Molecular Serotyping and Resistance of Clinical Strains of Haemophilus (Glaesserella) Parasuis in Chinese Pig Farms From 2016 to 2018.

Haemophilus parasuis (H. parasuis) is the etiological agent of Glässer's disease and brings great economic losses to the pig industry. The goal of our research is to reveal the serotypes of H. parasuis isolated from large-scale pig farms in China from 2016 to 2018. From 2016 to 2018, 8153 H. parasuis eld strains were isolated from 14610 clinical samples of sick pigs with clinical symptoms from 26 provinces and cities of China. Among them, 1386 strains were identied as H. parasuis by PCR, and the isolation rate was 9.49%. Through multiplex PCR, we showed that type 5/12 and type 4 strains had the highest separation rate, followed by type 13 and type 14 strains. Using disk diffusion method, we found cephalosporin antibiotics and peptide antibiotics all had good inhibitory effect on H. parasuis. Our conclusion may play a positive role in the prevention and treatment of H. parasuis. vitro sensitivity tests. studied the of of H. parasuis were determined by PCR (8). The results were conrmed by two repeated experiments. The PCR products were stained with GelRedTM (Biotium, Fremont, CA, USA) and performed gel electrophoresis in a 1.0% agarose gel in Tris-acetate-EDTA (TAE) buffer at 120 volts for about 20–30 min. isolates. The results showed that only 10% of the isolates could not be typed. The molecular method has the advantages of simple operation and less time-consuming in the mass identication of serotypes, but it is worth noting that this method cannot distinguish between serotype 5 and serotype 12. Using a multiplex PCR assay (8) and a specic PCR reaction for the H. parasuis serotyping were more precise.


Introduction
Haemophilus parasuis (H. parasuis) is a pleomorphic and a member of the family Pasteurellaceae. H. parasuis is a NAD-dependent, Gram-negative bacterium (1). H. parasuis could cause Glässer's disease, pneumonia and septicemia during different breeding periods (2). H. parasuis has brought serious economic losses to the pig industry (3). Scientists have to study the epidemiology and pathogenesis of H. parasuis. H. parasuis has 15 serovars and a large number of non-typeable (NT) isolates (4). The diversity of the H. parasuis genotype makes prevention and treatment particularly di cult, such as the low cross-protection of vaccines and antibiotic resistance (5).
The pathogenesis of H. parasuis is very complex, which is related to virulence genes, serum and bio lm production (6). There are many molecular serum-based methods for H. parasuis, including agar gel diffusion (AGD), indirect hemagglutination (IHA) (7) and a multiplex PCR (8). To date, a total of 15 serotypes of H. parasuis have been identi ed, including serovars 1 through 15. However, the scientists cannot discriminate between serovars 5 and 12 (8). The serovars 1, 5, 10, 12, 13, and 14 were regarded highly virulent; the serovars 2, 4, 8 and 15 were regarded moderately virulent; and the serovars 3, 6, 7, 9 and 11 were considered low virulence potential (7). The serovars 5 and 4 of H. parasuis are widely regarded as pathogenic serums and are the most common serovars isolated from clinically sick pigs worldwide (9).
The abuse of antibiotics and the lack of biosafety knowledge hinder the prevention and control of H. parasuis (1). And more Clinical studies have shown that the protection of inactivated vaccine is mainly against isolates of the same serovars, and its crossprotection is extremely limited (10). Therefore, the outbreak of H. parasuis due to vaccination failure is a major concern for researchers and pig farmers. In order to prevent and control H. parasuis safely and effectively, developing an effective vaccine is still the best choice at present (11,12). However, the key to the development of a vaccine is to nd a highly virulent and widespread strain in the country, so epidemiological investigation is a necessary process.
In this study, we conducted an epidemiological survey of more than 10,000 clinical samples in different provinces of China from 2016 to 2018, and identi ed 8153 H. parasuis strains from 14610 disease materials, among them, 1386 strains were identi ed as H. parasuis by PCR. Besides, we Serotyped 320 H. parasuis strains by multiplex PCR, and our results showed that type 5/12 and type 4 strains had the highest proportion, followed by type 13 and type 14 strains.
With the large-scale and intensive development of the pig industry the incidence of H. parasuis is increasing year by year, but the vaccine of H. parasuis has poor cross-protection between different serotypes. in order to avoid huge economic losses, pig farms have to rely on antibiotics to control this disease. Therefore, it is necessary to screen drugs based on the results of vitro drug sensitivity tests. We studied the drug resistance of H. parasuis to 18 common antibiotics by the K-B disc method, and the results showed that the cephalosporin and peptide antibiotics could signi cantly inhibit the growth of H. parasuis.
Our work studied the serotypes of H. parasuis isolated from large-scale pig farms in China from 2016 to 2018 and the drug resistance of 166 H. parasuis to 18 common antibiotics, which played a vital role for the research of vaccines against prevalent H. parasuis strains in our country.

