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Advances in the Development of Anti-Trichinella spiralis Vaccine, Challenges, and Future Prospective

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Muhammad Tahir Aleem, Ruofeng Yan, Asad Khan, Rida Asrar, Amna Shakoor, Areej Asif, Zhaohai Wen, Zhengqing Yu, Muhammad Abdullah Malik, Tauseef-ur-Rehman, Rao Zahid Abbas, Muhammad Mohsin, Xiaokai Song, Lixin Xu and Xiangrui Li

Submitted: December 19th, 2021 Reviewed: February 2nd, 2022 Published: May 11th, 2022

DOI: 10.5772/intechopen.103027

Parasitic Helminths and Zoonoses - From Basic to Applied Research Edited by Jorge Morales-Montor

From the Edited Volume

Parasitic Helminths and Zoonoses - From Basic to Applied Research [Working Title]

Prof. Jorge Morales-Montor, Dr. Víctor Hugo Del Río-Araiza and Dr. Romel Hernández Bello

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Trichinellosis is a food-borne, zoonotic disease that causes infection by a nematode parasite belonging to the genus Trichinella. This is an important disease, and its causative agent is prevalent throughout the world (cosmopolitan). More clinical awareness of trichinellosis is required due to its many outbreaks, increase in the consumption of pork meat and its by-products. Trichinellosis is an epizootic in nature and its economic burden is associated with the prevention of this disease from the human food chain. This disease is transmitted from animals to humans through the consumption of raw or undercooked meat containing encapsulated muscle larvae of Trichinella spiralis. This paper demonstrates the direct effect of progesterone (P4) and mifepristone (RU486) on the progesterone receptors of T. spiralis. Also, studied the challenges in the preparation of DNA and recombinant protein vaccination to control trichinellosis. It is simply done this study at different life cycle developmental stages of T. spiralis. Vaccines development against T. spiralis infection is the new paradime shift from prevention of trichinellosis to fulfilling the food safety requirements.


  • Trichinella spiralis
  • immune response
  • progesterone receptor
  • hormones
  • vaccine

1. Introduction

The phylum Nematoda consists of non-segmented invertebrates commonly known as roundworms that occur in a wide range of habitats around the globe and lack jointed appendages. The causative agent of trichinellosisis Trichinellaspecies including Trichinella spiralis(T. spiralis) which belongs to the superfamily Trichinelloidea of the phylum Nematoda. At present, the recognized number of species and genotypes in the genus Trichinellaare nine and three respectively based on the larvae appearance in muscle cells [1, 2]. The most pathogenic and prevalent pathogen in this genus is T. spiralis[3]. This is a most serious zoonotic food-borne parasite which can infect a wide range of hosts, is liable for trichinellosisdisease in humans, and can infect more than 150 species of animals such as carnivores and omnivores including human beings worldwide [4, 5]. Trichinellosisoccurs around many parts of the world and infects a huge number of human beings. Its ranges from Europe, North America, China, Japan, and Tropical Africa. However, China is the most affected country [1, 6].

The definitive hosts of nematode Trichinellainclude many domestic animals such as pigs, horses, and wild animals like bears, rats, and wild pigs. Each intermediate host of Trichinella spiralisis also its definitive host and serves as a source of infection for any other definitive host species by carnivorism [2]. Humans can acquire infection by the ingestion of undercooked, or raw meat of these animals contaminated with the larvae of T. spiralis[7]. The important preventive measure (to limit people from getting trichinellosis) is to disrupt the transmission of infective Trichinellalarvae encapsulated in meat to human beings [8, 9]. However, in some countries, this disease is also transmitted by wild animals [10]. Since an enormous amount of pork and its by-products are consumed in China, that results in increased issues in the country [5, 11]. Unlike many other helminth parasites, the survival of T. spiralisnematodes is only direct host-to-host transmission adapting normal cellular functions and host immunity at all the stages of infection [12, 13].

The life cycle of T. spiralisstarts when an adult female and male worms reproduce sexually in the small intestine of the host. The unique characteristic of T. spiralisamong other parasites is that it passes all the stages of its life cycle within a single host including all phases of adult worm, newborn larvae, and muscle larvae [10, 14, 15]. Humans acquire the infection when they ingest Trichinellalarvae that are encapsulated in the striated muscles of domestic or wild animals [5]. After the consumption of infected meat, parasitic larvae which are encysted in the meat are then released into the host stomach by acid-pepsin digestion [16]. Columnar epithelial cells of the intestine at the base of the villus are invaded by muscular larvae. Previously released into the stomach from meat and each molts four times to reach sexual maturity [7, 9, 17]. Approximately 1500 newborn larvae are produced by each fertilized female T. spiraliswithin 2–3 weeks and up to 10,000 over 4 months period. These larvae penetrate the intestinal lining with the help of unique sword-like stylet they possess and migrate to the striated (skeletal) muscles via the circulatory and lymphatic system [15]. These larvae can enter any type of cells but only survive in skeletal muscles. In the striated muscles, these previously migrated newborn larvae develop into infective muscle larvae and transform the skeletal muscle cells into a new type of cells known as nurse cells which maintain the larval life and for many species of Trichinellachanges into capsules or cysts made up of hyaline and collagen fiber [8]. These capsules containing the muscle larvae can persist for many years and calcification occurs in most of the cysts and dies within a few months (see Figure 1). The life span of live adult worms in the mucosa of the intestine is 4–6 weeks in human beings while the muscle larvae encapsulated in the striated muscle fibers persist for months to years [1].

Figure 1.

Life cycle ofT. spiralis[10].

Immunity is the defensive mechanism against any pathogenic organisms that invade the victimized host. When the host consumes contaminated meat containing nurse cells, an immune-mediated inflammatory response starts due to the development of the adult worms in the epithelium of the intestine to expel the parasites. The level of antibody IgE which defends the body against parasitic organisms starts to increase. The inflammatory infiltrates containing mast cells and eosinophils present there pathologically. Both these immune cells are involved in the clearance of parasites. Toxic oxygen molecules and major basic proteins are elaborated by eosinophil to kill invaded organisms but also cause to damage the host body tissues. While mast cell protease-1 (MCP-1) is produced by mast cells that are also lethal to worms. There is widespread inflammation, edema if worm load is high and cells death occurs frequently during the parenteral stage of infection [18, 19].

