In Vivo Effectiveness of Injectable Antibiotics on the Recovery of Acute Actinobacillus pleuropneumoniae -Infected Pigs

Abstract

The aim of this trial was to evaluate the in vivo effectiveness of injectable antibiotics of one- or two-dose administration on recovery of acute App (Actinobacillus pleuropneumoniae)-infected pigs. Ninety pigs with moderate general clinical score (GCS) of a commercial farm, suffering from acute App infection, were divided in two groups: (a) T1: one administration of gamithromycin injectable solution and (b) T2: two administrations of florfenicol injectable solution. Morbidity/mortality, clinical scores (clinical appearance score index-CAS, clinical respiratory score-CRS, clinical cough score index [CCS], general respiratory clinical score-GCRS, and general clinical score-GCS), body temperature score (BTS), and posttreatment interval were recorded. The carcass weight and lung scoring were estimated, based on slaughterhouse pleurisy evaluation system score, lung lobes score, and pneumonia area. The results of this study indicated that the tested antibiotics are efficacious for the recovery of acute App-affected pigs. Quicker improvement of BTS in sick pigs (at day 1 and 2) and quicker recovery of clinical signs, based on the improvement of clinical parameters (CAS, CCS, GCRS, GCS on day 2 and 3, and CRS on day 2), were noticed in T1 group. In conclusion, the use of tested antibiotics in acute App-affected pigs is an effective strategy for the control of an acute outbreak.

Introduction

Actinobacillus pleuropneumoniae (App) is the causative agent of porcine pleuropneumonia and one of the most important bacterial respiratory pathogens in pigs.¹ App is also involved in porcine respiratory disease complex (PRDC).² It can critically affect the economic productivity of pig farms. Porcine pleuropneumonia has severe economic impact on swine farms worldwide, due to the increased mortality and feed conversion, costs for antimicrobials and immunoprophylaxis, and increased fattening period.^3,4 App can induce an acute form of respiratory disease characterized by severe fibrinohemorrhagic and necrotizing pleuropneumonia, causing high morbidity and mortality. Pigs surviving the acute form often become asymptomatic carriers of the organism. Chronically infected herds suffer from growth retardation and decreased feed efficiency.⁴ The main clinical signs of the acute App outbreak are high fever, vomiting, diarrhea, anorexia, and severe respiratory distress (increased respiratory rate, coughing/sneezing, and dyspnea).⁴ Animals with the peracute presentation may show all these clinical signs for a very short period of time, often overlooked under farm conditions, together with a foamy bloody nasal or oral discharge just before death.⁵ The animals that survive the acute phase of the disease may become chronically infected, showing little or no fever, mild coughing, inappetence, and reluctance to move. Moreover, there are a proportion of animals that might remain infected without showing any apparent clinical sign. These subclinically infected animals are considered carriers of the infection.⁶ PRDC is characterized clinically by slow growth, decreased feed efficiency, anorexia, lethargy, fever, cough, and laborious breathing, and is common in pigs around 10–20 weeks of age.² However, there are large variations in morbidities and mortalities between herds.⁷

Control of porcine pleuropneumonia is mainly based on the improvement of management and housing conditions, by antimicrobial therapy, and by vaccination.¹ In acute outbreaks of porcine pleuropneumonia, mortality can be controlled mainly by antimicrobial therapy of affected animals.⁴

Variation in outbreaks with respect to size⁸ and severity⁹ is related to factors such as strain and serotype differences,^10,11 immunity of the host,^12,13 and coinfections.¹⁴ Currently, the association between disease and pathogen dispersion, and thereby the clinical course of App outbreaks, is poorly understood. Transmission of pathogens is dependent on the susceptibility and the infectiousness of the uninfected and infected individuals, respectively, and the contact rate.¹⁵ It could be that, already subclinically infected pigs develop clinical signs at the same time because of the presence of a risk factor (trigger) that may be either infectious or noninfectious.¹⁶ Alternatively, it may be that an outbreak starts with only a few diseased pigs, which rapidly spread a clinical form of the infection on the farm.¹⁷ However, recent studies reported that it is more likely that an outbreak occurs due to the development of clinical signs at the same time in already infected pigs due to some (non-) infectious trigger.¹⁸

Acute App outbreaks demand immediate parenteral antimicrobial treatment of affected animals. Commonly used agents used for prevention and/or treatment include injectable gamithromycin (one-dose administration) and florfenicol (two-dose administration). The aim of this trial was to evaluate the in vivo effectiveness of these injectable antibiotics on recovery of acute App-infected pigs, mainly on recovery of severe clinical signs (e.g., pyrexia), decrease of morbidity and mortality rates, as well as decrease of pneumonia and pleuritis lesions.

