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Abstract

  1. Top of page
  2. Abstract
  3. Acknowledgments
  4. References

A Mycoplasma gallisepticum–Escherichia coli mixed infection model was developed in broiler chickens, which was applied to pharmacokinetics of valnemulin in the present experiment. The velogenic M. gallisepticum standard strain S6 was rejuvenated to establish the animal model, and the wild E. coli strain O78 was injected as supplementary inoculum to induce chronic respiratory disease in chickens. The disease model was evaluated based on its clinical signs, histopathological examination, bacteriological assay, and serum plate agglutination test. The pharmacokinetics of valnemulin in infected chickens was determined by intramuscular (i.m.) injection and oral administration (per os, p.o.) of a single dose of 10 mg/kg body weight (BW). Plasma samples were analyzed by liquid chromatography–tandem mass spectrometry. The plasma concentration–time curve of valnemulin was analyzed using the noncompartmental method. After the i.m. administration, the mean values of Cmax, Tmax, AUClast, MRT, CLβ/F, Vz/F, and t1⁄2β, were 27.94 μg/mL, 1.57 h, 171.63 μg·h/mL, 4.51 h, 0.06 L/h/kg, 0.56 L/kg, and 6.50 h, respectively. By contrast, the corresponding values after p.o. administration were 5.93 μg/mL, 7.14 h, 47.60 μg·h/mL, 9.80 h, 0.22 L/h/kg, 3.35 L/kg, and 10.60 h. The disposition of valnemulin was retarded in infected chickens after both modes of extravascular administration as compared to the healthy controls. More attention should be given to monitoring the therapeutic efficacy and adverse effects of mixed infection because of higher required plasma drug concentration and enlarged AUC with valnemulin treatment.

Mycoplasma gallisepticum is a simple prokaryote that functions as the major pathogen of chronic respiratory disease (CRD; Levisohn & Kleven, 2000) in fowl. The disease is prevalent in commercial poultry farms with weak health control measures, CRD retards growth, decreases the laying rate, and lowers the feed conversion ratio (Glisson et al., 1989; Machado et al., 2012). The disease is difficult to eradicate because of its vertical transmission through the eggs (Lin & Kleven, 1982). The possible economic losses are aggravated when CRD is combined with Escherichia coli infection (Mohammed et al., 1987). The cardinal signs of the M. gallisepticum–E. coli mixed infection are cough, sneezing, runny noses, tracheal rattle, and tracheitis (Stipkovits & Kempf, 1996). To date, the infection is mainly controlled by biological isolation, immunization, and drug treatment. Among these treatments, pharmacotherapy plays a significant role in treating M. gallisepticum infection. Valnemulin is a prospective drug, as an alternative to enrofloxacin, tilmicosin, and tylosin (Forrester et al., 2011).

Valnemulin is a semisynthetic pleuromutilin antibiotic derivative that exhibits antibacterial activity by binding to the 50s ribosomal subunit of prokaryotes and inhibiting protein synthesis. The antibacterial efficacy of valnemulin against M. gallisepticum has been investigated in vitro and in vivo (Jordan et al., 1998).

The pharmacokinetics of valnemulin in healthy broiler chickens, Muscovy ducks, and pigs was studied in our previous reports (Wang et al., 2011; Zhang et al., 2011; Sun et al., 2012). However, the absorption, distribution, and elimination of valnemulin in diseased animals may be distinguished from that of healthy controls. Further study was necessary to obtain more clinically significant pharmacokinetic data. In the present study, a M. gallisepticum and E. coli mixed infection model was established. The pharmacokinetics of valnemulin in the infected chickens was determined by the intramuscular (i.m.) injection and oral administration (per os, p.o.) of a single dose of 10 mg/kg body weight (BW).

A total of 50 Sanhuang broiler chickens weighing 1.5–2.1 kg were supplied by the Guangdong Academy of Agricultural Sciences (Guangzhou, China). The birds were raised under controlled conditions at 25 °C as required, fed with antibacterial-free balanced feedstuff ad libitum, and provided access to clean water. The Frey medium, agar, cysteine, and nicotinamide adenine dinucleotide (NADH) were provided by Seabo Biological Technology Co., Ltd (Guangdong, China). The standard M. gallisepticum strain S6, the antigens, and positive/negative sera were purchased from the Chinese Veterinary Microorganism Culture Collection Center (Beijing, China). The wild E. coli strain O78 was previously isolated by our laboratory. Valnemulin hydrochloride (>99%) was provided by the Guangdong Dahuanong Animal Health Products Co., Ltd (Guangdong, China). The acetonitrile (Fisher, Fair Lawn, NJ, USA) used was of high-performance liquid chromatography grade. The remaining analytical-grade reagents were purchased from the Guangzhou Chemical Reagent Factory (Guangzhou, China). Penicillin, thallium acetate, 10% inactivated swine serum, 0.2% cysteine, and 0.2% NADH were added to the Frey medium; the said medium was used for the isolation, subculture, and inoculation of M. gallisepticum (Frey et al., 1968). The solid medium was prepared by adding 1% agar. The E. coli O78 strain was cultured in LB broth. All the reagents were stored at 4 °C and used within 1 month.

