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.