Detection of parasites in food of animal origin - Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasitic protozoa that has no specific host, and almost all animals, including various nucleated cells in the human body, can be infected, and the infection rate of humans and animals is very high. When toxoplasmosis (toxoplasmosis) occurs in pigs, the entire pig farm can become ill, and the case fatality rate is as high as 60%, causing huge economic losses and seriously affecting the development of the breeding industry. Toxoplasmosis of pigs is closely related to public health, pork is the most important animal-derived food in people's lives, toxoplasmosis infected pigs and their meat products are an important source of infection for human infection with Toxoplasma gondii, posing a great threat to human health.

1 Etiological features

In 1908, the pathogen toxoplasma was discovered in the rodent comb-toed rats of Tunisia, North Africa, by Nicollole and Manceaux, and officially named it. Toxoplasma gondii (T. gondii) is abbreviated as Toxoplasma gondii, belonging to apicomplexa, Sporozoasida, Eucocciida, Sarcocystidae, and Toxoplasma. Currently, most scholars believe that toxoplasma gondii has only one species, one serotype, but there are different strains.

Sexual reproduction of Toxoplasma gondii occurs in the small intestinal epithelial cells of the terminal host cat, and asexual reproduction occurs in the nucleated cells other than red blood cells in the middle host warm-blooded animals. The whole process of toxoplasma gondii development generally includes trophozoites (also known as tachygos), cysts, fissures, gametophytes and egg sacs, which are completed in the intermediate host and terminal host, respectively. The tachydonts are arched, crescent-shaped, or banana-shaped, with one end being pointed and the other end being blunt and rounded, with an average size of (4 to 7) μm× (2 to 4) μm. Oocysts are found in felines and are oval in shape and are (11 to 14) μm in size× (7 to 11) μm. The cyst is ovoid or oval in shape and 5 to 100 μm in diameter.

Cats swallow the cysts or oocysts of Toxoplasma gondii, the worm body is released, part of the lymphatic and blood circulation is carried to the organs and tissues of the whole body, invading the nucleated cells, and the other part of the worm body in the small intestinal epithelial cells undergo fission reproduction and gametogenesis similar to the development of coccidiosis, producing oocysts. The oocysts are excreted with feces and develop into infectious spore-shaped oocysts outside. After the intermediate host eats or drinks food and water contaminated with spore oocysts, the sporeized oocysts release sporospores, which enter tissues and organs through lymph and blood, invade nucleated cells and form infections. If the infected strain is very virulent and the host fails to produce sufficient immunity, it can cause an acute attack of toxoplasmosis; conversely, if the strain is weak, the host can quickly develop immunity, and the worm body turns into a slow breeder stage, slowly proliferating in the capsule, forming a continuous infection.

2 Epidemiological features

Toxoplasmosis is a worldwide zoonotic disease in which infections in both animals and humans are extremely common, with at least 200 species of mammalian infections. The main sources of infection are sick, sick animals and insectoids, of which blood, meat, internal organs, etc. may have Toxoplasma gondii. Toxoplasma gondii are found in secretions such as milk, saliva, sputum, urine and nose; toxoplasmosis is abundant in the aborted fetus, in the placenta and in amniotic fluid. The infection rate is not strictly regional and seasonal, but the incidence is highest in autumn, winter and early spring, which may be related to the resistance of the animal body. About one billion people in the world are infected with toxoplasmosis every year, and its infection rate is related to the culture and eating habits of local residents, and the infection rate of foreign populations is 0.6% to 94%, with an average of 25% to 58%, of which the infection rate of Toxoplasma gondii in Europe and the United States is higher, and France is the most endemic country, with an infection rate of 80% to 90%; the infection rate of the Dutch population is also higher, reaching 40%; the infection rate in Africa and Latin America is also higher; the infection rate in Southeast Asia, North America and Oceania is lower. The discovery of Toxoplasma gondii in China is relatively early in Asia. Before the founding of New China, there were no reports of toxoplasmosis in China. In 1964, Xie Tianhua first reported toxoplasmosis in Jiangxi. Almost every province in China has been reported toxoplasmosis, the infection rate is 0.09% to 34%, the average infection rate is 7.88%, people of all ages can be infected, the infection rate in ethnic minority areas may be higher, although the infection rate of Toxoplasma gondii in China is lower than the world average, but it shows an upward trend year by year.