Trichinellosis infection is classified into three stages depending upon the life cycle of the pathogenic worm; (1)as an invasive stage, in which larvae grow into adult worms and after fertilization females begin to release newborn larvae which then migrate to blood circulation via the Lymphatic system [9]. This stage is characterized by nausea, diarrhea, abdominal cramps, and seldom vomiting. Constipation is also seen in some of the individuals instead of diarrhea. All these symptoms appear within 2–30 h of post-eating infected food. (2)As migratory phase; characterized by the encapsulation of larvae in the muscles of the host. The main symptoms observed in this stage include fever, face edema, swelling, muscle pain, and weakness of the infected muscles [14, 16]. (3) As encystment stage; characterized by the calcification of cysts in the striated muscles only and results in everlasting injury [1]. As this parasite shows nonspecific signs and symptoms of the disease, its clinical diagnosis is difficult [15]. For the diagnosis of Trichinellosis, the digestion method is the best method reported by World Organization for Animal Health (OIE) but to better detect the Trichinellaparasites molecular biology and serologic methods have been developed [5]. Currently, Trichinellosisdiagnosis is based on larvae detection in muscle biopsy or immunodiagnostic tests which are highly specific. Many antigens are expressed during the developmental stages of the T. spiralisand are useful for the serodiagnosis of trichinellosis. However, due to limited T. spiralisantigens availability testing is not extensively available [15, 16].

Trichinellosisis not only responsible for public health casualties but also cause the economic problem in food safety and swine animal production. Due to a large number of people infected with T. spiralis, this disease is regarded as re-emerging in many regions of the world [2, 15]. If transmission of this disease is not under control, it can lead to serious public health problems [10].


2. Status of anti-T. spiralisvaccine

It is a promising method for the control of parasites in pigs to develop a vaccine against T. spiralisinfection. However, most of the studies for the development of vaccines against Trichinellahave been performed in lab animals (Mouse models) so far. Only a few studies are performed on pigs for the development of vaccines against Trichinellainfection. To prevent and control the transmission of T. spiralisinfection from pigs to humans vaccine exploitation is an important step [20]. Trichinellais a tissue-lodging, enteral, and multicellular parasite. Its life cycle is complex and has a diverse developmental phase. Trichinellaworms have stage-specific antigens [21]. It is necessary to develop an effective vaccine against Trichinellato interrupt the transmission of parasites among animals and the cycle of pathogen transmission from swine to humans [5].

Till now, various practices and strategies have been used in the prevention and eradication of parasites including the application of chemicals. The chemical methods are not well signed and have certain limitations such as continuous use of antiparasitic drugs resulting in rising resistance, have risks related to the environment, health, and their potential effects on the host or non-target organisms. Chemotherapy and antiparasitic drugs are used to prevent T. spiralisinfection, but when we compare the vaccination of animals with chemotherapy treatment, we found that it has several advantages. A single dose of vaccine can provide lifelong prevention of T. spiralisinfection, reduce the risk of drug residues in meat and other by-products, and decline the emergence of T. spiralisdrug-resistant parasites [2].

Anti-Trichinellavaccines will provide a substantial contribution to the control, prevention, and elimination of Trichinellosis. The eradication of Trichinellaspp. infections in animals is a difficult task as vaccines against Trichinellathat act as a preventive weapon are not currently available except for rats and pig models [6]. For the past three decades, significant improvements have been made for the recognition of several antigens from T. spiralis. It will lead toward a better understanding of the formulation of novel vaccine developments. A variety of vaccines such as subunit vaccines, recombinant proteins vaccines, inactivated vaccines, synthesized epitope vaccines, DNA vaccines, viral or bacterial vector vaccines can elicit an immune response against Trichinellaand provide effective protection. Scientists have used different antigens to formulate recombinant protein vaccines and many of them have provided some effective protection against Trichinellainfection [4, 10].

Preventive vaccine development against Trichinellainfection in domestic pigs is valuable to control and prevent this parasite [21]. The diseases can also be controlled in animals through a veterinary vaccine. To induce long-term intestinal immunity the appropriate route for immunization against Trichinellosisis oral as the infection occurs due to the ingestion of poorly cooked meat containing encapsulated infective larvae [20]. Proteases (enzymes) are widely distributed in viruses, prokaryotes, and eukaryotes participate in different events of the parasite’s life cycle. In the process of causing infection, parasites serine proteases are thought to be a key factor and exist in the T. spiralisexcretory-secretory (ES) products [20, 22]. The hydrolyzing enzyme Elastase (trypsin-like serine protease) helps the parasites in the penetration of host tissue through the hydrolysis of laminin, fibronectin, elastin, and type IV collagen. Elastases also participate in the immune evasion process. This enzyme is also involved in the digestion and molting of parasites and has an important role in parasitic worm intrusion of victimized hosts. It might be a target for a novel vaccine [21]. Recently, many anti-T. spiralisvaccines have been developed to interrupt the transmission of parasites from animals to humans. Many vaccine candidates which are effective against T. spiraliswere selected from ES products and recombinant proteins. Serine protease enzymes (from T. spiralis) provide partial protection against T. spiralislarvae challenges [20].

T. spiralisexerts an immunomodulatory effect through ES products on the immune response of the host [22]. T. spiralisNudix hydrolase (TsND) is a protein that binds to intestinal epithelial cells of normal mice (up-regulated gene). The size of this gene is about 1248 bp. A partial protective immunity against Trichinellainfection was observed when mice were vaccinated with recombinant TsND protein [22, 23]. Vehicle (delivery system) and antigen are important elements that are responsible for the protection level induced by the candidate vaccine [24].


3. T. spiralis-associated immune mechanism

T. spiralis(Helminth) establishes infection which is long-lasting in the striated muscles of the host. Depending on the longevity of the host. It can persist successfully until the end of life in rodents and in higher species including humans can persist over several months to years following infection. They do not kill the host striated muscles cells during their stay, unlike some other intracellular parasites. This characteristic makes them one of the most successful symbiotic parasites [25]. Parasitic nematode T. spiraliscompletes its entire life cycle in one host and each stage of its life cycle provokes the immune system of the host differently [14, 15]. For the establishment of the life cycle successfully, this parasite with the help of its defensive mechanism manages to escape the host immune system responses. The excretory-secretory products of T. spiralisplay a crucial role in the establishment of parasitism and modulation of host immune response to protect both host and the parasite [22]. When the host acquired the infection of T. spiralis, at early-stage cellular immunity of the host is inhibited but later recovery of host cellular immune function occurs, and humoral immunity starts its role in resisting the infection of T. spiralis.During the infection, both the cells Th1 (T helper cell) and Th2 play a major role in maintaining the immune system function. They are involved in the eradication of pathogens. When the maintenance of the host immune system disrupts it gets infected [18]. Nitric oxide (NO) is a molecule in the immune system which acts as an immunomodulator and immunotoxin. It is a gaseous molecule and has appropriate lipid membranes solubility. Without binding to any specific receptor of viruses and bacteria, it exerts lethal effects on them. NO is involved in the selective killing of parasites including infected cells and has a major role in the adult worm expulsion during Trichinella spiralisinfection in mice (see Figure 2) [12].