Materials and Methods

Trial farm

This study was a cohort study, which was carried out in a commercial farrow- to-finish swine unit, suffering from acute App infection. The capacity of the trial farm was around 400 sows under production, with its own feed mill. The breeding stock was commercial hybrids (TOPIGS^® hybrids). It has a production of around 10,000–11,000 pigs per year. A grandparent nucleus of 40 sows was kept in the farm for producing own gilts, and these animals are separately housed, but in the same premises as the commercial herd. The farm is located in Xanthi (Mandra), Greece. The farm had a previous history of PRRSV infection in weaning, growing, and fattening stage, and had suffered from serious losses due to App infection (mortality rate due to App more than 8–10% for 1.5 years), mainly at the end of growing stage and during the finishing stage.

The criteria to select the trial farm were as follows: (a) PRDC-affected site (farm) mainly due to App confirmed by necropsy results in three severely diseased animals before the start of the study and (b) isolation of target pathogens that is, App and Pasteurella multocida by nasal swabs, oral fluids, blood samples, and lung tissue samples in clinically diseased animals before the start of the study. The susceptibility of the isolated target pathogens to gamithromycin and florfenicol was verified by antibiogram before the start of the trial, (c) pretesting for rectal temperatures in healthy pigs: before the start of the study, the rectal temperature of 10 healthy pigs 65–70 days of age was monitored and reported for site qualification. It was expected that temperature in healthy pigs is ≤39.5°C.

The farm facilities included five farrowing houses, one flat-deck unit, two growing houses, two finishing houses, one mating-pregnancy (dry period) stable, one breeding stock house, a feed mill, and an artificial insemination laboratory. Records in the farm are electronically kept. Piglets were weaned at 24 ± 3 days of age and around 210–220 weaners were moved weekly into the flat-deck unit, grouped in pens of 25 pigs. The nursery period ended at ∼10 weeks of age. The nursery stable contained 1 flat-deck unit each of 24 pens with slatted floor. Each room was automatically air-conditioned/ventilated keeping the temperature between 22°C and 27°C. Each flat-deck unit was daily cleaned and, when vacant, was thoroughly washed and disinfected. The entrance of newly weaned piglets in the unit was not earlier than 2–3 days' time after disinfection. Then, pigs were moved into the grower's stables, grouped in pens of 15 pigs (same animals), and remained until ∼15 weeks of age. Finally, pigs were moved to the finishing stables until slaughter at ∼23–24 weeks of age. In both stables, controlled temperature (18–20°) and ventilation were kept stable by windows and electrical ventilators. All those pens had slatted floor. Control of endoparasites/ectoparasites is currently obtained by sows' treatment with a single ivermectin injection 14 days before farrowing. Ivermectin is also applied in boars twice a year. Moreover, the vaccination program of the farm is shown in Table 1.

Table 1.

Vaccination Program of the Farm

Gilts	Sows and gilts	Boars	Weaners
Age of 150 days: AD + ART	30 days before farrowing: ART	Every 6 months against ERY, AD, ATR, PPV	M. hyo: one shot at weaning day
Age of 150 days: PRRSV	15 days before farrowing: Escherichia coli + Cl. perfringens		M. hyo: one shot at weaning day
Age of 170 days: AD + ART	60th day of gestation + 6 days postfarrowing: PRRS		PCV2: 15th day
Age of 180 days: PRRSV	15 days postfarrowing: ERY + PPV		PCV2: 15th day
Age of 190 days: ERY + PPV
Age of 210 days (service >15 days later): ERY + PPV

AD, Aujeszky's disease; ART, atrophic rhinitis; PRRS, porcine reproductive and respiratory syndrome virus; ERY, erysipelas; PPV, porcine parvovirus; M. hyo, M. hyopneumoniae; PCV2, porcine circovirus type 2.