Materials And Methods
Ethics approval and consent to participate All animal experimental procedures were performed in accordance with the Hubei Regulations for the Administration of Affairs

Clinical isolates
From January 2016 to December 2018, a total of 14610 clinical samples were collected from 30 to 70 days old pigs suspected of being infected with H. parasuis. The clinical samples covered 26 provinces and municipalities. all isolates were from the lungs, heart, brain, joints, or trachea of pigs, and had clinical symptoms similar to H. parasuis, such as high fever, arthritis, tremor, incoordination, lying on the side and depression and so on. All clinical samples were stored at − 80 ℃. Detailed information was recorded for each clinical sample, such as viscera, time, location, and clinical symptoms.

Bacterial isolation and identi cation
Clinical samples were streaked onto tryptic soy agar (TSA) plates (TSA; Becton, Dickinson and Company, Franklin Lakes NJ, USA) containing 10 µg/ml NAD (NAD; Roche, Basel, Switzerland) and 5% newborn calf serum (Gibco, New York, USA), and then incubated at 37°C for 36 h. The suspect colonies should be translucent colonies of 1 mm in diameter and be subjected to further identi cation by gram staining and PCR (13). Sequences of primers used in the research were showed in table 2.

DNA preparation
For the serotyping typing of H. parasuis, the bacteria were grown in tryptic soy broth (TSB; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for overnight at 37°C and bacteria suspension was spinned down at 10,000 × g for 5 min and then resuspended in phosphate buffered saline (pH: 7.4). The suspension was heated at 100°C for 10 min and followed by a centrifugation under 10,000×g for 5 min. The supernatant was collected to a nucleic acid-free tube and stored at − 20°C.

Serotyping
The serovars of H. parasuis were determined by PCR (8). The results were con rmed by two repeated experiments. The PCR products were stained with GelRedTM (Biotium, Fremont, CA, USA) and performed gel electrophoresis in a 1.0% agarose gel in Tris This study refers to the standard CLSI M2 A12 Ed. 12 (2015) Performance Standards For Antimicrobial Disk Susceptibility Tests for drug susceptibility testing. The speci c method was picking a puri ed single colony in the ultra-clean workbench and inoculated in TSB medium and cultured in a shaker at 37 ℃ for about 12h forming bacterial suspension, then dipped a sterile cotton swab into the bacterial suspension and spread on a TSA agar plate containing 10 µL/mL NAD and 5% (v/v) inactivated bovine serum, after the plate dry for 3 min, using sterilized tweezers to pick up the drug sensitive disk and place it on the dried plate. There are no more than 6 drug sensitive disk on each plate. At last, placing the plate at 37 ℃ for 24 to 36 h, and measuring the diameter of the inhibition zone.

Data analysis
Data from all samples were analyzed as descriptive statistics. All data were statistically analyzed by GraphPad software (GraphPad software®, La Jolla, CA, USA).

Prevalence of H. parasuis
We isolated 8153 strains of bacteria from 14,610 samples in 26 provinces from January 2016 to December 2018. Through morphological observation, gram stain (Fig. 1A) and PCR identi cation (Fig. 1B), 1386 strains of H. parasuis were nally con rmed, and the isolation rate was 9.49%.