Figure 2.

Immune response againstT. spiralis.

Infection of T. spirilishas an immunosuppressive effect on the innate immune system of the host. Larvae release secretory antigens that elicit a protective strong immune response which is specific to invading parasites [20]. ES products reduce inflammation when parasites invade the muscle cells, modulate the host immune system response in a way to protective for both the host and the parasites. For survival inside the organism, T. spiralisbuild a unique place for their living and their niche contains a cyst or capsule composed of nurse cell (cellular components) and collagenous wall [22, 25, 26]. Both the wall and nurse cell are originated from the host, provide protection and maintenance of the parasite’s metabolism respectively [14].

Macrophages play a major role in the immune response of the host against various pathogens [22]. In vitro, ES products from different phases of the life cycle of T. spiraliscan modulate macrophage’s function by inhibiting cytokine production. In chronic helminth infections, macrophages are activated by Th2 cytokines such as interleukin-4 (IL-4) and interleukin-13 (IL-13). Many immune mediator molecules are released such as IL-6, IL-12, nitric oxide (NO), and tumor necrosis factor (TNF-α) when in macrophages the signaling pathways are triggered by Th2 cytokines [22, 25, 27].


4. Genomic and proteomic profile of T. spiralis

There are 12 species and genotypes of Trichinellawhich are distributed worldwide and cause serious disease Trichinellosisin humans which leads to morbidity and mortality [1, 2, 12]. Based on larvae appearance in the muscle cells of the host only encapsulated and non-encapsulated clades (morphological distinct) Trichinellais recognized. Based on molecular studies, nine species and three genotypes of Trichinellashow a wide biological diversity. Based on genetic data, only Trichinellaencapsulated clade infects mammals includes Trichinella spiralis(T1), Trichinella nativa(T2), Trichinella britovi(T3), Trichinella murrelli(T5), Trichinella nelson(T7), and Trichinella patagoniensis(T12). The three Trichinellagenotypes includes T6, T8, and T9. The Trichinellanon-encapsulated clade includes Trichinella pseudospiralis(T4), which infects birds and mammals only, Trichinella papuae(T10), and Trichinella zimbabwensis(T11), they infect reptiles and mammals [1].

Proteomics (because of bioinformatics and mass spectrometry) is an effective technique to examine the modifications after the translation of genes such as proteolysis or glycosylation. These are powerful techniques to examine the samples obtained from pathogens to find the possible proteins involved in the pathogenesis of the disease [12]. Trichinellais substantially different in molecular and biological characteristics from other crown groups. The assembly of Trichinellais 64 million bp in length and about 15,808 proteins are encoded by this genome assembly. In T. spiralisgenome, the estimation of repeat content is about 18% having low GC content (about 27%) relative to the overall genome (34%) and protein-coding region (43%) of Trichinella spiralis[14]. Microsatellites are present in the entire genome and many are distributed in the non-coding sequence of the genome. It leads to genetic diversity due to mutation [28]. During the early stage of Trichinellainfection, Trichinella spiralis14-3-3 protein is a strong immunogenic antigen [29]. Ts14-3-3 is an immunodominant antigen and this protein is also used to detect the whole period of infection with Trichinella.During the early phase of Trichinellainfection, HSP70, cysteine protease, and Ts14-3-3 play a crucial role in balancing the host–parasite relationship. Therefore, these proteins are a good target for the development of vaccines and early immunodiagnostic measures [15].

4.1 DNA based vaccine

DNA vaccines got a glare in the early 1990s and evoked both humoral and cellular responses, when tested and identified, particularly induced cytotoxic T cell response, and abolished the safety concerns associated with the live vaccine [17]. Such vaccines tend to sustain host immune system stimulation in comparison to the Recombinant protein-based vaccines [6]. DNA vaccines emerged as a strong way of eliciting a humoral and cellular immune response against many parasitological antigens in small animal models. Moreover, DNA vaccines produce a concurrent Th1 and Th2 immune response against T. spiralis[30, 31].

The TspE1gene encoding a 31 kDa antigen of T. spiralishas been cloned to an expression vector pcDNA3 and administered in a mouse as a DNA vaccine [31]. Naturally, T. spiralischallenge suppresses the type 2 immune system response which inhibits them [17]. The mice immunized with the TspE1-pcDNA3 presented a significant larval reduction rate and an increased serum anti-Trichinellaantibody level, hence this DNA vaccine proved to be partially protective against T. spiralischallenges [31]. Spleen cells after stimulation with the TspE1 recombinant protein exhibited a lymphoproliferative response, which is an indication of cellular response elicited by the DNA vaccine. Sequence of a serine protease (Ts-NBLsp) cDNA from newborn larvae of T. spiralis, cDNA sequence of recombinant TsNd (Trichinella spiralisnudix hydrolases) has been cloned to the plasmid pcDNA3.1 [17, 31, 32]. The antibody response against the serine protease of T. spiralisinhibits the protease activity thus hindering invasion of the parasite. The DNA vaccines Ts-NBLsp-pcDNA3.1 and pcDNA3.1-TsNd presented a balanced systemic Th1\Th2 immune response. The immunization with recombinant TsNd DNA vaccine resulted in an increased intestinal IgA and total IgG response with an exalted IgG1 than that of IgG2a [31]. To compare the recombinant nudix hydrolase DNA vaccine, the Ts-NBLsp-pcDNA3.1 vaccine showed a dominant IgG2a anti trichinellaantibody and a predominantly Th1 immune response [17]. DNA vaccines elevated IFN gamma, IL-2, IL-4, and IL-10 levels [31]. Secretory IgA causes a significant reduction in the female worm fecundity and this response is enhanced by cytokine IL-10 specifically. The intestinal mucosa of the infected animals produces a specific antibody response against T. spiralis. Ts-NBLsp-pcDNA3.1 and reduces the muscle larvae burden (77.93%) greater than that of the TsNd vaccine (53.9%).

