Effectiveness of doxycycline in the prevention of an experimental infection with Actinobacillus pleuropneumoniae in pigs

The effectiveness of medication with doxycycline in feed in the control of pleuropneumonia in pigs was tested using an Actinobacillus pleuropneumoniae serotype 1 aerosol challenge model. Two groups of 10 animals were used for the challenge, a 'medicated group' and an 'unmedicated group'. A third group of four animals was used as a 'control group'. Pigs from the medicated group were provided with feed containing 250 p.p.m. doxycycline (HIPRAMIX/DOXI) for 8 consecutive days and were challenged on the fifth day of treatment. No clinical signs were observed in pigs from the 'control group'. Four animals from the 'unmedicated group' died within the first 48 h after challenge with clinical and lesional evidence of an acute form of pleuropneumonia. Clinical signs of animals surviving the first 48 h were progressively less severe and showed lesions similar to those described for subacute-chronic forms of the disease. However, only one animal from the 'medicated group' showed clinical signs of a chronic form of pleuropneumonia. Reisolation of A. pleuropneumoniae was more evident from lung tissues of animals fed the doxycycline-free feed (70%), coinciding with the presence of both acute and subacute lesions. However, the micro-organism could be reisolated from only one animal which belonged to the 'medicated group'. It is concluded that the treatment of pigs with 250 p.p.m. doxycycline (HIPRAMIX/DOXI) prevents disease caused by A. pleuropneumoniae.     

Detection of Actinobacillus pleuropneumoniae in the porcine upper respiratory tract as a complement to serological tests.

Attempts were made to isolate Actinobacillus pleuropneumoniae from the nasal cavities and tonsils of 442 healthy pigs from 15 herds. Samples were streaked onto different media formulations. Serum samples were assayed for antibodies to A. pleuropneumoniae by enzyme-linked immunosorbent assay and complement fixation test. Actinobacillus pleuropneumoniae was isolated from the nasal cavities only in 24 pigs, from tonsils only in 90 pigs, and from both the nasal cavities and the tonsils in 11 pigs. A PPLO medium supplemented with lincomycin, bacitracin and crystal violet allowed recovery of A. pleuropneumoniae from more animals than a tryptic soy agar medium from both sites. Incubation of plates in an enriched CO2 atmosphere did not affect the recovery rate. Actinobacillus pleuropneumoniae belonging to serotypes 1, 2, 3, 5a, 5b, 7, 8, 10 and 12 were isolated, and, in several herds, more than one serotype were recovered. Serotypes of A. pleuropneumoniae were isolated from nine herds which were found seronegative to these. The isolation of A. pleuropneumoniae from the upper respiratory tract can be useful for detection of carrier pigs and complements serological screening.     

Mycoplasma hyosynoviae isolation from the upper respiratory tract and tonsils of pigs.

The occurrence of Mycoplasma hyosynoviae at different locations of the upper respiratory tract and tonsils of pigs was investigated in herds with problems of arthritis apparently caused by this microorganism. The isolation of M. hyosynoviae was facilitated by the use of a medium selectively suppressing the growth of Mycoplasma hyorhinis. M. hyosynoviae was cultured from 106 of 178 tonsils of slaughterhouse pigs from 8 herds but could not be isolated from the mucosa of the nasal cavity or the oral-pharyngeal area of 100 living, 10-20 weeks old pigs in 5 of the herds. The value of the selective principles in the medium appears from the circumstance that 86 of the 106 isolates were obtained despite the presence of M. hyorhinis. It is concluded that the tonsil is a reservoir for M. hyosynoviae and is probably the location of choice for an easy demonstration of the presence of this mycoplasma in a pig herd.     

Association between transmission rate and disease severity for Actinobacillus pleuropneumoniae infection in pigs

A better understanding of the variation in infectivity and its relation with clinical signs may help to improve measures to control and prevent (clinical) outbreaks of diseases. Here we investigated the role of disease severity on infectivity and transmission of Actinobacillus pleuropneumoniae, a bacterium causing respiratory problems in pig farms. We carried out transmission experiments with 10 pairs of caesarean-derived, colostrum-deprived pigs. In each pair, one pig was inoculated intranasally with 5 × 10⁶ CFUs of A. pleuropneumoniae strain 1536 and housed together with a contact pig. Clinical signs were scored and the course of infection was observed by bacterial examination and qPCR analysis of tonsillar brush and nasal swab samples. In 6 out of 10 pairs transmission to contact pigs was observed, but disease scores in contact infected pigs were low compared to the score in inoculated pigs. Whereas disease score was positively associated with bacterial load in inoculated pigs and bacterial load with the transmission rate, the disease score had a negative association with transmission. These findings indicate that in pigs with equal bacterial load, those with higher clinical scores transmit A. pleuropneumoniae less efficiently. Finally, the correlation between disease score in inoculated pigs and in positive contact pigs was low. Although translation of experimental work towards farm level has limitations, our results suggest that clinical outbreaks of A. pleuropneumoniae are unlikely to be caused only by spread of the pathogen by clinically diseased pigs, but may rather be the result of development of clinical signs in already infected pigs.     

