Porcine respiratory and reproductive syndrome virus

At the age of six weeks, ten conventional pigs from a PRRSV negative herd were inoculated oronasally with 10⁶ TCID₅₀/pig PRRSV (Lena) in 2 ml of phosphate buffered saline (PBS, 1 ml in each nostril). Six pigs from the same origin were inoculated with 10⁶ TCID₅₀/pig of a recently isolated Belgian PRRSV, designated Belgium A, and served as a reference group for comparison of the clinical picture and virological findings. The Belgian PRRSV had been isolated from lungs and spleen of a stillborn piglet during a PRRSV outbreak in 2007. The herd experienced the following problems: birth of dead and weak piglets, high mortality rate among newborn piglets and respiratory disorders in growing pigs. The herd had been confirmed to be PRRSV positive. Sequencing demonstrated that PRRSV (Belgium A) belongs to the European subtype 1 PRRSV. The animal experiment was approved by the Ethical committee of the Faculty of Veterinary Medicine, Ghent University (EC2008/057).
Clinical signs (body temperature, respiratory disorders, and general signs such as appetite and behaviour) were monitored daily in both groups starting from six days before inoculation until the day of death or euthanasia. Respiratory disorders were scored from 0 to 6 (for interpretation, see legend Figure 1).
From the Lena-inoculated pigs, sera and nasal swabs were collected at 0, 3, 7, 10, 14, 21, 28, 35 and 42 dpi and stored at -70°C for virus titration. Also, sera were stored at -20°C for PRRSV-specific antibody detection. Seven out of ten Lena-inoculated pigs were anesthetized at 3, 14 and 21 dpi, for collection of tonsillar scrapings and in vivo pulmonary lavage as previously described [30 (link)]. The other three pigs were not treated and served as controls to exclude negative effects of in vivo pulmonary lavage. The lung lavages were centrifuged at 1500 × g for 10 min. The BAL fluids were collected, PAMs were resuspended in PBS and the total number of PAMs was calculated. Tonsillar scrapings, BAL fluids and PAMs were stored at -70°C for titration. At 28, 35 and 42 dpi, two pigs were euthanized and the following samples were collected: right lungs (apical, cardial and diaphragmatic lobes), tonsils and inguinal lymph nodes. In the case of death, the same organs were sampled. All samples were frozen and stored at -70°C for virus isolation and titration. The PAMs and BAL fluids were collected by pulmonary lavage from the left lung as previously described [31 (link)].
From the pigs inoculated with PRRSV (Belgium A), only sera were collected at the same time points as from PRRSV (Lena)-inoculated animals. All samples were stored at -70°C for virus titration and at -20°C for PRRSV antibody detection.

Two commercial farrow-to-finish farms were included in the study based on the willingness of the farmer to participate. On both farms, circulation of M. hyopneumoniae was assumed based on the presence of the pathogen in tracheobronchial swabs (TBS) taken from fattening pigs in the past. Danbred breeding gilts were reared on farm A and purchased on farm B. On farm A, the breeding gilts were vaccinated once against M. hyopneumoniae four weeks prior to first insemination with Ingelvac MycoFLEX^® (Boehringer Ingelheim Vetmedica GmbH, Ingelheim am Rhein, Germany). On farm B, breeding gilts were vaccinated with Stellamune^®Mycoplasma (Elanco, Utrecht, The Netherlands) at six months of age upon arrival at the farm and a second time four weeks later. Furthermore, on farm B gilts were also booster vaccinated twice shortly before farrowing. On both farms, sows were not vaccinated against M. hyopneumoniae.
Farm A practiced a 5-week batch-farrowing-system and piglets were weaned and moved to the nursery unit at approximately 22 days of age. The piglets were vaccinated at 16 days of age against M. hyopneumoniae with an inactivated whole cell J strain-based bacterin (Ingelvac MycoFLEX^®, Boehringer Ingelheim Vetmedica GmbH, Ingelheim am Rhein, Germany), porcine circovirus type 2 (PCV2) (Ingelvac CircoFLEX^®, Boehringer Ingelheim Vetmedica GmbH) and porcine reproductive and respiratory syndrome virus (PRRSV) (UNISTRAIN^® PRRS, HIPRA, Amer, Spain). Pigs were moved to the fattening unit at 9 weeks of age.
Farm B worked in a 4-week batch-farrowing-system and piglets were weaned and moved to the nursery unit at approximately 22 days of age. They were vaccinated at 16 days of age against M. hyopneumoniae with the same vaccine as in farm A (Ingelvac MycoFLEX^®, Boehringer Ingelheim Vetmedica GmbH) and PRRSV (UNISTRAIN^® PRRS, HIPRA). The piglets were moved to the fattening unit at 10 weeks of age. On both farms, five breeding animals (two gilts and three sows of mixed parity) were included in the study. The farrowing process was monitored by the main investigator and from each litter, five healthy piglets (birth weight >1 kg) were selected, ear notched and followed up monthly from birth till slaughter (n = 25 piglets / farm). Cross-fostering of the ear notched piglets was not allowed and pigs did not receive antibiotics active against M. hyopneumoniae on both farms during the entire trial.

The reference complete genomic sequences of ASFV, PRV, PPV, PRRSV, and PCV2 were acquired from GenBank with accession numbers of MK333180.1, MH766894.2, KU056477.1, JQ249927.1, KU131565.1, and AF201897.1, respectively. To identify the gene-deleted type and wild type ASFV, we choose three genes (CD2v, MGF505-2R, and P72) as the target for the detection of ASFV. CD2v and MGF505-2R would not be detected if the sample contained gene-deleted type ASFV. The specific primers were designed based on the conserved nucleic acid fragments of ASFV CD2v/MGF505-2R/P72, PRV gB, PPV VP2, PCV2 ORF2, and PRRSV P83 genes and synthesized by Beijing Genomics institution, BGI (Shenzhen, China). To assure the success of obtaining optimal primer sets, three sets of primers were designed for each gene. All primer sequences were listed in Table 1.

For stability verification, 10-fold serial plasmids dilutions with high (10⁶ copies/μl), medium (10⁴ copies/μl) and low (detection limits of each microfluidic LAMP chip system) concentrations were used to assess coefficients of variation (C.V.) of the microfluidic-LAMP chip system. Synthetic ASFV DNAs and clinical positive samples of PRV, PPV, PCV-2 and PRRSV were also applied to evaluate the reproducibility of the microfluidic-LAMP chip system. The C.V. was calculated by testing microfluidic-LAMP chip system in three consecutive runs (inter-group) in different chips and four times in the same chip (intra-group). Threshold time (Tt) was estimated from the reaction time when the positive signals of a particular sample exceed the baseline during the real-time amplification.