In another study of the TsDNase II, the complementary DNA sequence of T. spiralisserine protease 2.1 has been cloned to the eukaryotic expression vector pcDNA3.1 and administered as a DNA vaccine through an attenuated Salmonella typhimuriumto avoid degradation [30]. To elicit a persistent systemic and mucosal immune response against T. spiralis, attenuated salmonella is an effective live carrier that gives an efficient mode of vaccination. T. spiralisDNase II is an excretory-secretory product associated with adult worms and IIL which is expressed in the cuticle of IIL. T. spiralisserine protease appeared to be present in the spliceosome and cuticle of adult worms and intestinal infective larvae. Both of these vaccine candidates against T. spiralisresulted in the significant rise of specific IgG responses. IgG1 titer after the first dose of vaccination and then an increased level of IgG2a after the second dose of vaccination, furthermore they produced mixed Th1\Th2 response which can be described through elicited cytokines response as Th1(IFN gamma) and Th2 cytokines (IL-4, IL10) [30]. TsSP 1.2-pcDNA3.1 vaccine resulted in a 71.84% reduction in the muscle larvae in comparison to the TsDNase II DNA vaccine which caused a 59.26% reduction in the muscle larvae [30].

T. spiralisadult-specific DNase II-1 (TsDNase II-1) and DNase II-7 recognized in the excretory-secretory proteins of the AW [30] has been analyzed for their immune response against the worm. Antibody-dependent cell-mediated cytotoxicity assay (ADCC) revealed that both recombinant anti-TsDNase II-1 and anti-TsDNase II-7 sera mediated the attachment of mouse peritoneal exudate cells (PECs) to NBL and finally killing of the NBL. Paramyosin is a thick myofibrillar protein [6, 30, 33], which is an immunomodulatory protein that evades host immune response by inhibiting complement C1q and C8\C9. TsPmy and Ts87 both are efficient vaccine candidates against T. spiralis. The DNA encoding TsPmy and Ts87 have been cloned in a eukaryotic vector pVAX1 and the recombinant DNA was transformed in the S. typhimuriumstrain SL7207. The resulting DNA vaccines produced protective immunity against T. spiraliswhen administered in mice, both resulted in mucosal sIgA response in the intestine and systemic anti TsPmyIgG response. The antibody-secreting cells from the spleen and mesenteric lymph nodes of the mice immunized with TsPmy vaccine expressed the intestinal homing receptors CCR9 and CCR10. [30] determined that SL7207\ pVAX1-TsPmy vaccine came out with a 44.8% reduction in muscle larvae and a 46.6% reduction in adult worms. While SL7207\ pVAX1-Ts87 caused a 34.2% reduction in muscle larvae and a 29.8% reduction in adult worm burden. By using B and T cell epitopes from TsPmy a novel multi-epitope vaccine has been designed which elicits an immune response more efficiently as compared to traditional vaccines, TsPmy MEP vaccine reduced the muscle larvae up to 55.4% [33].

DNA vaccines have many advantages as they are inexpensive, focused immune response against the antigen of interest, heat stable and a broad-spectrum vaccine can be developed by mixing plasmids.

4.2 Protein-based vaccine

In recent studies, it is reported that specific protein molecules from numerous T. spiralislife cycle stages have been considered and expressed properly, so that their immune protection was also estimated, such as paramyosin (Ts-Pmy) obtained from an adult cDNA library [21], TspGST and fructose-1,6-bisphosphate aldolase (Ts-FBPA) taken from the T. spiralisdraft genome utilizing high expression at the ML stage, Ts31 from the ML ES proteins, serine protease (TsSP) from IIL (intestinal offensive stage) and ML surface proteins and cathepsin B (TsCB) from the T. spiralisdraft genome [8]. On the other hand, when these recombinant proteins were used for vaccinating mice, they showed only 36.2–53.50% ML reduction following the T. spiralischallenge. In the current study, we determine the protective immunity persuaded by vaccination through a novel TsE protein. TsE is highly expressed and acts as a secretory protein at the T. spiralisintestinal offensive stage (IIL), TsE shows potency to be exposed first to the host’s intestinal mucosa and then produce the local immune response through its working. It is observed that vaccination with rTsE persuaded significantly high levels of TsE-specific sIgA, which can simplify adult worm removal from the intestine. TE immune protection having 64.06% ML reduction, with this novel TsE vaccination was considered superior to those of the above-mentioned other T. spiralisproteins act as candidate vaccine target molecules. This study also recognized a foundation to develop polyvalent anti-T. spiralisvaccines in the upcoming period.

The immune response stimulated by a vaccine based on an exclusive antigen and multi-epitope (that work more efficiently than the large protein molecules) vaccines against T. spiralishas now been proposed. Therefore, the amalgamation of three selected epitopes from Ts-Pmy and Ts87 from T. spiralisadult produced in immunized mice IgG and IgG1 production and higher protection of about 35% in contrast to the parasite challenge in comparison to that encouraged by individual epitope peptides [8]. To achieve higher shielding immune responses counter to T. spiralis, it will be essential to propose a vaccine with multi-epitopes from different parasite stages focusing on NBL and adult stages (Table 1).

AntigenDatabase IDStrainDevelopmental stageFunctionReference
Cathepsin B [T. spiralis]XP_003373289ISS 195Muscle larvae(ML) and adult worm (AW)Has important function in worm invading, migrating, molting and immune escape[5]
Cysteine protease ATG4C [T. spiralis]XP_003367319ISS 195Intestinal infective L1 larvae (IIL1)Participates in IIL1 intrusion of the enteral epithelium[5]
Putative serine protease [T. spiralis]XP_003369429ISS 195AWInvolved in the processes of immune evasion[17]
Paramyosin [T. spiralis]XP_003371652ISS 195AWPlays an important role in the evasion of the host complement attack[10]
Conserved hypothetical protein, partial [T. spiralis]XP_003369591ISS 195AW[10]
Serpin B4 [T. spiralis]XP_003375999ISS 195AW, NBL, MLInitiates acute inflammatory response[7]
Putative nudix hydrolase 6 [T. spiralis]XP_003378071ISS 195IILCatalyzes the hydrolysis of nucleoside diphosphate[23]
Cystatin-B [T. spiralis]XP_003379766ISS 195AWHas immunomodulatory functions and helps the parasites to evade the host immune responses[34]
Antigen targeted by protective antibodies [T. spiralis]AAA20539Library lambda ZAPMLRegulates host immune response[31]
Membrane-associated progesterone receptor component 2 [T. spiralis]XP_003375934ISS 195AW, MLPlay a role in adipocyte function and systemic glucose homeostasis[11]

Table 1.

