Among the 1362 samples collected randomly from 14 cities of Guangxi, 655 samples were detected Mhp positive by real-time PCR. The mhp positive rates in different regions are as shown in Fig. 1: Liuzhou 85.71% (36/42), Laibing 85.12% (143/168), Hezhou 80.00% (12/15), Hechi 68.00% (68/100), Guigang 64.65% (64/99) and Guilin 58.02% (47/81) all had the high Mhp positive rates. What is more, the overall Mhp positive rate in Guangxi is 48.09% (655/1362), indicating a relatively serious Mhp infection in the Guangxi region. Among them, Liuzhou, Laibin, Hezhou, Hechi, Guigang, and Guilin have a positive rate of over 50%, making them being severely affected areas of Mhp infection. Based on the small sample size from Hezhou (15 samples), it is not statistically significant and thus will not be discussed for this time. Liuzhou, Laibin, Hezhou, Hechi, and Guilin have high positive rates, which may be related to their geographical location. All the above areas belong to the northern mountainous region of Guangxi (Fig. 2). The areas located in north of Guangxi is more mountainous and the temperature is relatively cooler than in the south. Although vaccination has a beneficial effect on infected pigs, these effects vary among different pig populations. These differences may be caused by various factors, such as infection levels, infection age, and variations in different isolates of Mhp[9, 10]. However, vaccination is still considered the most effective approach for controlling this infection[11]. Although vaccination can significantly reduce clinical symptoms and lung lesions, it only has limited effect in reducing the transmission of Mhp[1, 10]. It is worth noting that this disease does not restrict commercial trade [6]. Guangxi is a major province in China's pig industry, and the pig trade radiates to the surrounding provinces such as Guangdong, Jiangxi, which promotes the spread of diseases. That may be the main reason of a high overall infection rate of Mhp in Guangxi Province. This disease is widely prevalent in Guangxi China now. Finally, Guigang, Chongzuo, and Nanning have relatively high Mhp positive rates, which may be due to the fact that these areas are the main producing areas of pigs in Guangxi.
In the present study, genotyping of Guangxi China Mhp strains was performed comprehensively. Among the 655 positive samples, a total of 61 samples were amplified with the adk, rpoB, and tpiA genes simultaneously. In the multilocus sequence typing (MLST) method for Mhp, it is necessary to amplify seven housekeeping genes simultaneously. However, Mayor et al.[12] have demonstrated that amplification of only three housekeeping genes (adk, rpoB, and tpiA) can yield identical genotyping results, and it is often impossible to successfully amplify and sequence all seven housekeeping genes for most samples, possibly due to the low nucleic acid content of Mhp in tissue samples or the presence of certain PCR inhibitors in the samples. Table 1 summarizes the allele types and ST types of successfully amplified samples. In independent analyses of each gene, 2 new adk alleles (56, 57), 10 new rpoB alleles (72, 73, 74, 75, 76, 77, 79, 80, 81, 82) and 2 new tpiA alleles (82, 83, 84) were identified. Moreover, novel ST types were found in all 14 cities of Guangxi. Some ST types (ST199 and ST203) were present in several cities, with the furthest distance (about 520 kilometers) between Guilin in the northeast and Chongzuo in the southwest. It can be concluded from this that long distance is not a barrier to the transmission of Mhp. From the study of Zhanghui et al.[12], it was shown that ST147 was discovered in Guangxi in 2019, and ST128 was the dominant type. Four years later, ST128 remained the dominant trend, but ST147 evolved to become one of the more prevalent types in Guangxi. In recent years, new STs have continued to appear in Guangxi, and some new STs have shown a trend of becoming dominant. For example, ST197, ST199, and ST203 have been discovered in different regions. Due to the limitations of the sample size, only these genotypes were found had a trend of prevalence, and in the future research a large number of samples need to be collected to prove whether multiple STs are already prevalent in Guangxi. The Simpson's index (0.939) analysis indicated that Mhp in China has a high discriminatory power and diversity.
According to the neighbor-joining tree analysis based on the ST sequences, the 61 isolates could be divided into five branches (I, II, III, IV, and V) just as shown in Fig. 3. And the distribution of different types was shown in Fig. 4. Branch I contained isolates from Switzerland, Hungary, and Thailand, and there are two isolates present in this experiment. Branch II contained 10 isolates from this experiment, covering seven cities (Nanning, Hechi, Chongzuo, Guilin, Wuzhou, Laibin, and Guigang), indicating a certain prevalence of type II Mhp in half of Guangxi. At the same time, there are evolutionary relationships between the UK J strain, US 232 strain and the isolates from this experiment, ST194, ST227, ST225, ST198, ST199, ST159 on this branch. However, there was no definite correlation in the alleles; some isolates had three different alleles. Coincidentally, the 4 isolates belonged to branch III were from the same pig farm in Nanning, Guangxi. Branch IV contained a total of eight isolates from this experiment, which have closer evolutionary relationships with the 2016 French strain, 2017 Thai strain, and 2020 Jiangsu strain in comparison to the Swiss strain on this branch. Branch V is a large category that includes the vast majority of isolates from this experiment and nearly covers all cities in Guangxi. This branch also includes the 2018 Jiangxi strain (JX486), 2019 Guangxi strains (GX23, GX8-2, GXF10), 2020 Guangdong strains (GD-22, GD-18), and 2020 Jiangsu strain (JS-10). It can be seen that this type had already appeared in other regions of China several years ago and showed a trend of prevalence in Guangxi and Guangdong regions. What is more, the close relationship between isolates from Guangxi and from other provinces may be related to the fact that Guangxi Province is a major province in China’s pig industry. The possibility of mhp spreading when selling pigs to Guangdong, Jiangxi and other provinces cannot be discharged. From Fig. 2, it can be seen that type II and type V cover the majority of prevalent strains in Guangxi. This indicated that Mhp has been presented in Guangxi for a long time and has formed individual clusters. It is speculated that high diversity of Mhp within Guangxi is the reason behind the high infection rate and difficult-to-control situation of Mhp in the region.