Toxoplasma gondii is very common in livestock, pigs, sheep, cattle, horses, etc. can be infected, the greatest harm to pigs, foreign reports, the disease occurs mostly in 3 to 4 months old piglets, its symptoms are similar to swine fever, the case fatality rate can be as high as 30% to 40%, of which the suckling pig case fatality rate is the highest, adult pigs have fewer diseases, most of them are hidden infections or mild symptoms. In 1955, Yu Enshu and others first isolated toxoplasma gondii from cats, pigs and other animals in Fujian, and in 1977, Wu Shuoxian isolated toxoplasma gondii from "nameless high-fever" pigs in Shanghai for the first time, proving the existence of an outbreak of toxoplasmosis in pigs in China. The infection rate of Toxoplasma pig in China is 4% to 71.4%, pigs of different breeds, ages and sexes can be sick, 7 to 9 months of age have more incidence, the highest incidence of fattening pigs with more than 25kg, pig month age does not have a certain regularity on the level of infection rate, and the gender difference is larger, sow infection rate is 36.4%, boar infection rate is 29.4%, according to the epidemic form can be divided into outbreak type, acute type, sporadic sporadic and latent infection.

Toxoplasma gondii infection in other animals in China is also very common, the infection rate of cats is 15% to 73%, the infection rate of dogs is 3% to 33%, the infection rate of sheep is 10% to 50%, and the infection rate of cattle is 0.2% to 43.3%. Poultry also has toxoplasmosis infection, and the infection rates of chickens, ducks and geese are 15.69%, 3.84% and 16.54%, respectively.

3 Hazards

People with normal immunity are usually infected with toxoplasma gondii, generally without obvious clinical symptoms, once the body's immunity declines or immunocompromised people are infected, it can cause systemic reactions and multi-organ damage, and in severe cases, cause death. Infection with Toxoplasma gondii during pregnancy can cause miscarriage, preterm birth, stillbirth, and Toxoplasma gondii can infect the fetus through the placenta, causing congenital infection of the fetus. After birth, the fetus is manifested by a series of central nervous system symptoms and congenital damage to the eyes and internal organs. Some newborns are born without special signs, only low weight, anemia, jaundice, etc., but gradually appear convulsions, meningitis and epilepsy and other neurological lesions, which can cause intellectual development disorders.

After animals infected with Toxoplasma gondii, they show slow growth, decreased feed utilization, low production performance, long-term insects, and death in severe cases; causing abnormal production of breeding animals, miscarriage, and stillbirth, resulting in serious economic losses. One of the most harmful is pigs. At present, toxoplasmosis in Pigs in China is widely distributed, and the occurrence of the disease has been reported in North China, East China, Northeast China, Central China, South China, Southwest China, Northwest China and other regions, and the infection rate and case fatality rate are generally high. Pork is the most important animal-derived food in people's lives, and toxoplasmosis-infected pigs and their meat products pose a great threat to human health. Pigs infected with Toxoplasma gondii usually have no obvious clinical symptoms, but in fact the tissues of latently infected pigs may contain toxoplasmosis sacs, which can trigger human toxoplasmosis infection if it is improperly processed during cooking or due to personal habits (such as undercooked or raw meat).

Mendonca et al. used PCR technology to amplify the treated pork sausage samples, and at the same time, mice and animals were tested with the treated pork sausage samples, and specific anti-Toxoplasma antibodies in mouse serum were detected by IFA, and the detection rate of PCR was 47.14% (33/70), and the mouse serum was negative, although the results showed that the pork sausage samples did not contain live Toxoplasma gondii, but the high PCR detection rate could also indicate that the pork sausage was more seriously contaminated with Toxoplasma gondii. If the salt concentration of the sausage during processing < 3.0% and the salting time < 3d, it is not effective to kill the toxoplasma sacs or rapid breeders in the sausage. Two outbreaks of 8 people in South Korea in 1997 were caused by eating the spleen and liver of unheated (wild) pigs. Dias et al. investigated 47 slaughterers' serum and 149 (8 manufacturers) fresh pork sausages in Londrina, Brazil, for mouse biological tests, and found that the seroprevalence rates were 59.6% (28/47) and 8.7% (13/149), respectively, and believed that fresh pork sausages play an important role in the spread of toxoplasmosis.

4 Diagnostic techniques

A preliminary diagnosis is made based on clinical symptoms, pathological changes, and epidemiology, and the diagnosis must be confirmed by finding the pathogen or specific antibody in the laboratory, mainly through pathogenic examination, serological testing, and molecular biology diagnosis.

