Zhong Quan Wang

Sensitivity and optimization of artificial digestion in the inspection of meat for Trichinella spiralis.

In many countries, the method of choice in inspecting meat for Trichinella spiralis infection is artificial digestion. We conducted a study of the sensitivity of the artificial digestion method recommended by the International Commission on Trichinellosis for detecting T. spiralis larvae in meat and of the effect of modifications of some procedures used in the method on its sensitivity. As part of this, we evaluated the effects on larval recovery of the vessels used for larval settling, sieve sizes, and temperatures at which larvae passed through the sieves, using larvae from T. spiralis-infected mice. We observed the effects on larval recovery of digestion duration and of modified artificial digestion by using 10-g samples of infected mouse muscle alone or mixed with uninfected pork. The percentages of larvae recovered with the respective use of separatory funnels and conical cylinders were 51.20% and 98.70%. The rates of recovery of T. spiralis larvae at 4 degrees C after passage through sieves of 425-microm mesh (No. 40), 250-microm mesh (No. 60), and 180-microm mesh (No. 80) were 98.42%, 90.59%, and 81.63%, which exceeded the 97.79%, 85.10%, and 61.12% rates of recovery of motile larvae at 40 degrees C and the 95.12%, 78.60%, and 44.16% rates of recovery of dead larvae at 90 degrees C. The larval recovery rate after digestion for 2 hours (96.18%) was greater than that after 0.5 hours (88.00%). We then examined a modified digestion method in which 10-g samples of pork mixed with 300 mL of digestive solution were digested for 2 hours at 43 degrees C followed by chilling of digest solution to 4 degrees C before passing it through a 425-microm mesh (No. 40) sieve and allowing it to settle in a 1-L conical cylinder. With this procedure, the modified method detected T. spiralis in samples of pork meat weighing 10 g and containing either 1 larva per gram or 0.1 larva per gram. Further validation of digestion method incorporating these modifications is required with the use of larger samples of infected muscle from species such as swine, which are routinely tested for T. spiralis for the purpose of food safety.

Cloning, expression and characterization of a Trichinella spiralis serine protease gene encoding a 35.5 kDa protein

Serine proteases are found in the excretory-secretory (ES) products from Trichinella spiralis muscle larvae, have collagenolytic and elastolytic activities, and may be related to the larval invasion of intestinal epithelial cells. In this study, the serine protease gene (TspSP-1.2, GenBank accession No. EU302800) encoding a 35.5 kDa protein from T. spiralis was cloned, and recombinant TspSP-1.2 protein was produced in an Escherichia coli expression system. An anti-TspSP-1.2 serum recognized the native protein migrating at 35.5 kDa by the Western blotting of the crude or ES antigens from muscle larvae at 42 days post infection. An immunolocalization analysis identified TspSP-1.2 in the cuticle and internal organs of the parasite. Transcription and expression of the TspSP-1.2 gene was observed at all developmental stages of T. spiralis (adult worms, newborn larvae, pre-encapsulated larvae and muscle larvae). An in vitro invasion assay showed that, when anti-TspSP-1.2 serum, serum of infected mice and normal mouse serum were added to the medium, the invasion rate of the infective larvae in an HCT-8 cell monolayer was 33.0%, 89.4%, and 96.2%, respectively (P<0.05), indicating that the anti-TspSP-1.2 serum partially prevented the larval invasion of intestinal epithelial cells. After a challenge infection with T. spiralis infective larvae, mice immunized with the recombinant TspSP-1.2 protein displayed a 34.92% reduction in adult worm burden and 52.24% reduction in muscle larval burden. The results showed that the recombinant TspSP-1.2 protein induced a partial protective immunity in mice and could be considered as a potential vaccine candidate against T. spiralis infection.

Proteomic analysis of Trichinella spiralis proteins in intestinal epithelial cells after culture with their larvae by shotgun LC–MS/MS approach

Although it has been known for many years that Trichinella spiralis initiates infection by invading intestinal epithelium, the mechanisms by which the parasite invades the intestinal epithelium are unknown. The purpose of this study was to screen the invasion-related proteins among the increased proteins of intestinal epithelial cells after culture with T. spiralis and to study their molecular functions. The proteins of HCT-8 cells which cultured with T. spiralis infective larvae were analyzed by SDS-PAGE and Western blot. Results showed that compared with proteins of normal HCT-8 cells, four additional protein bands (115, 61, 35 and 24 kDa) of HCT-8 cells cultured with the infective larvae were recognized by sera of the mice infected with T. spiralis, which may be the invasion-related proteins released by the infective larvae. Three bands (61, 35 and 24 kDa) were studied employing shotgun LC-MS/MS. Total 64 proteins of T. spiralis were identified from T. spiralis protein database by using SEQUEST searches, of which 43 (67.2%) proteins were distributed in a range of 10-70 kDa, and 26 proteins (40.6%) were in the range of pI 5-6. Fifty-four proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Out of 54 annotated proteins, 43 proteins (79.6%) had binding activity and 23 proteins (42.6%) had catalytic activity (e.g. hydrolase, transferase, etc.), which might be related to the invasion of intestinal epithelial cells by T. spiralis. The protein profile provides a valuable basis for further studies of the invasion-related proteins of T. spiralis.