Immunoregulatory kinetics of different T. spiralisbased protein after binding with host immune cells.


5. Role of progesterone receptor in trichinella spiralis

Progesterone (P4) is a sex steroid hormone that plays roles in the physiology of the reproductive system such as corpus luteum of the ovary and placenta in females, while testes and adrenal cortex in males also participate in many other functions such as brain activity, immune modulation, metabolism of bones heart and lungs physiology. P4 is also responsible for the maintenance of pregnancy and shows an immunosuppressive effect [35]. when a high level of progesterone is present during the luteal phase of the estrus/menstrual cycle in females. Recent studies showed that these hormones also influence the course of parasites infections and also restrict the invasion of parasites when a high level of P4 in female animals is produced. Restricts the invasion of parasites [11]. P4 has an immunomodulatory effect on fetal antigens during pregnancy by suppressing the maternal immune response. However, progesterone can be either an inhibitory or stimulatory effect on the immune response mechanism depending upon cell type, concentration, and exposure time to steroids. It has nematotoxicity against newborn larvae of T. spiralis. Progesterone is responsible for decreased parasite load during pregnancy [11].

Sex steroids are known as immune response modulators and play a major role in T. spiralissusceptibility at two levels viz. (1) protective immune response and (2) direct effect on the development of worms. Besides, P4 up-regulates many molecules expressions from major histocompatibility complex class I and it also participates in the down-regulation of genes that are responsible for the fecundity and oviposition of the worms and inhibits the nuclear factor kappa B (NFƙB) activation in innate immunity [11].


6. Role of progesterone and mifepristone against T. spiralis

Progesterone is a gonadal hormone primarily involved in the preparation of the endometrium for implantation of an embryo and necessary for the maintenance of pregnancy, while mifepristone is a drug that works as an antagonist of progesterone and glucocorticoid. It has an abortifacient effect and terminates early pregnancy by binding to intracellular progesterone receptors. Mifepristone has an antagonistic effect on the T. spiralis(Ts) membrane-associated progesterone receptor component-2 (Ts-MAPRC2). It also down-regulates the expression of the Ts-MAPRC2 gene and results in the abortion of the pregnant adult female worms [11].

Mifepristone (RU486) can be taken as an example that works as an antagonist in contrast to the progesterone receptor (PR) and glucocorticoid receptor (GR) with some lethal properties such as aborting agent and anticancer activities in the body. In the case of helminths, several research studies are concentrated on PGRMC receptors. Similarly, RU486 was one of the first medications accepted for surgical abortion and is frequently used to terminate an early or midterm pregnancy. Hereafter, PR and binding of P4 molecules (agonist) and RU486 (antagonist) can be helpful to elaborate T. spiralisspecies regarding differentiation and reproductive development as well as creating potential pharmacological targets that might be used as a drug therapy against Trichinellosis.

Progesterone is known for its immune-modulatory effects, which happen during pregnancy that is done by suppressing the response from the mother toward paternal antigens expressed in the fetus [11]. Taking into the description, we can conclude that progesterone is an adequate inducing activation of the effector cell populations responsible for cell death in an antibody-independent cytotoxic mechanism. This cytotoxicity should also be activated by soluble antigens released by the parasite because at constant self-aggression of tissues by these activated cells 0% NBL mortality 10 10 100 Progesterone (ng/ml) cells [35] .


7. Challenges to developing an efficient vaccine against T. spiralis

The control of helminths in animals is usually through anthelmintics. Vaccine development against T. spiralisinfection in pigs is an alternate method for the prevention of parasite T. spiralisfrom zoonosis. Effective vaccine development against Trichinellosisis conducted in mice instead of pigs. Effective development of a vaccine, is not only due to high price of experimental pigs but also due to poorly satisfied antigens detected from the mice. Moreover, the immune response induced by the same antigen in swine and mice is extremely different. So, [2]. concluded that in mice, poor immunogenic vaccine candidates are not capable to induce a strong protective immune response against T. spiralisinfection in pigs.

TsT was a T. spiralissomatic antigen and at adult-stage with specific surface antigen it had a good antigenicity. If vaccination of mice is done with TsT, it will induce a systemic mixed Th1/Th2 response and an intestinal local sIgA response, which can produce partial protection against T. spiralislarval challenge. Then these results suggested that TsT plays a role in T. spiralisgrowth and survival in the host, and it might be deliberated as a potential target antigen for anti-T. spiralisvaccines. However [9], revealed that oral anti-Trichinella vaccines comprised of multiple antigenic epitopes of various T. spiralislife cycle phases should be recognized.

7.1 Diversity within T. spiralisparasites

T. spiralisis a nematode parasite that is prevalent throughout the world and translocated by humans and their animals. They occupy well-defined geographic ranges [36]. There is a big diversity among the T. spiralisparasites present in different geographic locations [24]. T. spiralisnematode belongs to the clade that diverged early in the phylum Nematoda evolution [14]. T. britoviparasitizes many sylvatic mammals such as Felidae, Canidae, Ursidae, Mustelidae, Suidae, Viverridae and is endemic to Northern-western Africa and Eurasia while T. murrelliis the only present in wild animals in North America. Millions of years ago, Trichinellacould infect human beings evidenced by the ingestion of other parasites in meat [36].

The nematode T. spiralisis involved in the most common cause of human trichinellosis, which is considered a zoonotic disease worldwide. The heredity of T. spiralisgiving rise to the genus Trichinellaand reported that the last shared common ancestor was approximately 275 million years ago (Lower Permian Period) identified, however the modification of extant Trichinellaspecies happened about 16–20 million years ago [14].

We compare the molecular physiognomies of nematodes and former metazoans by using the T. spiralisgenome as standard. This comparative approach by using the T. spiralispermitted us to categorize conserved protein and gene sequences through the superficial model, particularly for the phylum Nematoda. We bring an approach that intrachromosomal modifications were common all over the phylum. However, this was in divergence to other features such as births and deaths of a protein family, which exhibited clear discrimination among the parasitic and non-parasitic nematodes. The identification of well-maintained physiognomies predicated based on this work will advance the more accurate research on pathogens from a phylum embracing thousands of pathogens that are mainly to infect humans, animals, and plants and behaves like infectious agent. The advances possibly will one day be responsible for complete strategies to prevent and control diseases that are caused by pathogens from across the Nematoda family around the globe [14, 36, 37].