The site and geographical distribution of H. parasuis isolation
Between 2016 and 2018, we made statistics on the separation rate of samples from different tissues in order to nd out the isolation of H. parasuis from different tissues. We found that the highest bacterial isolation rate of effusion was 27.27% (3/11), followed by 13.23% (1312/9914) of lung. a small amount of H. parasuis could also be isolated from joints, spleen, brain and liver, while kidney and heart could not be isolated ( Fig. 2A).
In order to nd out the situation of H. parasuis infection in different areas of China in recent three years, we counted the isolation rate of samples in various provinces. The majority were from Guangdong province (15.3%), Zhejiang province (11.82%) and Hunan province (9.38%) (Fig. 2B).
The separation rates of different H. parasuis serotypes From 2016 to 2018, we identi ed a total of 1386 strains of H. parasuis, including 446 strains in 2016, 561 strains in 2017 years, and 379 strains in 2018 years. We identi ed the serotypes of H. parasuis isolates by PCR (Fig. 3A), while we did not identi ed strains of serotype 8.

Drug resistance pro les of H. parasuis isolates
The drug sensitivity analysis of 166 H. parasuis isolates found that 18 tested drugs included a total of 94 drug resistance pro les. As shown in table 4, only 5 isolates were sensitive to all tested drugs, 10 isolates were resistant to 1 tested drug, 15 isolates were resistant to 2 tested drugs, 33 isolates were resistant to 3 tested drugs, and 39 isolates were resistant to 4 tested drugs resistance, 21 isolates were resistant to 5 tested drugs, 16 isolates were resistant to 6 tested drugs, 13 isolates were resistant to 7 tested drugs, 10 The isolates were resistant to 8 tested drugs, the tested isolates were mainly resistant to 3-5 tested drugs.