4.1 Etiological examination

4.1.1 Direct smear

Take blood, serum, ascites or liver, lung, lymph nodes and other disease materials directly smeared With Gimsa or Reeb's staining, microscopic examination, and the diagnosis of the worm body can be confirmed. This method is very accurate in diagnosing toxoplasmosis, but the detection rate is low and it is easy to miss tests.

4.1.2 Tissue section staining

Cryo-sectioning of diseased tissue, stained with immunases or fluorescence, can not only color the cysts, but also color the scattered toxoplasmosis tachypris and infected intracellular insect bodies, which has high sensitivity and specificity, and can improve the detection rate of insects.

4.1.3 Animal inoculation and isolation and cell culture

After the liver, lungs, lymph nodes and other disease materials are homogenized, add 5 to 10 times of normal saline to mix well, filter, centrifuge, take the supernatant peritoneal cavity inoculation in susceptible animal mice, observe the incidence, and blindly pass on for several generations to improve the detection rate. This method is highly sensitive and specific, but time-consuming. The above disease materials can also be inoculated in vivo cultured monolayer nucleated cells, which are less sensitive but require less time than animal inoculation.

4.1.4 Oocyst examination

Stool is floated with saturated sodium chloride or sucrose solution (30%) and oocystoscopy is collected, but the detection rate is generally low.

4.2 Serological testing

Serological testing has become a widely used and rapidly developed method for detecting Toxoplasma gondii infection due to its rapidity, accuracy and economy. The main methods include: staining test, indirect red blood cell agglutination test, ELISA, indirect immunofluorescence test, complement binding test and immunoglioidal gold technology.

4.2.1 Staining test (DT)

Toxoplasmosis tachycardia, complement cofactors and serum to be tested together at 37 °C incubation for 1h, and then stained with methylene blue, when there is a specific antibody, the parasite membrane permeability increases, cytoplasm outflow, tachyproduct can not bind to the dye and appear colorless; when no specific antibody is present, the tachyclopris can adsorb the dye and appear blue. This method is a classic serological test for toxoplasmosis with good specificity, sensitivity, and reproducibility. However, this method has great dangers due to the need to use live, toxic tachypris during inspection, and its application is limited.

4.2.2 Indirect erythrocyte agglutination test (IHAT)

The tachyzoite soluble antigen is adsorbed on the surface of erythrocytes, and then reacts with the corresponding antibody, and the erythrocytes are indirectly agglutinated through the specific reaction of the antigen and the antibody. Jacob and Lunde first applied the method to toxoplasmosis testing in 1957. In 1979, Yu Enshu of China applied IHAT to the detection of porcine toxoplasmosis. In 1982, the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences developed the IHAT diagnostic kit. Wilson et al. tested 100 serum specimens using three kits, ELISA, IHAT, and IFAT, and concluded that IHAT lacks sensitivity to serum in the early stages of acute infection. Cui Zhaojun et al. believe that IHAT specificity is high, false positive rate is low, diagnostic value is high and easy to operate, the disadvantages are that the sensitivity is not as good as ELISA, the repeatability is poor, the sensitized red blood cells are unstable, and the positive judgment criteria have a certain subjectivity.

4.2.3 ELISA

The enzyme-labeled secondary antibody (such as horseradish peroxidase-labeled anti-sheep IgG) is added dropwise, and the catalytic action of the enzyme and the principle of substrate amplification are used to conduct qualitative and quantitative analysis of the antibody to be examined. In 1976, Voller et al. applied this method to the detection of Toxoplasma gondii-specific antigens for the first time, and obtained a good compliance rate with DT and IHAT, and strong sensitivity. Peter et al. established an indirect ELISA to detect porcine toxoplasmosis with toxoplasmosis turbozoan antigens, and compared it with dt methods, the sensitivity and specificity of indirect ELISA were 94%.