Phage-displayed specific polypeptide antigens induce significant protective immunity against Trichinella spiralis infection in BALB/c mice.

Trichinellosis is a public health problem and is considered an emerging/re-emerging disease in various countries. The etiological agent of trichinellosis is the nematode Trichinella, which infects humans, domestic animals and wildlife. A veterinary vaccine could be an option to control the disease in domestic animals. Although several vaccine candidates have shown promising results, a vaccine against trichinellosis remains unavailable to date. Phage particles are especially ideal vaccine delivery vehicles because they do not interfere with the immune response against the displayed peptide antigens, and, if anything, are more likely to efficiently direct antigen expression to professional antigen-presenting cells. In this study, Tsp10 polypeptide, which was encoded by a cDNA fragment of Trichinella spiralis intestinal infective larvae and was found to bind to normal mouse intestinal cells, was displayed on the surface of T7 phage. Anti-Tsp10 antibodies were able to recognize the native Tsp10 protein mainly localized to the stichosome of T. spiralis. Mice immunized with the recombinant phage T7-Tsp10 showed a 62.8% reduction in adult worms and a 78.6% reduction in muscle larvae following challenge with T. spiralis muscle larvae. Our results demonstrate that the vaccination with Tsp10 polypeptide displayed by T7 phage elicits the Th2-predominant immune responses and produces a significant protection against T. spiralis infection in mice. These findings suggest that phage display is a simple, efficient, and promising tool to express candidate vaccine antigens for immunization against T. spiralis.

Identification of Differentially Expressed Genes of Trichinella spiralis Larvae after Exposure to Host Intestine Milieu

Although it has been known for many years that T. spiralis muscle larvae (ML) can not invade intestinal epithelial cells unless they are exposed to the intestinal milieu and activated into intestinal infective larvae (IIL), which genes in IIL are involved in the process of invasion is still unknown. In this study, suppression subtractive hybridization (SSH) was performed to identify differentially expressed genes between IIL and ML. SSH library was constructed using cDNA generated from IIL as the ‘tester’. About 110 positive clones were randomly selected from the library and sequenced, of which 33 T. spiralis genes were identified. Thirty encoded proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Out of 30 annotated proteins, 16 proteins (53.3%) had binding activity and 12 proteins (40.0%) had catalytic activity. The results of real-time PCR showed that the expression of nine genes (Ts7, Ndr family protein; Ts8, serine/threonine-protein kinase polo; Ts11, proteasome subunit beta type-7; Ts17, nudix hydrolase; Ts19, ovochymase-1; Ts22, fibronectin type III domain protein; Ts23, muscle cell intermediate filament protein OV71; Ts26, neutral and basic amino acid transport protein rBAT and Ts33, FACT complex subunit SPT16) from 33 T. spiralis genes in IIL were up-regulated compared with that of ML. The present study provide a group of the potential invasion-related candidate genes and will be helpful for further studies of mechanisms by which T. spiralis infective larvae recognize and invade the intestinal epithelial cells.

Normal Mouse Intestinal Epithelial Cells as a Model for the in vitro Invasion of Trichinella spiralis Infective Larvae

It has been known for many years that Trichinella spiralis initiates infection by penetrating the columnar epithelium of the small intestine; however, the mechanisms used by the parasite in the establishment of its intramulticellular niche in the intestine are unknown. Although the previous observations indicated that invasion also occurs in vitro when the infective larvae are inoculated onto cultures of intestinal epithelial cells (e.g., human colonic carcinoma cell line Caco-2, HCT-8), a normal readily manipulated in vitro model has not been established because of difficulties in the culture of primary intestinal epithelial cells (IECs). In this study, we described a normal intestinal epithelial model in which T. spiralis infective larvae were shown to invade the monolayers of normal mouse IECs in vitro. The IECs derived from intestinal crypts of fetal mouse small intestine had the ability to proliferate continuously and express specific cytokeratins as well as intestinal functional cell markers. Furthermore, they were susceptible to invasion by T. spiralis. When inoculated onto the IEC monolayer, infective larvae penetrated cells and migrated through them, leaving trails of damaged cells heavily loaded with T. spiralis larval excretory-secretory (ES) antigens which were recognized by rabbit immune sera on immunofluorescence test. The normal intestinal epithelial model of invasion mimicking the natural environment in vivo will help us to further investigate the process as well as the mechanisms by which T. spiralis establishes its intestinal niche.