Commencing from the time of the discovery of Trichinellawhich is in 1835 in anticipation of the middle of the next century. During the last decade, the use of molecular and biochemical methods in combination with experimental studies on biology, have resulted in the identification of seven Trichinellaspecies that have different epidemiological and topographical distributions. Even though these species are very difficult to differentiate morphologically, this can be done with the molecular and positive biological characters for further identification [16].

7.2 Genetic diversity related to multiple hosts

A total of 30 species of T. spiralishaving mtDNA genomes has 20 unique haplotypes that were observed containing 86 isolating sites. So, with four out of five shared haplotypes taking place in European and North American samples. Samples from North America had one haplotype, which is present in each geographic sampling site [38]. Out of the total, mostly the variations were limited to the Asian T. spiralissamples. There are about 7 Asian samples, and from these 8 haplotypes were identified; these differed on an ordinary by 24.9 nucleotides. In comparison to this, western samples are averaged with only 3.2 nucleotide differences per haplotype with only 13 haplotypes in 23 samples; the most different pair of western haplotypes differed by only 6 nucleotide differences between any two isolates. Similarly, nucleotide diversity (pi) was 0.00016 in western samples while Asian nucleotide diversity was 10-fold greater (0.00179). As a result, we can say that all Asian samples are different from the western samples by at least 45 bp and averaged 49 bp differences [24].

The most noticeable properties of this parasite’s epidemiology are its requisite transmission by mode of meat ingestion in consumers. There is another important feature, which is present in two normally isolated ecological systems, which are sylvatic and domestic. In certain situations, the two biotopes are connected from end to end man’s activities, which results in the revelation of humans to Trichinellaspecies [38]. Usually, it is restricted to sylvatic animals. The species furthermost often associated with human infection is T spiralis, which is the reported species that is usually found in the meat of domestic pigs. The life cycle of T spiralisincludes a multipart set of possible routes. Farm transmission can be the result of predation on or hunting other animals for food purposes (rodents), hog anthropophagy, and the feeding of uncooked meat leftovers [16].

Most outbreaks resulting from ingesting of T spiralisinfected pigs can lead to its outbreak through local single-source but, progressively, the mass marketing of meat can distribute the disease-causing parasite in the entire population. There has been a great increase in the reported cases due to Trichinella species, just because of having so many species that are involved in the food chain. The reason for having genetic diversity is also stated that we are lacking in vaccines to eradicate it. The main source is considered as the meat from the game and domestic animals. From recent reports, we can conclude that it also specifies that infected herbivores including horses, sheep, goats, and cattle have been the source of the outbreak [14, 16].

7.3 Multiple stage complexity of T. spiralis

T. spiralisalso has several stages of the complex life cycle that completes in two niches viz. intra-multicellular niche occurs in the intestine epithelium of host where adult male and female worms are involved with the help of (proteolytic digestive enzymes and become mature adult worms). Whereas intracellular niche occurs in the striated muscles of the organism where muscle larvae participate in the development of nurse cells [16], T. spiralislife cycle represents different antigens specific for a particular stage, where these antigens elicit immune responses and facilitate the developmental cycle of the parasites by modifying the host immune responses. To complete their life cycle, they skip the defensive mechanism of the host against invading the foreign body.

Once newborn larvae invade the lymphatic or circulatory system, they can drive anywhere in the organism and survive only in the skeletal muscles of the host. Humans can acquire T. spiralisinfection only if they consume undercooked or raw meat containing muscle larvae [36]. Several genes are differentially expressed among the life cycle stages and up-regulated genes in the newborn larvae. The genome of T. spiralisis regulated in the developmental stages [34].


8. Future perspective

Trichinella infection is an emerging zoonosis in many countries and where it become the reason for trichinellosisdisease. Due to its widespread prevalence and high amount of pork meat consumption more clinical awareness is required. The acute infection is characterized by two phases viz. enteral which disrupt intestinal functions and parenteral phases are associated with the inflammatory and allergic reaction. The diagnosis of this disease contains new specific serological tests such as immunoblot or ELISA. Anthelmintics and anti-inflammatory drugs are the drug of choice for Trichinellainfection [16]. Vaccines formulated for veterinary purposes have made a great impact not only on animal welfare, production, and health but also on human health. Vaccines are considered reliable, efficient, and sustainable for the control and prevention of parasitic infection.

In Thrichinellosis, induction of protective and therapeutic responses should evoke both innate and adaptive immune systems to prevent the establishment of parasites in the organism. The life cycle of T. spiralisis complex, and the immune response is not strong that induced by a vaccine containing specific antigen to overcome the challenging infection. Therefore, a vaccine containing multiple epitopes against T. spiralis induces higher immunity[24]. Probiotics such as Lactobacilluskeep the environment of the intestine healthy and prevent enteric infections. Probiotic Lactobacillus caseiis most commonly used for protection against Trichinellosis. L. caseiis involved in the production of IL-4, IgA, and IgG (anti-T. spiralisantibodies) and has a preventive role against high infection of T. spiralis. Some strains of L. caseiinclude L. caseiATCC 469, L. caseiATCC 7469, and L. caseiShirota have proven efficacy against T. spiralisinfection. For the control of Trichinellosis, Probiotics and plants-based veterinary vaccines are a new approach and can be used as treatment and edible vaccines for various parasitic diseases in animals. Due to the low cost of plants production, sterile delivery, and transportation at a suitable temperature, plants are considered as a suitable vehicle for veterinary vaccines [1].

Antigens in the vaccines administered orally are subject to proteolysis by the proteolytic enzymes present in the digestive tract of the organism. It will decrease the bioavailability of the vaccines and will induce a low immune response [1]. On the other hand, in plant-based vaccines antigens are protected from proteolytic enzymes by the cell wall of the plant cells and enable antigens to reach their desired destination (gut-associated lymphoid tissue). Various plants and vegetable species such as potato, tomato, tobacco, alfalfa, rice, spinach, beans, maize, strawberries, and carrots can be used in the biotechnology of plants for the expression and production of recombinant proteins.