In recent years, with the application of molecular biology technology, recombinant antigens have replaced natural antigens as diagnostic antigens, eliminating the cumbersome process of antigen preparation, so as to achieve economic, specific and safe purposes. Redlich et al. established ELISA with the expressed fusion protein GRA6-GST, and its sensitivity reached 98%; Aubert et al. mixed the recombinant rod protein 1 (ROP1) and surface antigen protein (P25 and P35) to establish ELISA for the detection of Toxoplasma gondii IgM, and its sensitivity, specificity and compliance rates were 93.1%, 95.0% and 94.5%, respectively, which can be used for the early diagnosis of Toxoplasma gondii Huang et al. established ELISA with surface protein P22 (SAG2), compared with IHAT, and found that the method had strong specificity and high sensitivity. In China, Lü Bin et al. established ELISA for detecting IgM in human serum with recombinant P35-GST, which can distinguish acute infection and chronic infection of toxoplasmosis. Zhang Donglin et al. established AG-ELISA, which can detect antibodies to a variety of animals, and detected anti-Toxoplasma antibodies in the serum of artificially infected pigs and 4 naturally infected animals (pigs, cattle, cats and dogs), indicating that the positive detection time of AG-ELISA was earlier than that of the improved agglutination test method, and its detection rate, sensitivity and accuracy were better than the latex agglutination test.

In short, the method is easy to automate, has a high degree of specificity and sensitivity, the results can be quantified, suitable for the detection of batch samples, and has good promotion and application value.

4.2.4 Indirect fluorescent antibody test (IFAT)

Incubate with the tested diluted serum with the fused antigen, add fluorescein (fluorescein isothiocyanate) labeled secondary antibody, and observe the results under a fluorescence microscope. Miller et al. used this method to detect the serum of 28 positive and 46 negative patients isolated by immunohistochemistry and pathogens, and found that their sensitivity and specificity were 96.4% and 67.3%, respectively. This method is used for the detection of IgM and IgG antibodies in early toxoplasma gondii infection, and has the advantages of high sensitivity, specificity and reproducibility, but rheumatoid factor can cause false positive response of IgM antibodies. Due to the need for fluorescence microscopy observation, the widespread use of this method is limited.

4.2.5 Immunoidal Gold Technology (ICT)

ICT is a novel immunomarking technology that uses colloidal gold as a tracer marker for antigen antibodies. Wang Yanhua et al. established ICT to detect Toxoplasma gondii serum, and compared with IHAT, the result compliance rate was 82.5%, and ICT could detect Toxoplasma antibodies 2d earlier than IHAT, indicating that the sensitivity of ICT was higher than that of IHAT. Rapid immunochromatography assay established by the University of Daihiro Animal Production in Japan to establish a rapid immunochromatography test to detect toxoplasmosis antibodies in cats. The results prove that ICT is a fast, easy, sensitive (sensitivity up to the level of ELISA) and specific, suitable for on-site diagnosis of Toxoplasma-specific antibodies and effective tools.

4.2.6 Latex agglutination test (LAT)

Using latex particles as the carrier for the agglutination reaction, Yu Enshu et al. pointed out that LAT is used to detect the initial sensitivity of Toxoplasma gondii infection. Chen Yatang et al. believe that LAT has the advantages of simple operation, small blood collection and stable and easy observation of results, which is suitable for large-scale seroepidemiological investigation. The London Public Health Laboratory uses LAT as a routine screening test, with a false-positive rate of 1% to 2%. Jiang Tao used the latex agglutination test (rMIC3-LAT) of recombinant Toxoplasma gondii microline protein 3 to detect the dynamic changes of serum-specific antibodies in pigs infected with Toxoplasma gondii, and compared with IHAT and ELISA, found that the sensitivity of LAT was higher than that of IHAT, the time of detection of antibodies was earlier than ELISA, and the time range of detection was wider.

In short, LAT has the characteristics of strong specificity, high sensitivity, good reproducibility, simple operation, easy determination of results, and can be used for early diagnosis, antibody monitoring, suitable for on-site detection and large-scale sero-epidemiological investigation. However, there are certain subjective factors in judgment and quantitative analysis cannot be done.

4.2.7 Complement binding test (CFT)

Nicolan and Rovello were the first to use the test to diagnose toxoplasmosis. Ondriska et al. consider it to be a reliable indicator for detecting Toxoplasma gondii infection, especially when examining individual samples, if cfTs are combined with IgM or IgA antibody levels or with affinity IgG antibody levels to reflect the course of the disease more objectively. The test is not applicable to certain animals such as cattle and goats.

4.3 Molecular biology diagnosis

At present, a variety of toxoplasmosis gene diagnosis methods have been established at home and abroad, mainly including PCR and DNA probe technology, which have the advantages of simple operation, fast, specific and sensitive.