Experimental products

The following injectable antibiotics were tested: (a) antibiotic of one dose: gamithromycin injectable solution, 150 mg/mL (Zactran^®; Boehringer Ingelheim/Merial), one administration, and (b) antibiotic of two doses: florfenicol injectable solution, 300 mg/mL (Nuflor^®; MSD Animal Health), two administrations in 48 hr.

Animals

The animals that are included in the trial come from a commercial farrow-to-finish farm. Totally, a number of 115 postweaners (age: 65–70 days to 100 days/weight range: 25–50 kg), vaccinated according to the vaccination protocol of the farm, were selected and ear tagged based on the following inclusion criteria applied for the experimental animals: (a) sick animals showing App-related clinical signs according to their general clinical score (GCS) (GCS ≥2 for at least 50% and GCS) >1 for the rest/less than 50% (Table 2), (b) vaccines (particularly PCV2, PRRSV, M. hyo) and concomitant medications were known and described, and (c) within test site, housed in the same building/pen that other recruited pigs.

Table 2.

Clinical Parameter Scoring System Used to Characterize Respiratory Disease in Pigs

Score	Depression classification (CAS)	Respiratory classification (clinical respiratory score index—CRS)	Cough classification (CCS)	BTS
0 = Normal	Alert, active, normal appetite, well hydrated, coat normal	Normal rate and pattern, no abnormal nasal discharge	No coughing	Normal <39.5°C
1 = Mild	Moves slower than normal, slightly rough coat, may appear lethargic, but upon stimulation appears normal	Slightly increased respiratory rate, some roughness in breathing	No coughing	Between 39.5°C–40.5°C
2 = Moderate	Inactive, may be recumbent or recumbent pig will rise upon stimulation, but remains depressed or lethargic gaunt, may be dehydrated, evidence of inappetance through gauntness and/or rough haircoat	Increased respiratory rate, some abnormal breathing	Occasional coughing or coughing is repetitive or episodic (several coughing episodes within a few minutes of rising)	Febrile ≥40.5°C
3 = Severe	Severely depressed to moribund, down or reluctant to rise or very resistant to stimulation but will rise, and immediately returns to or seeks recumbency	Increased respiratory rate with abnormal effort and dyspnea (open-mouth breathing, thumping, grunting, dog sitting)	Repeated, marked coughing, extensive, nonproductive, repetitive, or episodic periods of coughing	Febrile ≥40.5°C

The estimation of GCSs will be the following:

General respiratory clinical score (GCRS) = (CRS + CCS): 2.

GCS = (CAS + CRS + CCS + fever score) ÷ 4.

GCS, general clinical score; CAS, Clinical Appearance Score index; CCS, clinical cough score index; BTS, body temperature score.

The animals were not selected at once, but they were selected at the beginning of trial period from different pens and followed for the clinical signs to occur. Finally, 90 animals were included in this cohort study, as 20 animals were excluded, based on the following exclusion criteria: (a) any animal that was debilitated, suffering from systemic, disease other than App infection, or were injured, fractious, or otherwise unsuitable, (b) animals that have received medications that may impact treatment response (such as systemic corticosteroids, NSAIDs, and other systemic antimicrobials), and (c) animals that did not receive complete therapy program (e.g., only one shot of florfenicol or second shot at a different timeline than scheduled).

To control any bias with this study, we randomized pigs and blinded those collecting and analyzing data (high internal validity), whole keeping exclusion criteria to a minimum, thus making study groups closely related and allowing generalization of results (high external validity) (see the steps of treatment procedure Treatment section).

The animal experiment was approved by the Ethics Committee of the Faculty of Veterinary Medicine of University of Thessaly.

Study design

Treatment

A total of 90 animals were targeted with at least 50% of the pigs showing moderate GCS (GCS ≥2). After inclusion, the pigs were immediately treated with antibiotics according to the manufacturer's recommendations and divided into two groups (Table 3).