For the prevention and control of diseases in animals and their transmission from animals to humans, plant-based vaccines seem to be an excellent tool. More research is required to thoroughly understand the applications of medical plant extracts, probiotics, and other biological agents [24].


9. Conclusion

At least twelve species and genotypes of Trichinellagenus can cause veterinary or medical health hazards in a wide geographical range throughout the world. The main etiological agent of Trichinellosisin humans is only T. spiralisparasite and can result in mild to severe clinical signs and symptoms. Numerous antigens are used as candidate vaccines from different stages of T. spiralis andcan be used as DNA vaccines or recombinant protein vaccines. The role of progesterone and mifepristone against T. spiralisis also very helpful as they penetrate the vaccine into the target of T. spiralis.Altogether, we can get different strains for specific vaccines with molecular physiognomies of different Trichinella species.



The author wishes to thank all other co-authors for providing guidance and support.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1. Korhonen P, Pozio E, La Rosa G, et al. Phylogenomic and biogeographic reconstruction of theTrichinellacomplex. Nature Communications. 2016;7:10513. DOI: 10.1038/ncomms10513
  2. 2. Zhang N, Li W, Fu B. Vaccines against Trichinella spiralis: Progress, challenges and future prospects. Transboundary and Emerging Diseases. 2018;65(6):1447-1458. DOI: 10.1111/tbed.12917
  3. 3. Yr Y, Yf Q. Progress in Treatment and Prevention of Trichinellosis. Journal of Infectious Diseases & Therapy. 2015;03(06). DOI: 10.4172/2332-0877.1000251
  4. 4. Bai X, Hu X, Liu X, Tang B, Liu M. Current research of trichinellosis in China. Frontiers in Microbiology. 2017;8(AUG):1-7. DOI: 10.3389/fmicb.2017.01472
  5. 5. Cui J, Han Y, Yue X, Liu F, Song YY, Yan SW, et al. Vaccination of mice with a recombinant novel cathepsin B inhibits Trichinella spiralis development, reduces the fecundity and worm burden. Parasites and Vectors. 2019;12(1):1-12. DOI: 10.1186/s13071-019-3833-9
  6. 6. Qi X, Han Y, Jiang P, Yue X, Ren HN, Sun GG, et al. Oral vaccination with Trichinella spiralis DNase II DNA vaccine delivered by attenuated Salmonella induces a protective immunity in BALB/c mice 11 Medical and Health Sciences 1107 Immunology. Veterinary Research. 2018;49(1):1-12. DOI: 10.1186/s13567-018-0614-y
  7. 7. Jin X, Liu X, Ding J, Zhang L, Yang Y, Wang X, et al. Lentinan improved the efficacy of vaccine against trichinella spiralis in an nlrp3 dependent manner. PLoS Neglected Tropical Diseases. 2020;14(9):1-13. DOI: 10.1371/journal.pntd.0008632
  8. 8. Ehsan M, Hu RS, Liang QL, Hou JL, Song X, Yan R, et al. Advances in the development of anti-haemonchus contortus vaccines: Challenges, opportunities, and perspectives. Vaccines. 2020;8(3):1-18. DOI: 10.3390/vaccines8030555
  9. 9. Zhang Y, Zeng J, Song YY, Long SR, Liu RD, Jiang P, et al. Vaccination of mice with a novel trypsin from trichinella spiralis elicits the immune protection against larval challenge. Vaccines. 2020;8(3):1-20. DOI: 10.3390/vaccines8030437
  10. 10. Gu Y, Wei J, Yang J, Huang J, Yang X, Zhu X. Protective Immunity againstTrichinella spiralisInfection Induced by a Multi-Epitope Vaccine in a Murine Model. PLoS ONE. 2013;8(10):e77238. DOI: 10.1371/journal.pone.0077238
  11. 11. Aleem MT, Shi J, Yu Z, Wen Z, Zhang Y, Liang M, et al. Characterization of membrane-associated progesterone receptor component-2 (MAPRC2) from trichinella spiralis and its interaction with progesterone and mifepristone. Vaccines. 2021;9(8):934. DOI: 10.3390/vaccines9080934
  12. 12. Wang X, Li L, Wei X, et al. Proteomic analysis of the response of Trichinella spiralis muscle larvae to exogenous nitric oxide. Plos One. 2018;13(6):e0198205. DOI: 10.1371/journal.pone.0198205
  13. 13. White RR, Miyata S, Papa E, Spooner E, Gounaris K, Selkirk ME, et al. Characterisation of the Trichinella spiralis deubiquitinating enzyme, TsUCH37, an evolutionarily conserved proteasome interaction partner. PLoS Neglected Tropical Diseases. 2011;5(10):e1340. DOI: 10.1371/journal.pntd.0001340. Epub 2011 Oct 4
  14. 14. Mitreva M, Jasmer DP, Zarlenga DS, Wang Z, Abubucker S, Martin J, et al. The draft genome of the parasitic nematode Trichinella spiralis. Nature Publishing Group. 2011;43(3). DOI: 10.1038/ng.769
  15. 15. Yang J, Pan W, Sun X, et al. Immunoproteomic profile ofTrichinella spiralisadult worm proteins recognized by early infection sera. Parasites Vectors. 2015;8:20. DOI: 10.1186/s13071-015-0641-8
  16. 16. Bruschi F, Murrell KD. New aspects of human trichinellosis: the impact of new Trichinella species. Postgraduate Medical Journal. 2002;78(915):15-22. DOI: 10.1136/pmj.78.915.15
  17. 17. Xu D, Bai X, Xu J, Wang X, Dong Z, Shi W, et al. The immune protection induced by a serine protease from the trichinella spiralis adult against trichinella spiralis infection in pigs. PLoS Neglected Tropical Diseases. 2021;15(5):1-15. DOI: 10.1371/journal.pntd.0009408
  18. 18. Song Y, Xu J, Wang X, et al. Regulation of host immune cells and cytokine production induced by Trichinella spiralis infection. Parasite (Paris, France). 2019;26:74. DOI: 10.1051/parasite/2019074
