Duration of efficacy of ceftiofur crystalline free acid sterile suspension against clinical disease in grower pigs challenged with Actinobacillus pleuropneumoniae

Duración de la eficacia de la suspensión estéril de ácido libre de ceftiofur cristalino contra la enfermedad clínica en cerdos de crecimiento probados con el Actinobacillus pleuropneumoniae

Durée de l’efficacité d’une suspension stérile de ceftiofur cristalline “free acid” lors d’un épisode clinique chez des porcs en croissance soumis à une infection défi avec Actinobacillus pleuropneumoniae

John P. Crane, BSc; W. Lawrence Bryson, PhD; Young C. Anderson, PhD; J. Ken Callahan; Ellen S. Portis, BS; Cynthia J. Lindeman, BS; Merlyn J. Lucas, DVM, MS, Diplomate ACT; Edward J. Robb, DVM, MS, Diplomate ACVN

Pfizer Animal Health, Division of Pfizer Inc, Kalamazoo, Michigan. Corresponding author: John P. Crane, Pfizer Inc, 7000 Portage Rd, Kalamazoo, MI 49001; Tel: 269-833-2646; E-mail: john.p.crane@pfizer.com.

Cite as: Crane JP, Bryson WL, Anderson YC, et al. Duration of efficacy of ceftiofur crystalline free acid sterile suspension against clinical disease in grower pigs challenged with Actinobacillus pleuropneumoniae. J Swine Health Prod. 2006;14(6):302–306.
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Keywords: swine, respiratory disease, pleuropneumonia, ceftiofur, mortality
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Received: August 18, 2005
Accepted: October 21, 2005

Modern swine production is intensive. Very often, large groups of animals are housed in closed environments. This high stocking density favors transmission of airborne respiratory pathogens both within a herd and between herds. Respiratory disorders are regarded as the most serious disease problem in swine production.¹ The economic impact of acute bacterial swine respiratory disease at the herd level consists of mortality, decreased weight gain, decreased meat value (lung lesions), and disease-control expenditures, including medications and extra labor. Pathogens involved include Actinobacillus pleuropneumoniae, Haemophilus parasuis, Bordetella bronchiseptica, Mycoplasma hyopneumoniae, Pasteurella multocida, and Streptococcus suis. Very often, more than one of these species may be isolated in an outbreak. Secondary bacterial infections are also responsible for much of the illness associated with porcine reproductive and respiratory syndrome (PRRS).^2,3 The PRRS virus (PRRSV) is endemic throughout the world but does not tend to cause severe respiratory disease in the absence of bacterial coinfection.^2,3

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, which is associated with substantial economic losses throughout the world. Research suggests that this disease causes an average 34% decrease in weight gain and a 26% decrease in feed efficiency in affected herds.¹ Pleuropneumonia may also cause significant mortality or condemnation at slaughter, with losses as high as 10% to 20% for each of these outcomes. Total losses due to pleuropneumonia have been estimated at 2% of the value at slaughter.

Actinobacillus pleuropneumoniae can cause peracute disease outbreaks and is an important cause of severe lung lesions associated with Apx toxin production.⁴ Thus, A pleuropneumoniae is frequently used in bacterial respiratory disease models to investigate the efficacy of treatment regimens. Ceftiofur, a broad-spectrum, third-generation cephalosporin with excellent activity against the bacteria commonly involved in swine respiratory disease, is one of the most potent antibiotics against A pleuropneumoniae and is also highly active against P multocida and S suis.^5-12

Ceftiofur sodium and ceftiofur hydrochloride require daily injections for several days, which add to the cost of treatment as well as increasing stress to the animal. Dosing compliance is also a challenge. Ceftiofur crystalline free acid sterile suspension (CCFA-SS), a new injectable formulation in a specially treated vegetable-oil vehicle, provides sustained release of ceftiofur and prolonged therapeutic activity from a single dose. The objective of this study was to measure the duration of effectiveness of a single dose of CCFA-SS (Excede for Swine; Pfizer Animal Health, Kalamazoo, Michigan) against clinical disease caused by A pleuropneumoniae respiratory infection in swine.