Evaluation of serovar-independent ELISA antigens of Actinobacillus pleuropneumoniae in pigs, following experimental challenge with A. pleuropneumoniae, Mycoplasma hyopneumoniae and Pasteurella multocida

Objective To compare the sensitivity and specificity of six serological enzyme‐linked immunosorbent assays (ELISAs) based on serovar‐independent antigens of Actinobacillus pleuropneumoniae (App) and investigate cross‐reactivity in disease‐free pigs challenged with Mycoplasma hyopneumoniae and Pasteurella multocida. Design Five experimental pig trials using direct challenge with App serovars 1, 7 or 15 or direct challenge with M. hyopneumoniae and/or various dose rates of P. multocida. Procedure A 39‐kDa outer membrane protein antigen and five recombinant antigens from the apxIVA gene of App were evaluated. The latter were derived from the ApxIVA N‐terminus (ApxIVA‐N, ApxIVA‐NP, ApxIVA‐NPS) or C‐terminus (ApxIVA‐C, ApxIVA‐CP). Pigs were sampled after challenge and clinical and necropsy findings evaluated. Results The 39‐kDa ELISA had high sensitivity but lacked specificity, with significantly increased cross‐reactivity following P. multocida challenge. ELISAs based on ApxIVA N‐terminus antigens were significantly more sensitive than C‐terminus antigens for the detection of App‐induced disease. Although ApxIVA‐N and ApxIVA‐NP ELISAs had increased reactivity following P. multocida challenge, they retained high specificity for App‐induced disease (90–93%). Affinity purified ApxIVA‐NP antigen had marginally better specificity than ApxIVA‐N, without reduced sensitivity. Mycoplasma hyopneumoniae did not affect serological cross‐reactivity. In disease‐free pigs, the specificity of the ApxIVA‐NPS ELISA may be adversely affected by nasal carriage of apparently low‐virulence App strains. Conclusions ApxIVA‐N‐based ELISAs can be used for evaluating App status in commercial herds, but some appear limited by high carriage rates of low‐virulence App. The 39‐kDa antigen is only of merit in exclusion of App disease by negative serology.     

Efficacy of tulathromycin in the treatment of respiratory disease in pigs caused by Actinobacillus pleuropneumoniae

The efficacy of a single dose of tulathromycin, a novel triamilide antimicrobial of the macrolide class, given at 2.5 mg/kg or 5 mg/kg bodyweight, or three daily doses of ceftiofur, given at 3 mg/kg bodyweight, was evaluated in pigs with respiratory disease induced experimentally with Actinobacillus pleuropneumoniae. On day 0, 100 pigs with clinical signs of respiratory disease were randomly assigned to groups of 25 pigs, which were treated with either saline, one of the doses of tulathromycin, or ceftiofur. The pigs' rectal temperatures and clinical scores for respiratory signs and general attitude were recorded daily until day 10. Animals withdrawn from the study for welfare reasons were recorded. On day 10, the animals remaining in the study were weighed, euthanased and examined postmortem. Three of the animals treated with saline and one of those treated with 2.5 mg/kg tulathromycin were withdrawn from the study, but none of those treated with 5 mg/kg tulathromycin or ceftiofur were withdrawn. The least squares mean bodyweight gains of the pigs treated with the antimicrobial agents were significantly (P<0.05) higher than that of the saline-treated group, and the least squares mean percentages of the total lung involvement and incidence of respiratory disease associated with A. pleuropneumoniae were significantly (P<0.05) lower, but there were no significant differences between the three groups of pigs treated with the antimicrobial agents.     