  19. 19. Wakelin D, Lloyd M. Immunity to primary and challenge infections of Trichinella spiralis in mice: A re-examination of conventional parameters. Parasitology. 1976;72(Pt 2):173-182
  20. 20. Hafez EN, Kholy W. A. E. S. El, Amin MM, Hafez EN, Kholy W. A. E. S. El, & The, M. M. A. The potential protective role of gamma-irradiated vaccine versus Punica granatum treatment against murine trichinellosis. Journal of Radiation Research and Applied Sciences. 2020;13(1):560-567. Doi: 10.1080/16878507.2020.1777659
  21. 21. Zhang XZ, Sun XY, Bai Y, Song YY, Hu CX, Li X, et al. Protective immunity in mice vaccinated with a novel elastase ‑ 1 significantly decreases Trichinella spiralis fecundity and infection. Veterinary Research. 2020:1-12. DOI: 10.1186/s13567-020-00767-z
  22. 22. Han C, Yu J, Zhang Z, Zhai P, Zhang Y, Meng S, et al. Immunomodulatory effects ofTrichinella spiralisexcretory-secretory antigens on macrophages. Experimental Parasitology. 2018. DOI: 10.1016/j.exppara.2018.10.001
  23. 23. Liu P, Cui J, Liu RD, et al. Protective immunity against Trichinella spiralis infection induced by TsNd vaccine in mice. Parasites & Vectors. 2015;8:185. DOI: 10.1186/s13071-015-0791-8
  24. 24. Thompson PC, Bilska-zajac E, Zarlenga DS, Liu M, Cencek T. Divergence at mitochondrial and ribosomal loci indicates the split between Asian and European populations of Trichinella spiralis occurred prior to swine domestication. Infection , Genetics and Evolution. 2021;88(December 2020):4-10. DOI: 10.1016/j.meegid.2021.104705
  25. 25. Ilic N, Gruden-Movsesijan A, Sofronic-Milosavljevic L.Trichinella spiralis: shaping the immune response. Immunologic Research. 2012;52:111-119. DOI: 10.1007/s12026-012-8287-5
  26. 26. Han C, Yu J, Zhang Z, Zhai P, Zhang Y, Meng S, et al. Experimental Parasitology Immunomodulatory e ff ects of Trichinella spiralis excretory-secretory antigens on macrophages. Experimental Parasitology. 2019;196(March 2018):68-72. DOI: 10.1016/j.exppara.2018.10.001
  27. 27. Sun X, Li Y, Naqvi MA, Naqvi SZ, Chu W. Succinate coenzyme A ligase beta-like protein from trichinella spiralis suppresses the immune functions of rat PBMCs in vitro and inhibits the secretions of interleukin-17 in vivo. Vaccines. 2019;7:167. DOI: 10.3390/vaccines7040167
  28. 28. Zhang X, Han LL, Hong X, Jiang P, Niu YF, Wang ZQ, et al. Genotyping and Phylogenetic Position of Trichinella spiralis Isolates from Different Geographical Locations in China. Frontiers in Genetics. 2019;10(October):1-10. DOI: 10.3389/fgene.2019.01093
  29. 29. Love RJ, Ogilvie BM, Mclaren DJ. The immune mechanism which expels the intestinal stage of Trichinella spiralis from rats. Immunology 1976. 1976;30(1):7-15
  30. 30. Wang L, Wang X, Bi K, Sun X, Yang J, Gu Y, et al. Oral Vaccination with AttenuatedSalmonella typhimurium-DeliveredTsPmy DNA Vaccine Elicits Protective Immunity againstTrichinella spiralisin BALB/c Mice. PLoS Neglected Tropical Diseases. 2016;10(9):e0004952. DOI: 10.1371/journal.pntd.0004952
  31. 31. Wang ZQ, Cui J, Wei HY, Han HM, Zhang HW, Li YL. Vaccination of mice with DNA vaccine induces the immune response and partial protection against T. spiralis infection. Vaccine. 2006;24:1205-1212. DOI: 10.1016/j.vaccine.2005.08.104
  32. 32. Xu J, Bai X, Wang LB, Shi HN, Van Der Giessen JWB, Boireau P, et al. Immune responses in mice vaccinated with a DNA vaccine expressing serine protease-like protein from the new-born larval stage of Trichinella spiralis. Parasitology. 2017;144(6):712-719. DOI: 10.1017/S0031182016002493. Epub 2017 Jan 10
  33. 33. Gu Y, Sun X, Li B, Huang J, Zhan B, Zhu X. Vaccination with a paramyosin-based multi-epitope vaccine elicits significant protective immunity against trichinella spiralis infection in mice. Frontiers in Microbiology. 2017;8(August):1-9. DOI: 10.3389/fmicb.2017.01475
  34. 34. Liu X, Song Y, Jiang N, Wang J, Tang B, Lu H, et al. Global Gene Expression Analysis of the Zoonotic ParasiteTrichinella spiralisRevealed Novel Genes in Host Parasite Interaction. PLoS Neglected Tropical Diseases. 2012;6(8):e1794. DOI: 10.1371/journal.pntd.0001794
  35. 35. Nuñez G, Gentile T, Costantino S, Sarchi M, Venturiello S. In vitro and in vivo effects of progesterone on Trichinella spiralis newborn larvae. Parasitology. 2005;131(2):255-259. DOI: 10.1017/S0031182005007468
  36. 36. Rosenthal BM, Larosa G, Zarlenga D, Dunams D, Chunyu Y, Mingyuan L, et al. Human dispersal of Trichinella spiralis in domesticated pigs. Infection, Genetics and Evolution. 2008;8:799-805. DOI: 10.1016/j.meegid.2008.07.008
  37. 37. Liu X, Feng Y, Bai X, Wang X, Qin R, Tang B, et al. Comparative multi-omics analyses reveal differential expression of key genes relevant for parasitism between non-encapsulated and encapsulated Trichinella. Communications Biology. 2021:1-12. DOI: 10.1038/s42003-021-01650-z
  38. 38. Ren HN, Guo KX, Zhang Y, et al. Molecular characterization of a 31 kDa protein from Trichinella spiralis and its induced immune protection in BALB/c mice. Parasites & Vectors. 2018 Dec;11(1):625. DOI: 10.1186/s13071-018-3198-5

Written By

Muhammad Tahir Aleem, Ruofeng Yan, Asad Khan, Rida Asrar, Amna Shakoor, Areej Asif, Zhaohai Wen, Zhengqing Yu, Muhammad Abdullah Malik, Tauseef-ur-Rehman, Rao Zahid Abbas, Muhammad Mohsin, Xiaokai Song, Lixin Xu and Xiangrui Li

Submitted: December 19th, 2021 Reviewed: February 2nd, 2022 Published: May 11th, 2022