Molecular serotyping of Haemophilus parasuis isolated from diseased pigs and the relationship between serovars and pathological patterns in Taiwan

Bacterial isolate collection and identification

Haemophilus parasuis field isolates were collected from diseased pig herds between August 2013 and February 2017 in Taiwan (Table S2). Lesions suspected of being caused by H. parasuis in diseased pigs were located in the meninges, pleura, pericardia, peritonea, synovial cavities of joints and lungs. Lesions were swabbed and plated on chocolate agar (at 37 °C, 5% CO₂, 18–72 h for growth rate variation for various isolates), blood agar (at 37 °C, 16–24 h) and MacConkey agar (at 37 °C, 16–24 h). The bacterial isolates were identified by colony morphology, Gram stain (Gram negative bacillus), nicotinamide adenine dinucleotide dependence (only growing on chocolate agar) and virulence-associated trimeric autotransporter group 3 colony PCR (Pina et al., 2009).

Molecular serotyping mPCR

The molecular serotyping assay for H. parasuis isolates was modified from a previously published method (Howell et al., 2015). The species-specific (sp-sp) amplicon was used as an internal control. A loopful of bacteria from a passaged plate of pure culture was resuspended in 30 μL ultrapure H₂O, which was heated to 100 °C for 30 min then centrifuged at 4,000×g for 1 min. The supernatant was used in the mPCR reaction. Isolates from various lesions or pigs from the same herd were serotyped. If they belonged to same serovar, they were considered one isolate.

Each PCR reaction was performed in a total volume of 30 μL containing ultrapure H₂O, 1 × DreamTaq buffer, 250 μM dNTP, 0.2 μM concentrations of forward and reverse serovar-specific primers, 0.04 μM concentrations of forward and reverse sp-sp primers, 1.25 U of DreamTaq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and one μL of supernatant. The thermocycling conditions consisted of 5 min at 94 °C, 30 cycles of 30 s at 94 °C, 30 s at 58 °C and 1 min at 72 °C, and then a final extension at 72 °C for 5 min. The molecular serotyping mPCR amplicons were stained with ethidium bromide and analyzed using a 20 cm-long 2% agarose gel. A 50 bp DNA ladder RTU (GeneDireX, Las Vegas, NV, USA) and Bio-1D software (Vilber Lourmat, Collégien, France) were used to estimate molecular size. The electrophoresis conditions were an electric field six V/cm (300 V, 50 cm full-length electric field) and 3 h. The results were confirmed by twice repeating tests.

Sequencing and analysis of unexpected PCR-amplified products

Unexpected amplicons of the molecular serotyping mPCR products were cloned using a TA cloning kit (Yeastern Biotech Co., Ltd. Taipei, Taiwan) and sequenced using an ABI 3730XL DNA analyzer (Applied Biosystems, Foster City, CA, USA). Sequence data were analyzed using MEGA7 (Molecular Evolutionary Genetics Analysis Version 7.0) software and Basic local alignment search tool (BLAST) database.

Pathological examination

Cases of sick animals or fresh, complete carcasses were subjected to necropsy for gross morphological examinations and H&E staining. Histopathological examination focused primarily on meningeal, pleural, pericardial, peritoneal and synovial cavities of joints, and lungs. Typical meningeal gross lesions were characterized by yellow to white exudate accumulation in the subarachnoid space, on pia mater and in the sulci (Fig. S1). Meningeal histopathological lesions were principal neutrophils and few mononuclear inflammatory cells infiltration on pia mater with fibrin and cellular debris deposit (Fig. S2). Serosal lesions were characterized by yellow to white exudate accumulation in pleural, pericardial, abdominal and joint synovial cavities and yellow to white fibrin covering the visceral and parietal serosa (Figs. S3–S5). The histopathological lesions of serositis were principal neutrophils and few mononuclear inflammatory cells infiltration with fibrin deposit (Fig. S6). In typical cases, H. parasuis resulted in bronchopneumonia with numerous neutrophils, mononuclear inflammatory cells, erythrocytes, cellular debris and fibrinous exudate accumulation in alveoli (Figs. S7 and S8). Due to disease duration, lesions varied in field. Other lesions infected H. parasuis were also involved to determine pathological patterns, including chronic fibrous serositis with angiogenesis and mononuclear inflammatory cells infiltration (Fig. S9), and meningitis with principal macrophages infiltration (Fig. S10).