A mixed-model approach for the analysis of cDNA microarray gene expression data from extreme-performing pigs after infection with Actinobacillus pleuropneumoniae

We proposed a novel statistical approach for the analysis of cDNA experiments based on mixed-model methodology combined with mixtures of distributions. Our objective was to detect genes that may be involved in conferring heritable differences in susceptibility to common infections in intensive pig production. We employed a microarray expression profiling strategy and a mixed-model approach to the analysis of the expression data. A cDNA microarray of pig with 6,420 probes from immune tissues and cells was used to compare gene expression in peripheral blood leukocytes of two pigs showing extreme performance in their response to infection with Actinobacillus pleuropneumoniae. Principal components analyses were used to identify the two most extreme-performing pigs after infection (i.e., pigs whose measured responses to infection fell at the extremes). Blood samples and expression profiles from 0 to 24 h after infection were compared using a bivariate, mixed-model approach, in which the effect gene x immunological status interaction was treated as a random effect. Bayesian model-based clustering via mixtures of normal distributions of the resulting BLUP of the random interaction was approached and resulted in a list of 307 differentially expressed genes, of which 179 were down-regulated in the susceptible pig. The majority of the differentially expressed genes were derived from a cDNA library of leukocytes of A. pleuropneumoniae-challenged pigs that were subtracted against leukocytes before the challenge. These results provide evidence that the proposed statistical approach was useful in enhancing the knowledge of the mechanisms involved in the genetics of the immune response.     

Development and use of a multiplex polymerase chain reaction assay based on Apx toxin genes for genotyping of Actinobacillus pleuropneumoniae isolates.

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiological agent of a porcine pleuropneumonia that threatens the global swine industry. The major pathogenic toxins of A. pleuropneumoniae include ApxI, ApxII, ApxIII, and ApxIV, which are serotype or serovar specific. Several techniques have been developed for the identification and typing of A. pleuropneumoniae. Serological assays are used to identify and serotype A. pleuropneumoniae, but factors such as cross-reactivity limit their specificity. Labor, time, and the requirement for specific antibodies are also drawbacks of these assays. Multistep polymerase chain reaction (PCR) techniques based on apx genes have been reported for the identification and typing of A. pleuropneumoniae. This study developed multiplex PCR for the identification and genotyping of A. pleuropneumoniae based on apx genes. This multiplex PCR technique was successful in differentiating 11 of 15 reference serotypes. Five different primer sets were used to amplify the 4 apx genes from each serotype in a single-step reaction. The multiplex PCR reported in this study was further used in genotyping 51 field isolates of A. pleuropneumoniae from different regions of Korea. The concomitant amplification of all 4 apx genes makes multiplex PCR more specific and convenient for the diagnosis and genotyping of A. pleuropneumoniae.     

The influence of Actinobacillus pleuropneumoniae infection on tulathromycin pharmacokinetics and lung tissue disposition in pigs

A tulathromycin concentration and pharmacokinetic parameters in plasma and lung tissue from healthy pigs and Actinobacillus pleuropneumoniae (App)‐infected pigs were compared. Tulathromycin was administered intramuscularly (i.m.) to all pigs at a single dose of 2.5 mg/kg. Blood and lung tissue samples were collected during 33 days postdrug application. Tulathromycin concentration in plasma and lung was determined by high‐performance liquid chromatography with tandem mass spectrometry (LC‐MS/MS) method. The mean maximum plasma concentration (C_max) in healthy pigs was 586 ± 71 ng/mL, reached by 0.5 h, while the mean value for C_max of tulathromycin in infected pigs was 386 ± 97 ng/mL after 0.5 h. The mean maximum tulathromycin concentration in lung of healthy group was calculated as 3412 ± 748 ng/g, detected at 12 h, while in pigs with App, the highest concentration in lung was 3337 ± 937 ng/g, determined at 48 h postdosing. The higher plasma and lung concentrations in pigs with no pulmonary inflammation were observed at the first time points sampling after tulathromycin administration, but slower elimination with elimination half‐life t_1/2el = 126 h in plasma and t_1/2el = 165 h in lung, as well as longer drug persistent in infected pigs, was found.     

Evaluation of serum galactomannan detection for diagnosis of feline upper respiratory tract aspergillosis