Table 3.

The Experimental Groups of the Trial

Group	n	Treatment	Treatment schedule
T1	45	Zactran^® (gamithromycin, IM, 6 mg/kg-i.e. 1 mL/25 kg, max 5 mL per injection site)	On shot on D0
T2	45	Nuflor^® (florfenicol 300 mg, 15 mg/kg-i.e. 1 mL/20 kg max 3 mL per injection site)	Two shots: D0 and D0 + 48 hr

The animals were observed until D3 following the beginning of therapy to assess treatment success rate and speed of efficacy. Relapse will be evaluated on D7 and D14. Day 0 was the same day for all animals, but all animals in a block (block = 2 animals) were enrolled and treated the same day. If there was one animal eligible alone for enrolment, it could be left to the candidate pool and remained eligible on subsequent days. The trials included three replicates (15 animals per group/replicate).

Treatment allocation was blocked by farm. Pigs meeting the enrolment criteria were allocated to be enrolled, treated, and monitored daily for clinical signs: on each possible date for D0:

Step #1: identification of all pigs meeting the enrolment criteria (GCS ≥2 for at least 50% of the pigs and the rest with GCS >1).

Step #2: recording of ear tags, sex, and individual bodyweight.

Step #3: treatment allocation to balance for clinical signs between experimental groups, particularly pigs were allocated in treatment groups to balance for the severity of the clinical signs. Mandatorily, the same number of pigs showing GCS ≥2 was allocated in the different groups according to a randomized block design (block = 2 animals) in a 1:1 ratio.

Animals that did not meet all the inclusion criteria, if not in need of immediate treatment, were left to the candidate pool and remained eligible for enrolment on subsequent days for up to 3 days from the day of enrolment of the first block of animals. If there was one animal eligible alone for enrolment, it could be left to the candidate pool and remained eligible on subsequent days.

Blinding procedure

Ear tags (blue for the T1 group and white for the T2 group) were used to ease the execution experimental procedures, particularly to identify the inclusion day and particularly the need to give a second shot in treatment B (only applicable for Nuflor group-T2 group). The person(s) measuring bodyweights and rectal temperatures did not know the treatment assignment of the animals.

Clinical monitoring/routine herd monitoring

The animals were monitored as part of daily care and maintenance. Daily observations were noted according to the data of Table 3.¹⁹ Mortality, accident, abortion, and culling were recorded in all groups. At the discretion of the investigator, moribund animals were euthanized/culled. Necropsy was performed to confirm the cause of death as often as possible. Dates of events, the reason for culling, and cause of mortality were noted.

The primary objective of the trial relied on a diagnosis for clinical signs consisting of scoring for depression, respiratory sign, and coughing, as well as recording body temperature. For each pig enrolled in the study, clinical signs monitoring was performed on D0, D0 + 8 hr, D1 = D0 + 24 hr, D2 = D0 + 48 hr, and D3 = D0 + 72 hr, according to a moderated scoring system of Roberts et al.¹⁹ in Table 3. A clinical evaluation was also performed at 1 week (D7) and 2 weeks (D14) posttreatment to confirm clinical recovery.

Animals with PRDC or App infection and not responding to the treatment were humanely euthanized/culled at discretion of the investigator. The clinical status of these moribund animals was carefully recorded just before euthanasia to be compared with reference treatment failure criteria (Table 2): (a) depression score Clinical Appearance Score index (CAS) = 3 and/or Respiratory score CRS = 3, and (b) body temperature ≥40.5°C (febrile status). Pigs dead or culled due to App infection were necropsied. A sample of lung was collected for possible bacterial isolation and antibiograms.

Sampling and analyses

Pretrial survey

Before the start of the trial period, samples were collected for polymerase chain reaction (PCR) screening of respiratory pathogens. Oral fluids were collected from one pen at weaning stage, one pen from growing stage, and one pen from finishing stage. At growing and finishing stage, nasal samples were collected from five different animals at least. In addition, three animals with evident respiratory symptoms were humanely euthanized and necropsied for culture and isolation of organisms in the lungs, focused on the bacteria commonly involved in PRDC (App, M. hyo, Streptococcus suis, P. multocida, and Haemophilus parasuis). The susceptibility of App to gamithromycin and florfenicol was tested using relevant techniques.