Detection of porcine reproductive and respiratory syndrome virus

Nucleic acid extraction of pulmonary tissue was performed on a MagNA Pure LC 2.0 by using the MagNA Pure LC total nucleic acid isolation kit (Roche Applied Science, Indianapolis, IN, USA). Following cDNA synthesis was using PrimeScript^™ RT reagent kits (Takara, Kyoto, Japan). Porcine reproductive and respiratory syndrome virus (PRRSV) reverse transcription real-time PCR was performed as previously described (Lin et al., 2013).

Statistical analysis

Fisher’s exact test was used to compare the frequency of H. parasuis infected lesions and the percentage of various lesion patterns using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). Variables were considered significant at a 0.05 level (two-sided).

H. parasuis isolates, origins and pathological lesion patterns

A total of 133 isolates of H. parasuis were isolated from August 2013 to February 2017. The isolates were taken from 277 lesions on 155 diseased animals from 124 infected herds. Isolates from a herd serotyped as a single serovar were calculated as one isolate. Of 155 H. parasuis cases, 12 cases (7.7%) belonged to suckling pigs (≤3-week-old), 133 cases (85.2%) belonged to nursery pigs (4- to 12-week-old), seven cases (4.5%) belonged to growing pigs (13- to 26-week-old) and one case belonged to a breeding boar. Age information for two cases was unknown. A total of 86 cases (55.5%) had H. parasuis isolated from lung lesions with or without serosal lesions.

A total of 108 animals were necropsied with complete pathological examination and further correlated to pathological pattern and isolation proportion (Table S3). Of the H. parasuis infected animals, 54.6 % had serositis and pulmonary tissue lesions, 41.7% had serosal lesions only and 3.7 % displayed only pulmonary lesions (Fig. 1).

A total of 106 cases (98.1%) had bronchopneumonia, 64 cases (59.3%) displayed H. parasuis positive lung lesions. A total of 78 cases (72.2%) registered as positive for PRRSV via reverse transcription real-time PCR screening. The proportion of 204 H. parasuis infected lesions from 108 animals with complete pathological examination were meninges (10.3%), pleura (20.1%), pericardium (16.2%), peritoneum (13.7%), joint synovial cavity (9.3%) and lung (30.4%) (Fig. 2). The percentage of lung lesions showing H. parasuis infection was significantly higher than the percentage of serosal lesions (P < 0.05).

Serovar distribution by molecular serotyping assay

Of the 133 isolates, 91 (68.94%) isolates were typed using molecular serotyping mPCR. The most common serovars were serovar 5/12 (38.2%) and serovar 4 (27.5%) followed by serovar 14 (2.3%), serovar 1 (0.8%), serovar 2 (0.8%) and serovar 9 (0.8%) (Fig. 3). However, the product sizes of the serovar-specific primers analyzed by Bio-1D software were varied from the original publication (Howell et al., 2015). Furthermore, there were still 41 isolates (29.8%) classified as molecular serotyping non-typable (MSNT); these were divided into four groups based on the appearance of unexpected amplicons or the lack of serovar-specific amplicons. A total of 18 isolates (13%) only positive for a sp-sp marker were categorized as MSNT group 1. A total of 19 isolates (14.5%) were placed in MSNT group 2; these displayed amplicons of 300, 830 and 1,000 bp. Two isolates (1.5%) which showed unexpected amplicons at 500 and 660 bp were categorized as MSNT group 3. One isolate (0.8%), showing an amplicon of 300 bp was categorized as an MSNT group 4 isolate (Fig. 3; Figs. S11–S15).