Blood samples were collected from the chickens by puncturing the brachial vein; these were stored in tubes without the anticoagulant. The sera were used for the serum plate agglutination (SPA) test. The SPA-negative chickens were selected for the subsequent experiments. During the test period, the health status of the chickens was closely monitored by daily physical examination.

To rejuvenate the M. gallisepticum S6 strain, the pathogen was used to inoculate in the chickens and isolated from the infected air sacs. The disease mixed model was created by injecting 2 mL of the S6 culture at approximately 5 × 109 colony-forming units (CFU)/mL into the bilateral air sacs in the thoracic region. Another 1 mL of the inoculum was simultaneously administered using eye and nasal drops. The ammonia concentration should reach 0.5 mg/m3 to facilitate invasion. A 0.15 mL aliquot of the E. coli O78 culture at 1 × 108 CFU/mL was injected into the breast muscle at 3 days after the last pathogen inoculation. The chickens in the control group were treated with normal saline instead of the pathogen cultures.

Serum samples were collected from the chickens at 2, 4, 5, 6, 7, and 8 days after inoculation for the SPA test. Histopathological and bacteriological examinations were likewise performed. Swabs were collected twice from the terminal bronchiole and air sacs and used to inoculate the isolation medium. Subsequently, the pathogen subcultures were cultivated on solid media and incubated at 37 °C with 5% CO2 for 5 days. The medium was monitored daily for 14 days for color changes. Agar plates were identified as positive using an inverted microscope. Identical manipulations were performed on the control group.

A total of 24 infected chickens were randomly equally divided into two groups. Valnemulin hydrochloride powder was dissolved in normal saline to obtain a final concentration of 20 g/L. To inoculate group I, i.m. administration was applied through the chest muscle, whereas p.o. administration was used for group II via a mouth irrigation, using a thin plastic tube linked to a 2.5-mL syringe, followed by rinsing with 5 mL of water. A single dose of 10 mg/kg BW was used for both groups. The chickens exhibited jejunitis at 12 h before and 6 h after administration. Blood samples were collected and stored in heparin at 0.083, 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 16, 24, 36, 48, and 72 h after administration. Plasma samples were collected by centrifuging the blood samples at 2000 g for 10 min. The collected plasma was immediately frozen at −20 °C until further quantitative analysis. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the US National Institutes of Health.

The conditions for sample preparation and evaluation were identical to those of our previous study (Wang et al., 2011). The pharmacokinetic data of valnemulin were analyzed by the noncompartmental method with uniform weighting using the winnonlin software (version 6.1; Pharsight, St. Louis, MO, USA). The parameters apparent volume of distribution (V) and clearance (CL) were analyzed as V/F and CL/F because of their extravascular administration.

The cardinal signs of infection were observed, including depression, mouth breathing, eye closure, and wet rales. Pericarditis, perihepatitis, airsacculitis, and mucous hyperemia were noticeable in the trachea after dissection. The uninfected chickens had negative results in the SPA tests. By contrast, agglutination was detected in more than 80% infected chickens. The isolation rate of M. gallisepticum was higher than 90%, whereas the morbidity rate was 75%. The mortality rate was 15% at 4 days after infection but reached as high as 55% during subsequent sampling. Clinical signs were not observed in the control group, which always tested negative in the SPA tests. All evidence showed that the mixed infection model was successfully established.

Several infection models have been previously reported. M. gallisepticum at approximately 1 × 108 CFU/mL was used to infect laying hens to establish the transmission dynamics of M. gallisepticum and quantify the intervention measures for horizontal transmission (Feberwee et al., 2005). A wild M. gallisepticum strain G87/02 was intranasally inoculated into 14-day-old pheasants to study the pathogenicity of M. gallisepticum and estimate the therapeutic efficacy of tylvalosin against infectious sinusitis (Forrester et al., 2011). Another M. gallisepticum infection model was successfully developed in domestic canaries (Hawley et al., 2011). However, the pathogen in these experiments was solely M. gallisepticum. M. gallisepticum is generally a long-term underlying causative agent in most commercial poultry farms. The pathogen does not cause infection under normal circumstances. By contrast, E. coli usually facilitates the outbreak of CRD in chickens. Therefore, the combined infection of M. gallisepticum and E. coli usually observed in veterinary clinics. In this study, we used M. gallisepticum and E. coli to simulate the common mode of clinical infection. To produce pathopoiesis, the standard strain of M. gallisepticum S6 must be rejuvenated by using it to inoculate chickens and isolating the pathogen from the infected air sacs. M. gallisepticum targets the respiratory system of chickens, especially the air sacs (Gharaibeh & Hailat, 2011). Thus, the S6 was inoculated by direct injection into the bilateral air sacs of the thoracic region and by simultaneous eye and nasal drops. Moderate ammonia concentration in the henhouse can promote the incidence of CRD. The E. coli strain O78 was then supplementarily inoculated at 3 days after M. gallisepticum infection. Chickens infected with both M. gallisepticum and E. coli were selected for the pharmacokinetic study.