Trial survey

The sampling procedure included nasal swabs (two per each group) at 7 and 14 days after the observation of PRDC symptoms. Nasal samples/oral fluids were examined for the detection of respiratory pathogens by PCR (App, S. suis, P. multocida, H. parasuis, and M. hyo). Lung samples from each animal found dead or euthanized during the course of the study was collected and stored in appropriate conditions for bacterial isolation (App, S. suis, P. multocida, H. parasuis, and M. hyo).

Lung scoring

At slaughterhouse, 20 lungs from each experimental group were evaluated for lung lesions according to Sobestiansky and Barcellos (2007)²⁰ and Sibila et al.²¹ During the lung assessment, each lobe was divided into four equal parts to facilitate the recording of the affectation of each lobe. At the abattoir, each lobe was inspected, and its lesion percentage was established. The assessment was carried out with a careful palpation of the whole of the lungs in search of hepatization (“hardened”) areas, which belong to pneumonia lesions, and the percentage of involvement of each lobe was recorded on a card to correlate this with the weight and to obtain the pneumonia area percentage, according to Table 4. Also, any other kind of lesion seen (such as pleurisy, abscesses, adhesions, and parenchyma necrosis) was recorded.

Table 4.

Percentage Involvement of Each lobe with Respect to the Total Weight of the Lungs

Lung lobe	Percentage of lung weight
RA	11
RC	11
RD	34
LA	6
LC	6
LD	27
A	5

(Adapted from Sobestiansky and Barcello, 2007).²⁰

RA, right apical; RC, right cardiac; RD, right diaphragmatic; LA, left apical; LC, left cardiac; LD, left diaphragmatic; A, accessory.

Records

The parameters of morbidity and mortality due to respiratory failure and daily clinical scores (depression/attitude/anorexia-respiratory index-cough index) were recorded during the trial. Moreover, the body temperature was estimated on D0, D0 + 8 hr, D1 = D0 + 24 hr, D2 = D0 + 48 hr, and D3 = D0 + 72 hr, using infrared thermometer (Thermoscam, Zero one DT-886). Moreover, evaluation the impact of treatment protocol and posttreatment interval (PTI) concept on respiratory incidences/severity, including 7- and 14 day PTI. Treatment success was defined as the percentage of pigs that will be alive and not receive extra treatment with second im administration with Zactran or Nuflor for treatment following the PTI. Furthermore, the carcass weight at slaughterhouse was estimated. Finally, additional medications/treatments in feed of each group were recorded to estimate the total medication cost in each group.

For the evaluation of lung lesions, the following parameters were evaluated: (a) the Slaughterhouse Pleurisy Evaluation System (SPES, %) score grid for chronic pleuritis-CP [0 = Absence of CP lesions, 1 = Ventrocranial lesion: pleural adherence between lobes or at ventral border of lobes, 2 = Dorsocaudal monolateral focal lesion, 3 = Bilateral type 2 lesion or extended monolateral lesion (at least 1/3 of a diaphragmatic lobe), and 4 = Severely extended bilateral lesion (at least 1/3 of both diaphragmatic lobes)], (b) the lung lobes score (LLS, %), and (c) the pneumonia area (PA, %).

Statistical analysis

Data are expressed as mean ± standard deviation and normality was evaluated, using the Shapiro-Wilk test. Comparisons were performed using the nonparametric Mann-Whitney U test. Statistically significant differences were considered at p < 0.05 level. All analyses were performed using the IBM SPSS software version 25 (IBM Corp, Armnok, NY).

Results

Mortality/culling rate

The mortality rate in two groups did not present significant difference [T1 = 3/45 (6.66%), T2 = 4/45 (8.88%), p-value: 0.664]. Moreover, no significant difference was noticed in culling rate ([T1 = 2/45 (4.44%), T2 = 1/45 (2.22%), p-value: 0.656]).

