Development of a one-run real-time PCR detection system for pathogens associated with porcine respiratory diseases

Abstract

The etiology of Porcine respiratory disease complex is complicated by infections with multiple pathogens, and multiple infections increase the difficulty in identifying the causal pathogen. In this present study, we developed a detection system of microbes from porcine respiratory by using TaqMan real-time PCR (referred to as Dempo-PCR) to screen a broad range of pathogens associated with porcine respiratory diseases in a single run. We selected 17 porcine respiratory pathogens (Actinobacillus pleuropneumoniae, Boldetella bronchiseptica, Haemophilus parasuis, Pasteurella multocida, Pasteurella multocida toxin, Streptococcus suis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynovie, porcine circovirus 2, pseudorabies virus, porcine cytomegalovirus, swine influenza A virus, porcine reproductive and respiratory virus US strain, EU strain, porcine respiratory coronavirus and porcine hemagglutinating encephalomyelitis virus) as detection targets and designed novel specific primer-probe sets for seven of them. In sensitivity test by using standard curves from synthesized DNA, all primer-probe sets showed high sensitivity. However, porcine reproductive and respiratory virus is known to have a high frequency of genetic mutations, and the primer and probe sequences will need to be checked at a considerable frequency when performing Dempo-PCR from field samples. A total of 30 lung samples from swine showing respiratory symptoms on six farms were tested by the Dempo-PCR to validate the assay's clinical performance. As the results, 12 pathogens (5 virus and 7 bacteria) were detected and porcine reproductive and respiratory virus US strain, Mycoplasma hyorhinis, Haemophilus parasuis, and porcine cytomegalovirus were detected at high frequency. These results suggest that Dempo-PCR assay can be applied as a screening system with wide detection targets.

Seasonal Variation in Prevalence of Mycoplasma hyopneumoniae and Other Respiratory Pathogens in Peri-Weaned, Post-Weaned, and Fattening Pigs with Clinical Signs of Respiratory Diseases in Belgian and Dutch Pig Herds, Using a Tracheobronchial Swab Sampling Technique, and Their Associations with Local Weather Conditions

“…The disease, characterized by pneumonia and reduced growth performance, is an economically significant respiratory disorder of weaned piglets and finishing pigs, and remains a challenge to the swine industry worldwide. Multiple agents were reported to be associated with PRDC, including the major pathogens porcine reproductive and respiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae ( M. hyopneumoniae ), influenza A virus in swine (IAV-S), and porcine circovirus type 2 (PCV-2) [ 5 , 6 ]. Other pathogens associated with PRDC are porcine cytomegalovirus (PCMV), porcine respiratory coronavirus (PRCV), and Actinobacillus pleuropneumoniae ( A. pleuropneumoniae ) [ 2 , 5 , 6 ].…”

“…Multiple agents were reported to be associated with PRDC, including the major pathogens porcine reproductive and respiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae ( M. hyopneumoniae ), influenza A virus in swine (IAV-S), and porcine circovirus type 2 (PCV-2) [ 5 , 6 ]. Other pathogens associated with PRDC are porcine cytomegalovirus (PCMV), porcine respiratory coronavirus (PRCV), and Actinobacillus pleuropneumoniae ( A. pleuropneumoniae ) [ 2 , 5 , 6 ]. Infection with each single pathogen does not necessarily result in appearance of symptoms, but complex infections with a variety of pathogens can develop severe conditions.…”

Detection and genetic analysis of a novel atypical porcine pestivirus from piglets with congenital tremor in Japan

“…Tissues of the CNS (cerebellum), mesenteric lymph node, and tonsil from two 3-day-old piglets that showed severe signs of CT and moribund were collected and examined by reverse transcription (RT)-polymerase chain reaction (PCR) for pathogens of neurological disorders, such as porcine teschovirus (Krumbholz et al, 2003), porcine sapelovirus (Zell et al, 2000), porcine astrovirus (Chu et al, 2008), and APPV (Postel et al, 2016), and by real-time RT-PCR (qRT-PCR) for porcine circovirus 2 (Sunaga et al, 2019), pseudorabies virus (Ma et al, 2008), porcine hemagglutinating encephalomyelitis virus (Sunaga et al, 2019),Haemophilus parasuis (Turni et al, 2010), andStreptococcus suis (Bonifait et al, 2014). Tissues were minced by scissors, diluted 1:10 in phosphate-buffered saline, and homogenized for 20 sec at 3,200 rpm in the presence of three stainless steel beads (φ4 mm) by using a bead crusher μT-12 (TAITEC, Inc.).…”

“…Step PrimeScript RT-PCR Kit (Takara Bio) for RNA viruses and Premix Ex Taq (Takara Bio) for DNA viruses and bacteria, and performed as described previously (Sunaga et al, 2019).…”

Elevated Mhp462 antibody induced by natural infection but not in vitro culture of Mycoplasma hyopneumoniae

“…Mycoplasma hyopneumoniae is the respiratory pathogen of porcine enzootic pneumonia (PEP), a chronic infectious disease of pig, which results in decreased feed conversion, reduced daily weight gain, and significantly delayed time to market, and prevalent in almost all pig-rearing countries [ 1 , 2 ]. More importantly, infected pigs are vulnerable to secondary infection by other pathogens, such as porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, swine influenza A virus, Pasteurella multocida , Actinobacillus pleuropneumoniae ; these pathogens contribute to porcine respiratory disease complex (PRDC) [ 3 , 4 ].…”