A symmetrical peak shape with no interfering substance was obtained, and valnemulin was analyzed by liquid chromatography–tandem mass spectrometry under optimized conditions. The matrix-matched standard curve was linear from 5 to 500 ng/mL with correlation coefficients (r2) >0.99. The recovery rates ranged from 85.5 to 101.0% at the spiked concentrations of 5, 100 and 500 ng/mL. The intra- and interday coefficients of variation were <6.0%.

The semilogarithmic plots of the mean plasma concentration–time curves of valnemulin in infected chickens are shown in Fig. 1. The pharmacokinetic parameters are summarized in Table 1.

Table 1. Pharmacokinetic parameters of valnemulin following i.m. and p.o. administration at a single dose of 10 mg/kg BW in M. gallisepticumE. coli infected chickens (n = 12)
Parametersi.m. (Mean ± SD)p.o. (Mean ± SD)
  1. BW, body weight; i.m., intramuscular; p.o., per os.

Cmax (μg/mL)27.94 ± 6.145.93 ± 2.31
Tmax (h)1.57 ± 0.347.14 ± 7.90
AUClast (μg·h/mL)171.63 ± 39.0447.60 ± 14.11
AUC0–∞ (μg·h/mL)171.76 ± 39.1647.78 ± 14.08
MRT (h)4.51 ± 0.819.80 ± 4.73
CLβ/F (L/h/kg)0.06 ± 0.010.22 ± 0.04
Vz/F (L/kg)0.56 ± 0.163.35 ± 1.21
T1/2β (h)6.50 ± 1.8410.60 ± 3.72
image

Figure 1. Plasma mean concentration-time semilograthimic profile of valnemulin following intramuscular (i.m.) and per os (p.o.) administration at a single dose of 10 mg/kg body weight in Mycoplasma gallisepticumEscherichia coli infected chickens (n = 12).

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To develop the optimal dosage regimen, the absorption, distribution, metabolism, and excretion of drugs in the target organism are monitored. Most research has generally been performed using healthy individuals. However, the disposition of a drug may significantly differ between healthy and infected individuals because of the possible damage to hepatonephric functions during illness. Consequently, the pharmacokinetic study of infected animals may be of greater clinical significance. After i.m. administration, a Cmax of 27.94 ± 6.14 μg/mL was achieved at 1.57 ± 0.34 h in infected chickens, which was more than tenfold that of healthy fowls at an identical dose (Wang et al., 2011). The results of the present study are consistent with previous report (Kirch et al., 1987). The observed Tmax was postponed from 0.43 to 1.57 h. Reduced body clearance and enlarged AUCs were likewise observed in infected birds as compared to healthy ones. The same phenomenon was observed after p.o. administration. Similar results were reported in the pharmacokinetic study of cetirizine, which had a Tmax of 1.09 h in the healthy volunteers and 2.50 h for the patients (Horsmans et al., 1993). The function of the cardiac pump could be weakened by pericarditis, thereby disturbing the microcirculation (Dudzinski, et al. 2012; Humphreys, 2006) and preventing the absorption of valnemulin after i.m. or p.o. administration. Hepatic microsomal enzyme activity could be reduced by perihepatitis (Farrell, 1999). All these pathological factors may prolong the absorption and metabolism of the drug in diseased chickens. Thus, valnemulin could be determined until 72 h in both administration routes. The longer elimination half-life, lower clearance, and lower elimination rate constants contributed to retarding the elimination of midazolam in patients of a previous study (Pentikainen et al., 1989). In the current study, the elimination half-life of infected chickens was similar to that of the healthy controls after the two extravascular administrations. This phenomenon could be attributed to the main excretion pathway of valnemulin, which was through the bile (Horkovics-Kovats & Schatz, 1997). The Vz/F of 0.56 L/kg after i.m. administration was much less than the 3.35 L/kg after p.o. administration because of the retarded distribution in the tissue. The multiple-peak phenomenon was observed in the plot of p.o. administration. The possible enterohepatic recycling may have delayed the excretion of valnemulin and increased the observed AUC.

Not all drugs display retarded disposition during infection. Different drugs may exhibit diverse pharmacokinetic characteristics. No conspicuous differences were observed when xamoterol was applied to patients with cardiac failure and hepatic dysfunction after p.o. administration (Nicholls et al., 1989). The pharmacokinetics or pharmacodynamics of various drugs is usually altered by liver or kidney diseases. The pathological state and degree of impairment may vary, thereby making these effects difficult to quantitatively measure. The appropriate dosage of these drugs in different animals is required (Rodighiero, 1999).

In conclusion, a combined M. gallisepticumE. coli infection model was developed in broiler chickens. The infected fowls were used in a pharmacokinetic study of valnemulin. The absorption, distribution, and elimination of the drug were retarded in the infected animals, with considerable changes to the Cmax values following both extravascular administrations. The therapeutic efficacy and adverse effects of drugs should be monitored more closely because of the higher drug concentration in plasma and enlarged AUC during valnemulin treatment.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Acknowledgments
  4. References

This study was supported by National Science Fund for Distinguished Young Scholars of China grants 31125026.

References

  1. Top of page
  2. Abstract
  3. Acknowledgments
  4. References
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