Clinical parameters

PRDC-specific clinical parameters in two groups are presented in Table 5. Based on the results, the body temperature score (BTS) was significantly lower on day 1 and 2 after the administration of Zactran (T1 group) in comparison to T2 group (Nuflor). In addition, in T1 group, in comparison to T2 group, the CAS was significantly lower on day 2 and 3, while the Clinical Respiratory Score index (CRS) was lower on day 2. Finally, the Clinical Cough Score index (CCS), the General Clinical Respiratory Score (GCRS), and the GCS were significantly lower on day 2 and 3 in T1 group in comparison to T2 group.

Table 5.

Porcine Respiratory Disease Complex-Specific Clinical Parameters in Experimental Groups (Mean ± SD)

	Parameters
Groups	CAS	CRS	CCS	BTS	GCRS	GCS
Time: 0
T1^*	2.00 ± 0.67	1.84 ± 0.47	2.22 ± 0.42	1.87 ± 0.79	2.03 ± 0.34	1.98 ± 0.41
T2^*	1.87 ± 0.63	1.98 ± 0.45	2.13 ± 0.55	1.93 ± 0.69	2.06 ± 0.39	1.98 ± 0.41
p-Values^**	0.340	0.174	0.464	0.620	0.915	0.977
Time: 0 + 8 hr
T1	1.80 ± 0.55	1.80 ± 0.40	2.16 ± 0.37	1.78 ± 0.74	1.98 ± 0.30	1.88 ± 0.33
T2	1.69 ± 0.51	1.82 ± 0.49	2.13 ± 0.55	1.93 ± 0.69	1.98 ± 0.43	1.89 ± 0.41
p-Values	0.351	0.882	0.926	0.275	0.932	0.726
Time: day 1
T1	1.42 ± 0.50	1.69 ± 0.47	2.00 ± 0.21	1.40 ± 0.54	1.84 ± 0.26	1.63 ± 0.28
T2	1.51 ± 0.51	1.76 ± 0.43	1.98 ± 0.40	1.71 ± 0.59	1.87 ± 0.34	1.74 ± 0.36
p-Values	0.401	0.483	0.733	0.01	0.478	0.077
Time: day 2
T1	1.16 ± 0.37	1.29 ± 0.46	1.78 ± 0.42	1.13 ± 0.46	1.53 ± 0.31	1.34 ± 0.26
T2	1.56 ± 0.55	2.00 ± 0.48	2.13 ± 0.46	1.71 ± 0.59	2.07 ± 0.41	1.85 ± 0.39
p-Values	<0.001	<0.001	<0.001	<0.001	<0.001	<0.001
Time: day 3
T1	0.78 ± 0.42	1.04 ± 0.30	1.42 ± 0.50	0.84 ± 0.47	1.23 ± 0.29	1.02 ± 0.31
T2	0.96 ± 0.37	1.20 ± 0.50	1.89 ± 0.38	0.98 ± 0.45	1.54 ± 0.38	1.26 ± 0.33
p-Values	0.039	0.066	<0.001	0.174	<0.001	0.001
Time: day 7
T1	0.42 ± 0.54	0.76 ± 0.48	1.20 ± 0.40	0.64 ± 0.61	0.98 ± 0.37	0.76 ± 0.41
T2	0.44 ± 0.76	0.80 ± 0.55	1.18 ± 0.53	0.62 ± 0.81	0.99 ± 0.48	0.76 ± 0.58
p-Values	0.653	0.742	0.522	0.448	0.802	0.481
Time: day 14
T1	0.27 ± 0.54	0.40 ± 0.58	1.07 ± 0.39	0.40 ± 0.69	0.73 ± 0.43	0.53 ± 0.48
T2	0.38 ± 0.81	0.44 ± 0.76	1.11 ± 0.53	0.49 ± 0.82	0.78 ± 0.59	0.61 ± 0.66
p-Values	0.850	0.861	0.763	0.727	0.876	0.831

Mean (±SD), ^**nonparametric → Kruskal-Wallis H test.

T1, Zactran, T2, Nuflor; CRS, clinical respiratory score index; GCRS, general respiratory clinical score; SD, standard deviation.

Lung scoring

The results of lung scoring are presented in Table 6. Based on the results, no significant differences were noticed between two groups for SPES score, as well as percentage of LLS and PA.