Materials and methods

Study animals

One hundred and ninety (190) castrated male Landrace × Yorkshire piglets, 3 to 4 weeks of age, were randomly assigned to 19 pens (10 piglets per pen) on arrival at a research facility in Michigan. Pigs were obtained from a local specific-pathogen-free commercial source that was free of both Mycoplasma hyopneumoniae and PRRSV, and had no recent history of A pleuropneumoniae infection. Serum samples collected from three randomly selected pigs per pen on arrival were submitted to Biovet Inc (St Hyacinthe, Quebec, Canada) for testing by A pleuropneumoniae ELISA (APP serotype 5). A sample:positive (S:P) ratio = 0.50 was considered positive and an S:P ratio = 0.40 and < 0.50 was considered suspect. Sera from all three pigs per pen were negative for antibodies to A pleuropneumoniae serotype 5.

Prior to arrival, all pigs had received chlortetracycline in drinking water for 2 to 3 days postweaning, but no other routine treatments or vaccinations. No antibiotics were administered during the 7-day acclimation period before the study began.

Housing and management

All pens were located in the same isolated room, which contained 20 segregated, raised pens divided into two equal rows by a central alley. Each pen measured approximately 1.5 m × 2.4 m, which provided 0.36 m² of space per pig. Pens were separated by solid partitions to prevent pig-to-pig contact between pens. Temperature, lighting, and negative-pressure ventilation were kept relatively uniform throughout the study by use of automatic environmental controls. Unpelleted feed and municipally sourced drinking water were provided ad libitum. Feed was manufactured on site and contained no antimicrobial additives.

Study design

Pens were randomly assigned to six groups (Table 1), which included five CCFA-SS treatment groups and a negative (untreated) control group, with 30 animals (three pens) per treatment. An additional pen of 10 animals was retained as sentinels that received neither treatment nor bacterial challenge.

All pigs allocated to CCFA-SS treatment received the label dosage of 5 mg ceftiofur equivalents (CE) per kg body weight (BW) as a single intramuscular (IM) injection in the neck. To maintain blinding, personnel who administered treatments were not involved with animal removals, recording clinical observations, or pathological assessments.

All animals were observed daily prior to challenge. Pigs were excluded at randomization or during the pre-challenge observation period if they met any of the following three criteria: manifested visible abnormalities that could affect study conduct or outcome (eg, hernia or lameness); had a rectal temperature > 40.1°C on at least one occasion within 2 days before challenge and showed clinical signs of disease; or had received systemically active antibiotic treatment (other than the test drug) within 18 days before challenge.

Immediately before A pleuropneumoniae challenge on Day 0, three pigs per pen were randomly selected for collection of blood to determine the concentration of ceftiofur and desfuroylceftiofur-related metabolites in plasma. Each pig (excluding the 10 sentinels) was then inoculated with A pleuropneumoniae serotype 5 (Pfizer strain collection #200–18062). Pigs were observed for clinical signs of abnormal demeanor and respiration at 4- to 5-hour intervals for the 24-hour period immediately post challenge and at least twice daily for 9 days thereafter. All observations were scored according to the system shown in Table 2 by trained study personnel who were blinded to treatment.

Rectal body temperatures of all pigs were taken once daily from Day -2 to Day 9 at approximately the same time of day. Feed consumption by pen was measured daily from Day -4 to Day 9.

Pigs were removed and humanely euthanized if they exhibited either a demeanor score of 3 or a respiratory index score of 3 on two consecutive observations, or a score of 3 for both signs concurrently on any single observation. On euthanasia (either at removal or at the end of the study), all animals were necropsied by a pathologist blinded to treatment. In addition to gross pathology, microbiological confirmation by culture of affected tissue was used to support the diagnosis of A pleuropneumoniae pneumonia.

Serum samples for serological testing were obtained from all pigs that survived to study termination. Samples were submitted to Biovet Inc for testing by A pleuropneumoniae ELISA (APP serotype 5).

This study was conducted under close veterinary supervision and was pre-approved by an Institutional Animal Care and Use Committee.

