosterone propionate in the caprine.

Nine sexually mature intact grade does were injected intramuscularly with testosterone propionate and subsequent plasma steroid concentrations determined and male-like behavior recorded. The does received either 100 mg testosterone propionate every three days for six treatments, total dose 600 mg (N = 5); 50 mg testosterone propionate daily for eighteen days, total dose 900 mg (N = 2) or 10 mg testosterone propionate daily for eighteen days, total dose 180 mg (N = 2). The treatments induced male-like sex behavior, the intensity of which was related to the dose of exogenous testosterone used, the regimen of administration, and the plasma levels of testosterone. Exogenous testosterone treatment had minimal effect on the subsequent reproductive activity of the does.     

Evaluation of differentially expressed proteins following serum exposure in avian pathogenic Escherichia coli

Colibacillosis, caused by avian pathogenic Escherichia coli (APEC), is an extraintestinal disease that causes great economic loss to the poultry industry each year. APEC must overcome host defenses, such as immune system components found in serum, in order to establish infection; however, the mechanism of such serum resistance has been elusive. In the present study, a proteomic approach was used to evaluate APEC proteins that were differentially expressed after exposure to chicken serum to identify specific proteins that may be involved in serum resistance of APEC isolates. Proteins were isolated and separated by two-dimensional (2D) gel electrophoresis, and 10 protein spots corresponding to differentially expressed proteins were chosen for sequencing using electrospray ionization tandem mass spectrometry. Eight proteins were identified among the spots, some of which have previously been associated with the virulence of E. coli. Significantly, an outer-membrane protein previously associated with serum resistance, OmpA, was among those proteins identified, further indicating that differential regulation of this protein may be involved in serum resistance. This study opens the door to future research using a proteomic approach to identify the key players in serum resistance of APEC.     

The clinical, pathological and microbiological outcome of an Escherichia coli O2:K1 infection in avian pneumovirus infected turkeys

The purpose of this study was to evaluate the effect of an Escherichia coli infection in avian pneumovirus (APV)-infected turkeys. One group of 2-week-old specific pathogen-free (SPF) and two groups of 3-week-old conventional (CON) turkeys were inoculated oculonasally with virulent APV subtype A alone, with E. coli O2:K1 alone or with both agents at varying intervals (1, 3, 5 or 7 days) between the two inoculations. The birds were followed clinically and examined for macroscopic lesions at necropsy. Titres of APV were determined in the turbinates, trachea, lungs and air sacs. The number of E. coli O2:K1were assessed in the turbinates, trachea, lungs, air sacs, liver and heart. In both SPF and CON turkeys, dual infection resulted in an increased morbidity and a higher incidence of gross lesions compared to the groups given single infections, especially with a time interval between APV and E. coli inoculations of 3 and 5 days. APV was isolated from the respiratory tract of all APV-infected groups between 3 and 7 days post inoculation. E. coli O2:K1 was isolated only from turkeys that received a dual infection. It was recovered from the turbinates, trachea, lungs, heart and liver. These results show that APV may act as a primary agent predisposing to E. coli colonization and invasion.     

TonB is essential for virulence in avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) strains have multiple iron-uptake systems that facilitate adaptation to iron-restricted environments and are believed to assist in colonisation of the host. These systems include several TonB-dependent transporters of ferri-siderophores encoded by the chromosome and the large virulence plasmid common to APECs. The tonB gene of the virulent APEC strain E956 was replaced with a selectable antibiotic resistance marker using Lambda Red recombinase mutagenesis. The phenotype of the ΔtonB E956 mutant was compared to the parent strain under various culture conditions and in chickens experimentally infected via the respiratory route. The mutant was resistant to streptonigrin, impaired in its ability to adapt to growth in iron-depleted medium and had greater tolerance of oxidative stress than the parental strain. The mutant was avirulent in chickens, did not affect the growth of chicks and colonisation was mostly limited to the trachea. This study has demonstrated that TonB is essential for virulence in APEC.     

Analysis of immune responses induced by avian pathogenic Escherichia coli infection in turkeys and their association with resistance to homologous re-challenge

Avian pathogenic Escherichia coli (APEC) cause severe respiratory and systemic disease in poultry yet the nature and consequences of host immune responses to infection are poorly understood. Here, we describe a turkey sub-acute respiratory challenge model and cytokine, cell-mediated and humoral responses associated with protection against homologous re-challenge. Intra-airsac inoculation of turkeys with 10⁵ colony-forming units of APEC O78:H9 strain χ7122nal^R induced transient and mild clinical signs of colibacillosis followed by clearance of the bacteria from the lungs and visceral organs. Upon re-challenge with 10⁷ χ7122nal^R, primed birds were solidly protected against clinical signs and exhibited negligible bacterial loads in visceral organs, whereas age-matched control birds exhibited high lesion scores and bacterial loads in the organs. Levels of mRNA for signature cytokines suggested induction of a Th1 response in the lung, whereas a distinct anti-inflammatory cytokine profile was detected in the liver. Proliferative responses of splenocytes to either Concanavalin A or soluble χ7122nal^R antigens were negligible prior to clearance of bacteria, but APEC-specific responses were significantly elevated at later time intervals and at re-challenge relative to control birds. Primary infection also induced significantly elevated χ7122nal^R-specific serum IgY and bile IgA responses which were bactericidal against χ7122nal^R and an isogenic Δrfb mutant. Bactericidal activity was observed in the presence of immune, but not heat-inactivated immune serum, indicating that the antibodies can fix complement and are not directed solely at the lipopolysaccharide O-antigen. Such data inform the rational design of strategies to control a recalcitrant endemic disease of poultry.     