Measurement of serum galactomannan (GM), a polysaccharide fungal cell-wall component, is a non-invasive test for early diagnosis of invasive aspergillosis in humans. Feline upper respiratory tract (URT) aspergillosis is an emerging infectious disease in cats. Diagnosis requires biopsy for procurement of tissue specimens for cytological or histological detection of fungal hyphae and for fungal culture. The aim of this study was to evaluate serum GM measurement as a non-invasive diagnostic test for URT aspergillosis in cats. A one-stage, immunoenzymatic sandwich ELISA was used to detect serum GM in 4 groups of cats; Group 1 (URT aspergillosis) – confirmed URT aspergillosis (n = 13, sinonasal aspergillosis (SNA) n = 6 and sino-orbital aspergillosis (SOA) n = 7), Group 2 (URT other) – other URT diseases (n = 15), Group 3 (β-lactam) – cats treated with β-lactam antibiotics for non-respiratory tract disease (n = 14), Group 4a – healthy young cats (≤1 y of age, n = 28), Group 4b – healthy adult cats (>1 y of age, n = 16). One cat with SNA and two cats with SOA caused by an Aspergillus fumigatus-mimetic species, tested positive for serum GM. For a cut-off optical density index of 1.5, the overall sensitivity and specificity of the assay was 23% and 78% respectively. False positive results occurred in 29% of cats in Group 3 and 32% of cats in Group 4a. Specificity increased to 90% when Groups 3 and 4a were excluded from the analysis. Overall, serum GM measurement has a poor sensitivity but is a moderately specific, non-invasive screening test to rule out infection in patients with suspected feline upper respiratory tract aspergillosis.     

Evaluation of a single dose versus a divided dose regimen of danofloxacin in treatment of Actinobacillus pleuropneumoniae infection in pigs

A single versus a divided dose regimen of danofloxacin was evaluated in treatment of porcine Actinobacillus pleuropneumoniae infection using clinical observations combined with biochemical infection markers: C-reactive protein, zinc and ascorbic acid. Twenty hours after experimental infection, the 18 pigs received danofloxacin intravenously as a single dose of 2.5mg/kg or four doses of 0.6 mg/kg administered at 24h intervals. These dosage regimens resulted in similar AUCs of the plasma danofloxacin vs time curve. The maximum concentration was 3.5-fold higher using the single dose regimen, while the time with concentrations above the MIC was 2.5-fold longer using the fractionated regimen. Using the single dose regimen, temperature was normalised 32 h post-infection. In contrast, normalisation was delayed until 44 h post-infection using four low doses and a relapse with elevated temperatures at 52 and 68 h was observed. No other significant differences between the treatments were found, neither regarding clinical, haematological nor biochemical observations. The use of the more convenient single dose regimen was appropriate, as it was at least equivalent to the fractionated regimen.     

Systemic and local antibody responses after experimental infection with Actinobacillus pleuropneumoniae in piglets with passive or active immunity

The objectives of the present study was to describe different dynamics of humoral immune responses to experimental infection in piglets of different stages of infection and immunity. Two groups of piglets originating from non-immune (group 1) and immune (group 2) sows at the age of 3 weeks were subdivided as follows: a half of each group of piglets was exposed to a low-dose infection with Actinobacillus pleuropneumoniae (APP) strain 9. At the age of 8 weeks, all four groups of piglets were challenged with a high infection dose of APP of the same strain. Isotype characterization of the specific antibodies in sera and in bronchoalveolar lavage fluids (BALF) to a lipopolysaccharide was carried out, besides monitoring clinical signs and post-mortem examinations. A typical primary immune response was observed in specific antibody-free piglets infected with a challenge infection. Colostrum-derived immunoglobulin-G (IgG) antibodies persisted in sera and BALF of piglets up to the age of 8 weeks. However, they did not prevent induction of specific-primary antibody response, either in 8 or 4 weeks of age, when levels of specific colostrum-derived antibodies were still high. It was demonstrated by the increase of specific IgM antibodies in sera. The infection induced an increase in the levels of IgA antibodies in BALF regardless the severity of infection and presence of specific colostrum-derived antibodies. The specific antibodies of IgG isotype increased only in BALF from piglets without colostrum-derived antibodies.     

Evaluation of a single dose versus a divided dose regimen of amoxycillin in treatment of Actinobacillus pleuropneumoniae infection in pigs

The theory of a time-dependent effect of amoxycillin was examined in a model of porcine Actinobacillus pleuropneumoniae (Ap)-infection using clinically relevant dosage regimens. Twenty hours after infection of fourteen pigs, when clinical signs of pneumonia were present, one group of pigs received a single dose of amoxycillin (20 mg/kg, i.m.), whereas another group received four doses of 5 mg/kg injected at 8-h intervals. A similar AUC of the plasma amoxycillin concentration versus time curve was obtained in the two groups, whereas the maximum concentration was threefold higher using the single high dose. Plasma amoxycillin was above the MIC for twice as long using the fractionated dosage scheme. The condition of the animals was evaluated by clinical and haematological observations combined with quantification of biochemical infection markers: C-reactive protein, zinc and ascorbic acid. Within 48 h of treatment, the pigs in both treatment groups recovered clinically. No significant differences in the time-course of clinical observations or plasma concentrations of the biomarkers of infection were observed between the two treatments. In conclusion, the efficacy of these two dosage regimens of amoxycillin was not significantly different in treatment of acute Ap-infection in pigs.     