Preparation of challenge inoculum

The A pleuropneumoniae challenge strain was revived from lyophilization by streaking onto brain-heart infusion (BHI) plus 2% Supplement C (Difco; Becton Dickinson Company, Franklin Lakes, New Jersey) agar plates. Supplement C, a desiccated yeast concentrate containing hematin yeast extract, L-glutamine, coenzyme, growth factor, and cocarboxylase, is used to supplement media for cultivating fastidious organisms with exacting growth requirements, and provides for a more robust growth of A pleuropneumoniae than BHI with nicotinamide adenine dinucleotide alone. After incubation for 18 to 24 hours at 37°C in an atmosphere of 5% CO2, colonies from the area of individual growth were selected with a sterile loop and inoculated into 9 mL BHI broth containing 2% Supplement C. The culture was vortexed, then added to 41 mL of BHI broth (containing 2% Supplement C) and incubated at 37°C (without CO2) in a rotary shaker at 200 rpm until it equated to approximately 2 × 10⁹ colony forming units (CFU) per mL (by predetermined optical density). The culture was then diluted 1:250 (to approximately 8 × 10⁶ CFU per mL) in BHI broth without Supplement C. Final inoculum concentration was verified by viable plate counts both before and after inoculation of pigs. This strain of A pleuropneumoniae is sensitive to ceftiofur, with a minimum inhibitory concentration value of < 0.03 µg CE per mL.

Administration of inoculum

Approximately 4 × 10⁷ CFU of A pleuropneumoniae in 5 mL of BHI broth was administered by intratracheal inoculation. Delivery was performed using a 10-mL syringe and a rubber catheter (approximately 40 cm long with a single-eye opening at the distal end). Upon manual restraint of each pig and aided by a speculum, the catheter was inserted into the mouth and advanced caudally, following the line of the ventral side of the soft palate, directly into the trachea. The inoculum was injected with sufficient pressure to ensure delivery to the trachea. The catheter was then closed by crimping the rubber tubing and the syringe was removed from the tube. Using the same syringe, air was expressed into the catheter to ensure full delivery of the inoculum deep within the lung. The catheter was then removed and the pig released.

Assay for ceftiofur and desfuroyl-ceftiofur-related metabolites in plasma

Approximately 10 mL of blood from each selected pig was collected in heparin-treated tubes, then immediately placed on ice until centrifuged. The plasma was divided into two vials of approximately equal quantities and stored frozen at or below -20°C pending liquid chromatographic assay.¹³

Data analysis

This study was a one-way randomized controlled clinical trial, with pigs nested within pens. Therefore, the experimental unit was pen, with three replicates per treatment. The primary outcome of interest was mortality. Ancillary variables included clinical demeanor, respiratory index, rectal temperature, and feed intake. Mortality was analyzed as the proportion of pigs within each treatment-pen combination that died or required euthanasia due to A pleuropneumoniae disease. This proportion was transformed using a Freeman-Tukey arcsine transformation, then analyzed by weighted ANOVA. The weights were n + 0.5, in which n equals the number of pigs in each treatment-pen combination. ANOVA was performed using the MIXED procedure in SAS Version 6, release 6.12 (SAS Institute, Cary, North Carolina), with a fixed treatment effect and a random residual error. Statistical significance was set at α = 0.05.

Results

Due to several pigs meeting the study exclusion criteria, Groups 1, 2, 3, 5, and 6 started the A pleuropneumoniae challenge and subsequent observation period with 29 pigs instead of 30. One pig was later removed from Group 4 during the 9-day postchallenge period because of an injury. Therefore, all mortality analyses were based on 29 pigs per group.

The A pleuropneumoniae challenge caused a peracute, severe pleuropneumonia (including substantial respiratory distress and clinical depression) within approximately 4 hours of challenge. As a result, 89.3% of the untreated control pigs (Group 6) died or were euthanized within 24 hours of challenge (Table 3). Mortality rates were lower for pigs that had been treated with CCFA-SS on Days -1, -4, and -7 (Table 3; P < .001), with no pigs lost among the 58 pigs treated on Days -1 and -4. Mortality rates for pigs treated on Days -10 and -13 were similar to that of controls. One of the 10 sentinel pigs also died. Gross pathology and microbiological culture of the lungs confirmed that all deaths were caused by A pleuropneumoniae infection.