Isolation and characterization of ZZ1, a novel lytic phage that infects Acinetobacter baumannii clinical isolates.

BackgroundAcinetobacter baumannii, a significant nosocomial pathogen, has evolved resistance to almost all conventional antimicrobial drugs. Bacteriophage therapy is a potential alternative treatment for multidrug-resistant bacterial infections. In this study, one lytic bacteriophage, ZZ1, which infects A. baumannii and has a broad host range, was selected for characterization.ResultsPhage ZZ1 and 3 of its natural hosts, A. baumanni clinical isolates AB09V, AB0902, and AB0901, are described in this study. The 3 strains have different sensitivities to ZZ1, but they have the same sensitivity to antibiotics. They are resistant to almost all of the antibiotics tested, except for polymyxin. Several aspects of the life cycle of ZZ1 were investigated using the sensitive strain AB09V under optimal growth conditions. ZZ1 is highly infectious with a short latent period (9 min) and a large burst size (200 PFU/cell). It exhibited the most powerful antibacterial activity at temperatures ranging from 35°C to 39°C. Moreover, when ZZ1 alone was incubated at different pHs and different temperatures, the phage was stable over a wide pH range (4 to 9) and at extreme temperatures (between 50°C and 60°C). ZZ1 possesses a 100-nm icosahedral head containing double-stranded DNA with a total length of 166,682 bp and a 120-nm long contractile tail. Morphologically, it could be classified as a member of the Myoviridae family and the Caudovirales order. Bioinformatic analysis of the phage whole genome sequence further suggested that ZZ1 was more likely to be a new member of the Myoviridae phages. Most of the predicted ORFs of the phage were similar to the predicted ORFs from other Acinetobacter phages.ConclusionThe phage ZZ1 has a relatively broad lytic spectrum, high pH stability, strong heat resistance, and efficient antibacterial potential at body temperature. These characteristics greatly increase the utility of this phage as an antibacterial agent; thus, it should be further investigated.

Sparganosis, Henan Province, Central China

To the Editor: Sparganosis is a parasitic zoonosis caused by invasion of the spargana, the plerocercoid larvae of various diphyllobothroid tapeworms belonging to the genus Spirometra (1). Although human sparganosis is cosmopolitan, it is most frequently found in eastern and southeastern Asia (2). During 1927–2009 in the People’s Republic of China, >1,000 cases in humans in 27 provinces were reported; most cases were in southern China, where human infections were mainly acquired by eating raw or insufficiently cooked meat of frogs and snakes or by placing frog or snake flesh on open wounds for treatment of skin ulcers or on eyes to treat inflammation (3,4).

Comparative proteomic analysis of surface proteins of Trichinella spiralis muscle larvae and intestinal infective larvae.

The critical step for Trichinella spiralis infection is that muscle larvae (ML) are activated to intestinal infective larvae (IIL) and invade intestinal epithelium to further develop. The IIL is its first invasive stage, surface proteins are directly exposed to host environment and are crucial for larval invasion and development. In this study, shotgun LC-MS/MS was used to analyze surface protein profiles of ML and IIL. Totally, 41 proteins common to both larvae, and 85 ML biased and 113 IIL biased proteins. Some proteins (e.g., putative scavenger receptor cysteine-rich domain protein and putative onchocystatin) were involved in host-parasite interactions. Gene ontology analysis revealed that proteins involved in generation of precursor metabolites and energy; and nucleobase, nucleoside, nucleotide and nucleic acid metabolic process were enriched in IIL at level 4. Some IIL biased proteins might play important role in larval invasion and development. qPCR results confirmed the high expression of some genes in IIL. Our study provides new insights into larval invasion, host-Trichinella interaction and for screening vaccine candidate antigens.

Survey of Trichinella infections in domestic pigs from northern and eastern Henan, China

The aim of this work was to investigate the current situation of Trichinella infections in swine in the cities of Anyang and Shanqiu in the Henan province historically designated as trichinellosis-free. A total of 475 diaphragm muscle samples were collected from 2010 to 2011 and examined by trichinelloscopy and artificial digestion. No Trichinella larvae were detected by trichinelloscopy; however, using the digestion method, 3.79% (18/475) of domestic pigs were deemed positive for Trichinella. Among the 475 pigs examined, 112 from an industrialized pig farm were negative. However. Trichinella larvae were detected in 10% (9/90) of pigs from small pig farms, which was significantly higher than the 3.3% (9/273) of pigs found positive from backyard farms (P<0.05). The larval burdens in infected animals ranged from 0.1 to 1.58 larvae per gram. The larvae were identified by multiplex PCR as Trichinella spiralis. Our study confirms the existence of porcine trichinellosis in northern and eastern parts of Henan. The results will be useful for evaluating the risk of infection for humans. Given this new found data, public health officials should consider implementing strategies to eliminate human transmission.