Table 6.

Lung Scoring at Slaughterhouse in Experimental Groups (Mean ± SD)

		LLS %							PA %							Total
Values	SPES score	RA	RC	RD	LA	LC	LD	A	RA	RC	RD	LA	LC	LD	A	PA %	LLS %
T1 group
Mean	0.75	10.50	6.00	3.60	3.00	2.05	1.65	0.30	1.15	0.66	1.22	0.18	0.12	0.44	0.01	3.80	27.10
SD	0.85	3.59	2.05	3.20	4.10	2.32	2.13	0.73	0.39	0.22	1.08	0.24	0.13	0.57	0.03	1.86	11.43
T2 group
Mean	0.80	11.75	6.50	4.10	3.75	3.25	2.15	0.35	1.29	0.71	1.39	0.22	0.195	0.58	0.01	4.41	31.85
SD	0.89	4.94	2.85	4.08	4.25	2.44	1.89	1.18	0.54	0.31	1.39	0.25	0.14	0.51	0.05	2.48	14.50
p-Value	0.971	0.402	0.480	0.860	0.567	0.124	0.277	1.000	0.402	0.480	0.860	0.567	0.124	0.277	1.000	0.663	0.378

SPES score grid for CP: 0 = absence of CP lesions, 1 = ventrocranial lesion: pleural adherence between lobes or at ventral border of lobes, 2 = dorsocaudal monolateral focal lesion, 3 = bilateral type 2 lesion or extended monolateral lesion (at least 1/3 of a diaphragmatic lobe), 4 = severely extended bilateral lesion (at least 1/3 of both diaphragmatic lobes).

LLS, lung lobes score; PA, pneumonia area; SPES, slaughterhouse pleurisy evaluation system.

Weight of carcass at slaughterhouse

The mean carcass weight (Kg) of at slaughterhouse did not differ significantly between two groups (T1 = 58.41 ± 1.043, T2 = 58.45 ± 1.038, p-value: 0.899).

PTI/extra treatments

The 7- and 14-day PTI for the two groups are presented in Table 7. Based on the results, no significant differences were noticed between the two groups.

Table 7.

7-and 14-Day Posttreatment Interval in the Experimental Groups

Parameters	T1 7 day	T1 14 day	T2 7 day	T2 14 day
Animals enrolled	45	45	45	45
PTI/treatment success (%) no	95.55%^a (43/45)	95.55%^a (43/45)	93.33%^a (42/45)	93.33%^a (42/45)
Extra treatments	2	2	3	3

Percentages within each row with same superscripts do not differ significantly (p > 0.05).

PTI, posttreatment interval.

Laboratory results

The results of PCR screening of respiratory viral pathogens in oral fluids during pretrial period are as follows: (a) PRRSV positive in weaning, growing, and finishing stage, (b) PCV2 negative in all stages, and (c) SIV negative in all stages.

The results of PCR screening of respiratory bacterial pathogens in nasal samples of treated animals are presented in Table 8.

Table 8.

Pretrial and Trial survey Laboratory Results for Respiratory Bacterial Pathogens

Sampling	App	Streptococcus suis	Pasteurella multocida	M. hyo	Haemophilus parasuis
Pretrial period
Lung tissue 1: growing stage	Positive	Negative	Negative	Positive	Positive
Lung tissue 2: finishing stage	Positive	Positive	Negative	Positive	Positive
Lung tissue 3: finishing stage	Positive	Positive	Negative	Positive	Positive
Nasal swabs 1: growing stage	Positive	Negative	Negative	Positive	Positive
Nasal swabs 2: growing stage	Positive	Positive	Negative	Positive	Positive
Nasal swabs 1: finishing stage	Positive	Positive	Negative	Positive	Positive
Nasal swabs 2: finishing stage	Positive	Positive	Negative	Positive	Positive
Trial period
Nasal swabs: replicate 1	Negative	Negative	Negative	Negative	Positive
Nasal swabs: replicate 2	Negative	Negative	Negative	Negative	Negative
Nasal swabs: replicate 3	Negative	Negative	Negative	Negative	Negative