Tissue and serum concentrations of amikacin after intramuscular and intrauterine administration to mares in estrus.

Concentrations of amikacin in endometrial tissue and plasma were studied in mares in estrus after intrauterine infusion of 1.0 or 2.0 g once a day for 3 consecutive d, and after 9.7 or 14.5 mg/kg body weight (BW) had been injected intramuscularly once a day for 3 consecutive d to determine concentrations of amikacin sulfate in plasma and endometrial tissues, and whether parenteral administration provides any advantages over intramuscular infusion. No amikacin was detected in serum at the 1.0 g dose. At the infusion dose of 2.0 g once a day, very low levels of serum amikacin were detected at 1 and 4 h postinfusion on the 1st treatment day. Amikacin was found to penetrate the endometrium after intramuscular injection; however, the levels attained were not as high as those achieved following intrauterine infusion. Based on the tissue and serum concentrations of amikacin, an intrauterine infusion at a dose of 4.4 mg/kg BW/d would appear to be an appropriate therapeutic regimen for the treatment of gram-negative endometritis.     

Virulence factors of Escherichia coli associated with colisepticemia in chickens and turkeys.

Four hundred twenty turkey and 80 chicken Escherichia coli isolates from colisepticemic birds were examined for the following properties: heat-labile toxin (LT), heat-stable enterotoxin, verotoxin, colicinogenicity, hemolysin, and hydroxamate/aerobactin production. Twenty-four (5.7%) of the 420 turkey isolates and six (7.5%) of the 80 chicken isolates produced an LT that was cytotoxic for both Vero and Y-1 cells. In contrast, 48 (11.4%) of the turkey isolates and 18 (22.5%) of the chicken isolates produced a distinct LT that was cytotoxic only for Vero cells. In addition, 64 (80.0%) of the chicken isolates and 309 (74.0%) of the turkey isolates produced aerobactin. Colicinogenicity occurred in 51 (64.0%) of the chicken isolates, with 41 (51.0%) producing colicin V. By contrast, 254 (61.0%) of the turkey isolates produced a colicin, of which 176 (42.0%) produced colicin V. None of the chicken and turkey isolates produced hemolysin or heat-stable enterotoxin.     

Gentamicin sulfate in the horse: serum, synovial, peritoneal, and urine concentrations after single dose intramuscular administration

Ten healthy adult mares were given a single intramuscular dose (2.2 mg/kg) of gentamicin sulfate. Over a 48-h period, gentamicin concentrations were measured serially in the serum of all ten mares and in synovial fluid, peritoneal fluid, and urine of six of the mares. The mean peak serum gentamicin concentration was 5.73 μg/ml at 1 h. Gentamicin was detected in synovial fluid and peritoneal fluid, with mean peak gentamicin concentrations of 2.41 μg/ml and 3.92 μg/ml, respectively, observed at 2 h. These concentrations declined in parallel with serum concentrations and were not measurable at 48 h. Urine gentamicin concentration was relatively high, with a mean peak concentration of 424.9 μg/ml at 1 h after drug administration.     

Characteristics of ciprofloxacin and cephalosporin resistant Escherichia coli isolated from turkeys in Great Britain

1. A field study was performed to investigate the presence and characteristics of ciprofloxacin-resistant, extended spectrum β-lactamase (ESBL) and AmpC Escherichia coli from turkeys in Great Britain. E. coli were isolated from ～9000 boot swab samples from 27 different farms owned by four different companies. Between 1 and 14 visits were made to each farm (mean 3) at between 0 and 15?m intervals (mean?～?5?m).

2. CHROMagar ECC with and without ciprofloxacin or cephalosporin antibiotics was used as selective isolation media. Representative isolates with different phenotypes were tested for mutations in gyrA and for: qnrA, B, S, qepA and aac(6′)-Ib genes, for ESBL phenotype, the presence of bla _CTX-M genes and plasmid type, and for ampC genes_. Representative ciprofloxacin-resistant and CTX-M isolates were further tested for serotype and PFGE type. On ciprofloxacin selective media 55% of samples yielded ciprofloxacin resistant E. coli and of those further analysed, most had ciprofloxacin MICs >4 mg/l and mutations in gyrA.

3. For the different companies, the mean number of samples per farm with cefoxitin- or cefotaxime-resistant isolates ranged from 1·0% to 61·9% and 4·7% to 31·7% respectively. Cefotaxime-resistance was most commonly associated with an ESBL phenotype, a CTX-M-1 or CTX-M-14 sequence type and an I1-γ or K plasmid inc type. The mechanism of cefoxitin resistance was not determined for most isolates, but where determined it was bla _CMY-2.

4. PFGE and serotyping showed clonally-related isolates persisting over multiple visits suggesting both more prudent use of antibiotics and improved farm hygiene are needed to address the issue of antimicrobial resistance in isolates from turkeys.