The substantial mortality within the first 24 hours of challenge among the control and Day -10 and Day -13 treatment groups limited the available data on ancillary variables, precluding statistical analysis. However, by 24 hours after challenge, prevalence of abnormal respiration or clinical demeanor (ie, scores > 0) was numerically lower in the surviving pigs that had received CCFA-SS on Days -1, -4, and -7 than in the Day -10 and -13 groups. The percent of controls with abnormal respiration or demeanor was similar to that seen in the Day -1, -4 and -7 groups, but this was based on data from only four surviving control pigs. For the period Day -4 to Day 9, average feed consumption was numerically lower as the time between treatment and challenge increased, ranging from 0.99 kg per pig per day in the Day -1 group to less than half that value in the Day -10 and -13 groups.

Eight of 11 surviving pigs from the Day -13, -10, and control groups (approximately 73%) were seropositive for antibodies to A pleuropneumoniae. In contrast, 52 of the 80 survivors in the Day -1, -4, and -7 groups (65%) were seronegative, with S:P ratios < 0.40. Four of the nine surviving sentinels (approximately 44%) had positive or suspect ELISA results. The range of S:P ratios across all animals was 0.04 to 1.47.

The mean concentration of ceftiofur and related metabolites at challenge ranged from 3.96 µg CE per mL in pigs treated with CCFA-SS on Day -1 to below the limit of detection (0.04 µg CE per mL) in the Day -13 group (Table 3).

Discussion

This challenge model caused peracute, severe pleuropneumonia, resulting in nearly 90% mortality or removal of pigs in the control group within 24 hours. The most likely cause of this peracute response was endotoxin release from the A pleuropneumoniae in the lung.⁴

The challenge administered to pigs in this study was more severe than would be expected under natural field conditions. Not only was the challenge dose very high and administered directly into the trachea, but all pigs were housed in the same room with pens close to each other, increasing the possibility of lateral exposure. The death of one of the sentinel pigs, as well as the positive or suspect ELISA results for A pleuropneumoniae antibodies in four of the nine surviving sentinels, suggests transmission of infection between pens.

The available data for ancillary variables suggest a pattern of less severe clinical signs of disease and greater feed consumption among the Day -1, -4, and -7 treatment groups. These findings are consistent with the lower mortality observed for these three treatment groups, suggesting a milder form of clinical disease compared with other groups.

ELISA results suggest lower seroconversion among the Day -1, -4, and -7 treatment groups than the other groups, consistent with previous reports that effective treatment blunts seroconversion.⁹ However, the 9-day observation period may have been insufficient for maximal seroconversion. In addition, seronegative animals may harbor subclinical A pleuropneumoniae infection (carrier state), which often goes undetected by serological testing.¹⁴

Plasma pharmacokinetic results for the Day -1, -4, and -7 treatment groups validate pharmacodynamic-pharmacokinetic analyses¹⁵ which suggested that plasma levels of ceftiofur and desfuroylceftiofur metabolites > 0.2 µg per mL subsequent to a single dose of CCFA-SS provide efficacy against respiratory disease caused by sensitive bacterial pathogens. Actinobacillus pleuropneumoniae is highly sensitive to ceftiofur.^5-7,9,10,12 Multiple daily doses of either the sodium or hydrochloride formulation of ceftiofur significantly reduce mortality caused by swine respiratory disease associated with this pathogen.^9,16-18 However, the current study used only a single dose of CCFA-SS, suggesting obvious economic advantages in terms of reduced labor and treatment costs, as well as improved animal welfare and treatment compliance.

Implications

• A single IM dose of CCFA-SS (5 mg CE per kg BW) can provide 7 days of therapeutic activity against respiratory disease caused by A pleuropneumoniae in feeder pigs.

• Use of a single dose of CCFA-SS rather than multiple daily doses of other ceftiofur formulations would lower treatment costs, improve treatment compliance, and enhance animal welfare.

Acknowledgement

All authors were employed by Pfizer Animal Health while this study was being conducted.

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* Non-refereed references.