4.3.1 PCR

The target genes used for PCR detection mainly include B1 gene, P30 (SAG1) gene, ITS1, rDNA (18S rRNA and 5.8S rRNA) and 529bp repeat sequence. The B1 gene is a multi-copy gene with a highly conserved sequence and is currently the most ideal detection gene. Burg is equivalent to the first B1 gene as the target gene of PCR in 1989, and has successfully detected only 1 toxoplasmosis contained in cell lysates; Li Jian et al. selected the B1 gene as the target gene, compared the PCR detection of whole blood DNA with ELISA detection of serum IgM, and the earliest detection time of PCR positive was earlier than ELISA, which can be used for the early diagnosis of toxoplasmosis. Xie Dehua et al. established a specific PCR diagnostic method for toxoplasmosis swine based on the sequence of the first internal transcriptional space zone (ITS1) of toxoplasma gondii DNA, which can detect the DNA of up to 10 toxoplasmosis tachygoids, and there is no cross-reaction with the 8 related protozoans and nematodes. Yu Li et al. used 529bp repeat sequence as the target gene to establish a pork PCR detection method, which can detect 100 fast breeders per 10g of pork, while the B1 gene can detect 1000 fast breeders per 10g of pork, and its sensitivity is 10 times that of the B1 gene.

In order to improve the sensitivity and specificity of the test, on the basis of ordinary PCR, nested PCR, sleeve PCR, RT-PCR, etc. have been developed. Johnson et al. designed two pairs of primers based on the SAG1 gene, first amplified with DS29 and DS30 primers, and then amplified with DS38 and DS39 primers, with a sensitivity of up to 0.05 p. of Toxoplasma gondii DNA. Chen Xiaoguang et al. established nested PCR, which can detect at least 1 pg of Toxoplasma toxoplasma DNA; Sawa et al. used nested PCR to detect 0.05pg of P30DNA, which is theoretically equivalent to the genomic DNA of a fast progenitor; ana Hurtado et al. established a single-tube sleeve PCR detection method, which obtained results that were basically consistent with the results obtained with IFAT, and the sensitivity reached 0.1 pg of Toxoplasma toxoplasma rhh strain DNA.

4.3.2 Ring-mediated isothermal amplification (LAMP)

LAMP is a new nucleic acid amplification technique reported by Notomi equal in 2000, which replaces DNA classical synthesis with the automatic circulation of strands by BstDNA polymerase. LAMP only needs to synthesize 10 to 20 μg of DNA under isothermal conditions (63 ~ 65 ° C), 30 ~ 60 min, compared with PCR, no staining is required, and the naked eye can observe the results. Yang Qiulin et al. designed a set of specific primers for LAMP according to the gene sequence of Toxoplasma gondii-specific gene B1, and the test showed good specificity and sensitivity. Zhang et al. designed lamp primers with a highly conserved repeat sequence of Toxoplasma gondii with a sensitivity of 85.7% compared to 76.9% PCR sensitivity. Krasteva et al. used the toxoplasmosis single-copy gene SAG1 to design lamp primers to amplify Toxoplasma gondii DNA, which was more sensitive than PCR. Sotiriadou et al. detected toxoplasmosis in water with lamp amplified Toxoplasma gondii B1 and OOP genes, and 0.1 tachycardia can be detected.

4.3.3 DNA probes

Blanco et al. established a gene library of RH strains of Toxoplasma gondii, screened out a repeat fragment, used a 32P marker as a probe, and spot hybridization can detect more than 80 pg of purified Toxoplasma gondii DNA. Xia Aidi et al. also established a toxoplasmosis gene library of toxoplasma gondii strain (ZS-2) strain, screened a 1.1 kb specific DNA fragment and used a 32P marker as a probe to detect Toxoplasma gondii with a sensitivity of 500pg to purify Toxoplasma gondii DNA. Angel et al. used ABGTg7-labeled probe hybridization to detect DNA in patients with acute Toxoplasma gondola encephalitis with a sensitivity of 66.7%. At present, the probes used for toxoplasma gondii detection are mostly specific DNA clone fragments or synthetic oligonucleotide probes, although the sensitivity is high, but its preparation requires certain conditions, and the cost is high, and it is not easy to promote.

Molecular biology diagnostics are very sensitive and specific methods, but they are highly technically demanding on the test environment and operators, and there are many factors that affect their results, and the high cost is not conducive to popularization. In practice, serological tests are still the mainstay.