4. Discussion
In recent years, cultivation of leeches has been growing China. Among the leeches being cultured, H. nipponia is the important representative species(Zhang., 2009). As Hirudin harbors anticoagulation, antithrombotic, anti-inflammatory, anti-tumor and anti-fibrosis activities, it plays an important role in the prevention and treatment of disease(Ahmad, 2006; Rowghani et al., 2007). Therefore, the market demand of Hirudin continues to expand and lead to the rapid development of the leech breeding industry. However, with the continuous expansion of the leech breeding industry, disease rates have also increased. Although, a small number of leech parasitic diseases were identified (Gouda, 2006; Hamilton et al., 2005), there are only a few reports discussing bacterial pathogens of leeches, such asEscherichia coli , Proteus mirabilis , Salmonella andAeromonas hydrophila (Mo et al, 2003; Zhang et al., 2009; Zhang et al., 2006). In this study, we first isolated a dominant SZ01 bacteria strain from H. nipponia and identified it as K. pneumoniaeusing biochemical identification, 16S rRNA sequence analysis and a phylogenetic tree. According to the phylogenetic tree analysis, the isolated bacteria and K. pneumoniae (GenBank login No. KF733734.1) were clustered into the same branch(Figure 3)and showed the highest homology. However, K. pneumoniae (GenBank login No. KF733734.1) was isolated from the human oral cavity. Results of the pathogenicity experiment revealed that K. pneumoniae was the main pathogen of this disease in H. nipponia . K. pneumoniae was found to infect H. nipponia and lead to a large number of deaths.
K. pneumoniae includes many virulence genes such as uge(encodes uridine diphosphate galacturonate4-epimerase), wab G (is involved in the biosynthesis of the outer core lipopolysaccharide),urea (related to the urease operon), mag A (mucoviscosity-associated gene A), mrk D (type 3 fimbriae adhesion), all S (activator of the allantoin regulon),kfu BC (iron-uptake system), rpm A (regulator of mucoid phenotype) and fim H (fimbrial gene encoding type 1 fimbrial adhesion), all which play prominent roles in bacterial pathogenesis (Gao et al., 2014; Lascols et al., 2013). K. pneumoniae widely exists in water, soil and grain, and is a parasitic pathogen in the respiratory and intestinal tracts of terrestrial animals. K. pneumoniae is ubiquitous in nature and has the ability to infect a wide range of mammalian organisms, including Homo sapiens and the California black langur (Holden et al., 2016; Jang et al., 2010; Magill et al., 2014). In humans, K. pneumoniae infects the human respiratory tract and is associated with pneumonia, liver abscesses and wounds (Bubeck et al., 2007; Clegg et al., 2016). K. pneumoniae had also been reported in aquatic animals such as fish, shrimp and crabs (Singh et al., 1992) and can cause bacterial diseases in soft shelled turtles, Eel, Nemipterus japonicus , Cyprinus carpio and Labeo rohita (Das et al., 2018; Xu et al., 2002; Deng et al., 2009; Diana et al., 2012; Oliveira et al., 2014). In this study, K. pneumoniaewas isolated from the body of H. nipponia and caused symptoms of ”edema and congestion” in H. nipponia , with a high mortality and high pathogenicity. The SZ01 strain that was isolated showed the highest homology with K. pneumoniae from humans, indicating that it is likely to cause human disease. Based on drug sensitivity studies, SZ01 appeared to only be sensitive to ciprofloxacin and sulfisoxazole. Otherwise, due to a large number of antibiotics and other drugs used as treatment, K. pneumoniae isolated from H. nipponia was a strong resistant strain, which makes it difficult to prevent and control of this disease. When cultivating H. nipponia , to treat noticeable disease as quickly as possible, a large number of antibiotics and other drugs are used, which may lead to resistance.
Modern biology supports the identification, isolation and synthesis of anticoagulant substances from leeches, among which Hirudin is the most representative. Hirudin is a medicinal substance extracted from leeches, with a peptide chain containing 65-67 amino acid residues. It is slightly acidic and has a molecular weight of 7 kDA. It can also exist stably in a dry state. Compared to other anticoagulants, Hirudin is the most efficient and does not lead to allergic reactions, making it ideal as an anticoagulant(Pan et al., 2006). Hirudin is now widely used as an antithrombotic, anti-congestion, anti-edema, anti-inflammatory and analgesic. Some studies show that Hirudin can be used to inhibit tumorigenesis(Lv et al., 2003). Extraction methods of Hirudin mainly include sun drying, low-temperature drying and freeze-drying(Elmadhun et al., 2013). During the Hirudin extraction process, the leech may contain K. pneumoniae but extraction conditions are not able to destroy bacterial toxins. Therefore, toxins from K. pneumoniaewill exist in extracted Hirudin. Furthermore, this will affect therapy and may cause a threat to humans.
To solve this issue, close attention should be paid to the prevention of leech diseases, which may occur through improved feeding conditions and methods. We have identified many issues in the cultivation of H. nipponia in Hubei Province. The cultivation of leeches has shown a state of disordered development and lacked government management and guidance, with a supply exceeding demand. Due to disordered development of leech breeding, the number of wild leech seedlings decreased resulting in a decline in the quality of the whole seedling. On the other hand, human pathogenic bacteria, such as K. pneumoniae, may also cause leeches to get sick. Human pathogens might release toxins and remain in leeches. Thus, the toxin would further pollute Hirudin and other pharmaceutical products, causing major medical issues. In addition, the direct consequence of frequent occurrence of disease is drug abuse. The abuse of drugs, especially antibiotics, leads to the emergence of drug-resistant strains such as highly resistant K. pneumoniae . At last, leeches in the Hubei province, especially H. nipponia , are mainly fed snails that are important hosts forSchistosoma (Lv et al., 2019). Therefore, Schistosomaeggs may be mixed in leeches, which endanger the safety of farmers and also contaminate products related to Hirudin, and then endanger patient safety.
To this end, we suggest: 1. To designate the government regulatory department for leech breeding, strengthen the guidance of its breeding industry and perform orderly breeding of leeches; 2. Measures should be taken to reduce the density and incidence of disease in leech culture to avoid contamination by bacterial toxins; 3. To reduce the use of drugs in the process of leech breeding, perform green scientific prevention and control of leech diseases to reduce the use and residue of drugs; 4. Strengthen the inspection and quarantine of leech related products, resolutely stop the sale of problematic products and destroy them. In conclusion, we propose that there are some factors threatening human safety in products that are related leeches. K. pneumoniae is an important representative pathogenic bacterium that deserves attention.