Identification of serovar-specific amplicons

The amplicons generated from molecular serotyping mPCR analyzed using Bio-1D software were not precisely the same sizes as previously indicated in the original description of this assay (Howell et al., 2015). The product size of a specific amplicon found in serovar 4 was mentioned at 320 bp in the original publication but the PCR generated an amplicon of nearly 350 bp which might be confused with serovar 6. In serovar 5, the PCR results generated an amplicon larger than 450 bp mentioned in the original publication which might be confused with serovar 7. The product size of serovar 9 serovar-specific primers, mentioned at 710 bp in the original publication, was smaller than the 700 bp ladder marker and might be confused with serovar 8. In light of these conflicting results, the isolates were serotyped again to confirm the sizes, and the amplicons were subsequently sequenced. Comparisons of the molecular serotyping original publication described, Bio-1D software analyzed, and BLAST product sizes are shown in Table 1 (Howell et al., 2015). The product sizes analyzed by Bio-1D software, BLAST and sequence are more consistent.

The unexpected PCR products of the MSNT isolates were cloned for sequencing (Table 2). The MSNT group 2 amplicon was 297 bp; this product was generated with the serovar 13 specific forward and the serovar 14 specific reverse primers targeting gltP gene as a marker of serovar 13 in the polysaccharide biosynthesis locus. These primers were paired because the target sequences in the respective serovars shared homologous segments. The other PCR generated an amplicon product of the MSNT group 2 isolate determined to be 836 bp, and was identified as a serovar 13 specific product. The Bio-1D software measured a 500 bp product of the MSNT group 3 isolate as 499 bp; this amplicon was identified as a serovar 7 specific product. The 300 bp product found in the MSNT group 4 isolate, (sequencing results indicated it was 297 bp) was generated by pairing the serovar 13 specific forward primer with the serovar 14 specific reverse primers. This result was the same as that generated using the same primer pair of DNA isolated from MSNT group 2.

Serovar distribution based on molecular serotyping assay and sequencing

The molecular serotyping assay combined with sequencing results reduced the percentage of isolates classified as MSNT from 30.1% to 13.5% (Fig. 3). The dominant serovars were serovar 5/12 (37.6%), serovar 4 (27.8%) and serovar 13 (15%) followed by serovar 14 (2.3%), serovar 7 (1.5%), serovar 1 (0.8%), serovar 2 (0.8%) and serovar 9 (0.8%). Combining the sequencing results showed serovar 13 is a common serovar.

Relationship between pathological lesion patterns and serovars

The distribution of H. parasuis serovars in lesions from necropsied animals were serovar 5 (42.6%), serovar 4 (21.3%), serovar 13 (20.4%) and MSNT group 1 (13%). These categories were further subdivided into animals displaying both serosal and pulmonary lesions, those with only pulmonary lesions, and those with lesions found only in serosa. The respective percentages of lesions vs. serovars, and the pattern of lesions in infected animals were showed in Fig. 1 and Table S4. Necropsied animals with both serosal and pulmonary lesions were the most frequent; animals with pulmonary lesions alone were the least frequent (p < 0.0001). Serovars 4 and 5/12 showed similar results, the MSNT group 1 both serosal and pulmonary lesions were more frequent than serosal lesions alone. Serovar 13 had more serosal lesions than the combination of serosal and pulmonary lesions.

A nine herds (7.3%) had populations infected with two H. parasuis serovars. One herd contained a population with lesions infected with serovars 1 and 4. Three herds were infected with serovars 4 and 5. Serovar 5, 13 and 5, 14 co-infections were seen in single herds. The infected lesions were located in animals displaying a variety of tissue lesion patterns. Other four herds contained individuals co-infected with two H. parasuis serovars. One clinical case showed pleural and pulmonary lesions coinfected with H. parasuis serovars 4 and 7, respectively. A separate herd contained one case of pulmonary lesions with serovar 5, as well as pleural, pericardial and peritoneal lesions infected with H. parasuis serovar 13. One case was co-infected with serovars 5 (pulmonary) and 13 (pleura and pericardium). A fourth case showed coinfection with serovar 4 and an MSNT group 1 isolate taken from separate pulmonary lesions.