CSFV、PRV和PRRSV多重PCR检测方法的建立及其应用

由猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)和猪繁殖与呼吸综合征病毒(PRRSV)引起的繁殖障碍性疫病在中国的不断暴发,增加了猪的发病率和死亡率。为了建立同时检测这3种病毒的多重PCR方法,本研究根据GenBank中这3种病毒的参考基因序列设计引物,并对反应中的影响因素进行优化,建立了同时检测CSFV、PRV和PRRSV的多重PCR方法。该方法扩增的基因片段大小分别为570(CSFV),232 (PRRSV)和173bp(PRV)。敏感性和特异性试验结果显示,该方法对3种病毒的核酸最低检出量分别为23.88(CSFV),13.10(PRRSV)和14.60pg(PRV),对大肠杆菌(Ec.oli)、沙门菌(Salmonella)、猪乙型脑炎病毒(JEV)及猪圆环病毒2型(PCV2)的检测结果均为阴性。对2015年5月至2016年1月收集的168份临床样本检测结果显示,CSFV阳性率为24.4%,PRRSV阳性率为21.4%,提示河北省该段时间内引起猪繁殖障碍性疫病的主要病原为CSFV和PRRSV。经多重PCR检测为PRRSV、PRV或CSFV阳性的临床样本,再次使用商品化的试剂盒进行检测,符合率为100.0%。这些结果表明,本试验建立的多重PCR方法省时、高效,可用于PRRSV、PRV和CSFV感染的临床诊断。     

Application of polymerase chain reaction and virus isolation techniques for the detection of viruses in aborted and newborn foals

The occurrence of two important pathogens, equine herpesvirus 1 (EHV1) and equine arteritis virus (EAV) causing abortions, perinatal foal mortality and respiratory disease, was investigated by polymerase chain reaction (PCR) and virus isolation to demonstrate the presence of abortigenic viruses in samples from 248 horse fetuses in Hungary. We found 26 EHV1- and 4 EAV-positive aborted or prematurely born foals from 16 and 4 outbreaks, respectively, proving that despite the widely applied vaccination, EHV1 is a far more important cause of abortions in the studs than EAV. We compared the virus content of different organs of the fetuses by PCR and isolation to identify the organ most suitable for virus demonstration. Our investigations indicate that the quantity of both viruses is highest in the lungs; therefore, according to our observations, in positive cases the probability of detection is highest from lung samples of aborted or newborn foals. Both the PCR and the virus isolation results revealed that the liver, though widely used, is not the best organ to sample either for EHV1 or for EAV detection. From the analysis of the epidemiological data, we tried to estimate the importance of the two viruses in the Hungarian horse population.     

A comparison of virus isolation, polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine circovirus 2 and porcine parvovirus in experimentally and naturally coinfected pigs.

Virus isolation, polymerase chain reaction (PCR), immunohistochemistry, and in situ hybridization were compared for the detection of porcine circovirus 2 (PCV2) and porcine parvovirus (PPV) from experimentally and naturally coinfected pigs. All coinfected pigs developed postweaning multisystemic wasting syndrome (PMWS), characterized by sudden onset of depression and anorexia. Microscopically, granulomatous inflammation with intracytoplasmic inclusion bodies was present in lymph node from all coinfected pigs at 32 days postinoculation. Of the 200 tissues from 20 experimentally coinfected pigs evaluated, 99 and 58 tissues were positive for PCV2 and PPV, respectively, by 4 techniques. Virus isolation, PCR, immunohistochemistry, and in situ hybridization identified PCV2 infection in 137, 148, 103, and 129 tissues and PPV infection in 107, 132, 59, and 94 tissues. Of the 200 tissues from 20 naturally coinfected pigs evaluated, 109 and 45 tissues were positive for PCV2 and PPV, respectively, by 4 techniques. Virus isolation, PCR, immunohistochemistry, and in situ hybridization identified PCV2 infection in 144, 155, 113, and 139 tissues and PPV infection in 93, 109, 45, and 82 tissues. Because the characteristic microscopic lesions are important criteria for the diagnosis of clinical PMWS, immunohistochemistry and in situ hybridization for the detection of PCV2 and PPV in formalin-fixed, paraffin-embedded tissues provide confirmation of a histopathological diagnosis of PMWS.