Discussion
H. parasuis is a Gram-negative, nicotinamide adenine dinucleotide dependent bacterium. Who can cause Glässer's disease in pigs, (14). H. parasuis usually appear in swine respiratory tract, causes systemic infections, pneumonia, brin polyserositis, polyarthritis and meningitis (15). This bacterial infectious disease can infect pigs of any age, which brings serious in uences to the pig breeding industry. Although the mortality rate of pigs is relatively low, it will seriously affect the disease resistance of the pig herd, resulting a decline of the immune abilities, and it is easy to be infected with a variety of infectious diseases, causing complex clinical symptoms, and bringing great di culty to disease diagnosis. At present, prevention and control of H. parasuis are di cult, the trend of large-scale and intensive development of the pig industry in our country is becoming more and more obvious recent years, and the prevalence of H. parasuis diseases is becoming diversi ed and complicated, which often presents as a secondary or mixed infection, and brings great di culties to the diagnosis and treatment of diseases. In order to reduce the economic damage caused by Glässer's disease, a kind of inactivated whole cell vaccine is widely used in the world (16). While inactivated whole cell vaccine does not produce local immunity and cell-mediated immunity ability is weak, so the immunity is slower, and good immunity is usually obtained 2 weeks after vaccination. Now, subunit vaccine is currently the best research direction (14).
Our work studied the serotypes of H. parasuis isolated from large-scale pig farms in China from 2016 to 2018, which played a vital role for the research of vaccines against prevalent H. parasuis strains in our country. H. parasuis isolates accurately would help to prevent and control Glässer's disease through appropriate vaccination in speci c geographic areas.
we rst identi ed 8153 strains of H. parasuis from 14610 disease materials derived from 26 provinces and cities of China, our sample size and typing method are larger and more representative. We Serotyped 320 H. parasuis strains by multiplex PCR. Molecular typing is an excellent alternative test compared to regular serotyping (gel immunodiffusion, Kielstein and Rapp-Gabrielson scheme), which is very cumbersome to perform because of the necessity of producing speci c anti sera (16). The Kielstein-Rapp-Gabrielson agar diffusion method for serotyping of H. parasuis is classic serological typing method, which can identify 15 serotypes, but 15 high immune serums are required, and about 20% of strains cannot be typed.
This study used the multiple PCR molecular serotyping method established by Howell et al in 2015 to serotype 320 H. parasuis isolates. The results showed that only 10% of the isolates could not be typed. The molecular method has the advantages of simple operation and less time-consuming in the mass identi cation of serotypes, but it is worth noting that this method cannot distinguish between serotype 5 and serotype 12. Using a multiplex PCR assay (8) and a speci c PCR reaction for the H. parasuis serotyping were more precise.
Our research also showed that annual isolation rate of H. parasuis ranged from 7.36-12.34%. The months with higher isolation rates were December, January, February, March, and April, and the isolation rates showed a clear upward trend in October and November.
Combined with data analysis over the past three years, the incidence of H. parasuis in coastal areas of high temperature and humidity such as Guangdong, Fujian, Zhejiang and Guangxi were relatively high. This may be due to the local climate was suitable for bacterial growth and reproduction, because climate was an important factor affecting the growth of bacteria (22).
Most Clinical studies had shown that the protection of inactivated vaccine was mainly against isolates of the same serovars, and its cross-protection was extremely limited (10), for this reason, choosing inactivated vaccines of local epidemic serotypes was more bene cial to defend against H. parasuis. In different regions and a combination of protective antigens might be able to provide effective protection against multiple H. parasuis serovars (23).
The susceptibility testing results showed that H. parasuis was more sensitivity to macrolide antibiotics, polypeptide antibiotics, chloromycetin, and β-lactam antibiotics (except ampicillin) among all 18 selected drugs tested, H. parasuis showed the highest sensitivity to polymyxin B and cefradine, followed by ceftriaxone, orfenicol, cefotaxime, ceftazidime and azithromycin. At the same time, H. parasuis was resistant to cipro oxacin, streptomycin, ampicillin, nor oxacin, amikacin and levo oxacin. The drug sensitivity analysis of 166 H. parasuis isolates found that 18 drugs tested included a total of 94 drug resistance pro les. Only 5 isolates were sensitive to all tested drugs. Now, abuse of antimicrobials in farmed animals was a hazard to humans, besides, H. parasuis clinical isolates had been reported to exhibit resistance to several antibiotics (24). So, vaccines were preferred method in control and elimination of Glässer Disease.
However, the current vaccines of H. parasuis were too poor, and cannot prevented H. parasuis disease. Recently, it was reported that subunit vaccines were better choice for treating diseases, However, in contrast to other diseases of similar importance, there were few effective subunit vaccines (25), in the light of our work could provide reference and basis for prevention and control of H. parasuis, the follow-up researcher could combine the prevailing trend of the dominant strains in this article, and prepared targeted vaccines.
Our work studied the serotypes of H. parasuis isolated from large-scale pig farms in China from 2016 to 2018 and the resistance characteristics of isolates, which played a vital role for the research of vaccines against prevalent H. parasuis strains in our country, and serotype H. parasuis isolates accurately would help to prevent and control Glässer's disease outbreaks through appropriate vaccination.

Conclusion
Haemophilus parasuis (H. parasuis), the causative agent of Glässer's disease, which seriously affected the global pig breeding industry. Our research showed a national trend of H. parasuis in China, our study was carried on from 2016 to 2018 and 8153 H. parasuis eld strains were isolated from 14610 clinical samples collected from sick pigs with clinical symptoms in 26 provinces and cities of China, among them, 1386 strains were identi ed as H. parasuis by PCR, and the isolation rate was 9.49%.
We Serotyped 320 H. parasuis strains by multiplex PCR, and our results showed that type 5/12 and type 4 strains had the highest proportion respectively were 38.44% and 25.31%, followed by type 13 and type 14 strains respectively were 7.81% and 6.56%, besides, 10% of isolates cannot be typed by this method.
Our antimicribial susceptibility test showed that H. parasuis was very sensitivity to polymyxin B and cefradine, then ceftriaxone, orfenicol, cefotaxime, ceftazidime and azithromycin. At the same time, H. parasuis was resistant to cipro oxacin, streptomycin, ampicillin, nor oxacin, amikacin and levo oxacin.
In general, our results revealed the diversity and distribution of different serotypes of H. parasuis across the country and the resistance characteristics of isolates, which were essential for the prevention and treatment of H. parasuis in our country.