Discussion

Porcine pleuropneumonia caused by App is a contagious disease reported to cause economic losses worldwide.⁴ In some cases, finding dead pigs is the first observation. The disease can also take on a chronic form where production losses are affected and lesions at slaughter, such as adherence, pleuritis, and lung abscesses, are usually seen.⁴ Under field conditions, the high mortality of acute outbreaks of porcine pleuropneumonia can be controlled mainly by antimicrobial therapy of affected animals. The use of antibiotics in feed is also a treatment strategy in practice, but the anorexia and high fever of affected pigs may influence negatively the success of treatment protocol in feed. Throughout Europe, the older classes of antibiotics have progressively been shown to be ineffective at treating App infection—penicillin, ampicillin/amoxicillin, and older tetracyclines are generally of limited value. As any exposure of bacteria to antibiotics may lead to selection of resistance, antimicrobial susceptibility should be carefully monitored. Knowledge about trends over time is important to ensure long-term efficacy of the antibacterial products. Recent reviews^22,23 and research findings in cattle and pigs^24,25 highlight the importance of validated, harmonized, and continuous monitoring of antimicrobial susceptibility of veterinary pathogens as a component of good stewardship.

In our study, we tested antibiotics of one (gamithromycin, Zactran)- and two-dose (florfenicol, Nuflor) administrations to evaluate their efficacy to recover the acute clinical signs of App infection and to prevent the high mortality rate of affected pigs. Based on the results of our trial, the tested two antibiotics are efficacious for the treatment of App in pigs with severe clinical signs, with treatment success more than 90%. The administration of gamithromycin had slightly better treatment success—PTI at 7 and 14 days, but without significant statistical differences in comparison to T2 group. Moreover, the parameters SPES, LLS, and PA of lung scoring, as well as the mortality rate, were better in T1 group in comparison to T2 group, but without significant statistical differences. In our study, we noticed that the use of gamithromycin injectable solution offered quicker improvement of BTS in sick animals, as the parameter of BTS was decreased significantly on day 1 and 2 after the injection in comparison to T2 group (use of florfenicol). The administration of gamithromycin offered quicker reduction of severity of clinical signs and thus quicker recovery in sick animals based on the parameters of CAS, CCS, GCRS, and GCS on day 2 and 3 after the injection, as well the parameter of CRS on day 2 after the injection. The aforementioned clinical parameters were decreased significantly after the injection in comparison to T2 group. Previous studies indicated much higher lung concentrations of gamithromycin than in plasma over the time after treatment and a distinct tissue penetration.^26–28 In addition, other studies reported that florfenicol was absorbed quickly and completely, distributed widely in tissues, and maintained the effective therapeutic concentrations, especially in the respiratory tract tissues that are the target organs of App.^29,30

Transmission of pathogens is dependent on the susceptibility and the infectiousness of the uninfected and infected individuals, respectively, and the contact rate.¹⁵ The association between disease and App dispersion and thereby the clinical course of App outbreaks is poorly understood. There is the hypothesis that already subclinically infected pigs develop clinical signs at the same time because of the presence of a risk factor (trigger) that may be either infectious or noninfectious.¹⁶ Alternatively, a study described that an outbreak may start with only a few diseased pigs, which rapidly spreads a clinical form of the infection on the farm.¹⁷ However, more recent studies did not support the hypothesis that outbreaks start with only a few diseased pigs that rapidly spread a clinical form of the infection. It is therefore more likely that an outbreak occurs due to the development of clinical signs at the same time in already infected pigs due to some (non) infectious trigger.⁹ For this reason, the use of injectable antibiotics in affected pigs during acute App outbreaks could be not only a treatment but also a preventive tool to control the outbreak, as it offers quick reduction of severity of clinical signs and thus quicker recovery of sick animals.

In conclusion, the results of this trial indicated that the use of injectable antibiotics in acute App-affected pigs is an effective strategy for treatment and control of acute outbreak in the farm.

Footnotes

Acknowledgments

This work was supported by Boehringer Ingelheim/Merial through the Research Committee of the University of Thessaly (Scientific Responsible: Assist. Professor V. Papatsiros).

Disclosure Statement

No competing financial interests exist.

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