Detection of parasites in food of animal origin - cysticercosis

Cysticercosis is the larvae of tapeworm, parasitizing the host's rhabdomy muscles and connective tissues, in the form of a cyst, so it is commonly known as "cysts". There are many kinds of cysticercosis that are parasitic in animals, and the pig cysticercosis and the bovine cysticercosis are transmitted to humans through meat products, of which the pig cysticercosis is more common. Cysticercosis is a global distribution and is more common in developing countries. Due to the imperfect health infrastructure in developing countries, the incidence of cysticercosis in pigs and populations is high, causing huge economic losses. In recent years, the incidence of cysticercosis has also increased in industrialized developed countries due to increased migration and international tourism. In 1993, the WHO listed cysticercosis as one of the six major diseases to be eradicated.

1 Etiological features

Cysticercosis is mostly parasitic in the tongue, masseter, gluteal, deep psoas and diaphragm. The appearance is oval vesicle-like, the size is (6 to 10) mm×5 mm, the sac is filled with liquid, the sac wall is a film, there is a round millet large opalescent inverted head segment on the wall, and there are 4 suction cups on the head segment. Some cysticercosis has a parietal process at the apex of the cephalic segment, and there are many small hooks at the tip of the apical process, arranged in two circles. Cysticercosis is called "rice pork" or "bean pork" because of its visible appearance to the naked eye, milky white, translucent, mung bean or soybean size.

2 Epidemiological features

Cysticercosis is mainly a zoonotic disease transmitted between pigs and humans, also known as porcine cysticercosis, and the prevalence of the disease has a lot to do with people's living habits. The corresponding adult worm of the pig cystic tail cyst is the tapeworm pig. Humans are the only terminal hosts, so the only source of infection for cysticercosis is in patients with tapeworm parasitism in the small intestine. Pigs are intermediate hosts, and the source of infection for taeniasis in pigs is sick pigs carrying cysticercosis. In addition, humans can also be infected with cysticercosis as an intermediate host. The main cause of cysticercosis in pigs is the free-range breeding of pigs or the lax management of human feces, etc., causing pigs to eat pregnant nodes or eggs in the feces of tapeworm patients, thus infecting cysticercosis. There are two main causes of human infection with cysticercosis: one is oral infection. People who eat food and water contaminated with tapeworm eggs, such as watering vegetables with human feces, people eat unwashed vegetables stained with insect eggs and become ill, or patients with taeniasis do not wash their hands or do not wash themselves after defecation, and mistakenly eat eggs contaminated on their hands and become infected with cysticercosis. The other is autoinfection. For patients with tapeworm parasitism in the small intestine, in some cases, tapeworm shedding segments retrograde on the stomach, under the action of digestive juices, the egg shell of the eggs is digested, and the larva migrates to various tissues and organs, making people infected with cysticercosis.

Cysticercosis is distributed globally, but is mainly endemic in some countries and regions in Asia, Africa, and Latin America. In China, except for the northeast, north China and northwest regions and parts of Yunnan and Guangxi, the rest of the areas are sporadic, the south of the Yangtze River is less, and the infection rate in the northeast region is high. Cases of tapeworm infection have been reported in provinces except Zhejiang Province. In recent years, the number of infected people in various places has shown an increasing trend, the infection rate has increased by 52.47% compared with 1990, of which the infection rate in Tibet and Sichuan provinces has increased by 97% and 98% respectively. Taeniasis of pigs has the highest infection rate in Heilongjiang, while taeniasis of cattle is led by Tibet, with an infection rate of more than 70% in some areas.

3 Hazards

Cysticercosis can infect both pigs and people. The infection rate of cysticercosis is low, but the infection rate of pigs is high, which occurs and is prevalent in various provinces in China, showing a sporadic situation, and some areas are endemic, of which the northeast, north China and southwest are the most important endemic areas. The harm of cysticercosis to pigs is mainly to hinder the growth and development of pigs, especially the growth of young pigs. Mild porcine cysticercosis is not clinically perceptible, and symptoms are present only when pigs are severely infected, such as stiff muscles, edema of the shoulder muscles, widened shoulders, bulging buttocks, and appearing unusually obese, while the middle of the body is narrow and thin, lion-like or dumbbell-shaped. If the cysticercosis parasitizes on the tongue, it has difficulty chewing and swallowing; parasitizes to the throat, the voice is hoarse: parasitizes the eyeball, the vision is blurred; parasitizes the brain, it has spasms, or sudden death due to acute encephalitis. The disease not only seriously affects the development of the pig industry, causing huge economic losses to the pig industry, but also threatens the health of human beings. On the one hand, human cysticercosis can greatly improve the probability of human infection with tapeworm pig, tapeworm parasitizes in the small intestine, resulting in symptoms such as anemia, emaciation, abdominal pain, indigestion, diarrhea and other symptoms; on the other hand, cysticercosis parasitizes in various tissues and organs of the human body, causing harm to the human body, and its degree of harm is related to its parasitic number and parasitic site. Cysticercosis parasitism in human muscles can appear sore, stiff; parasitism in the brain can appear neurological symptoms, convulsions, epilepsy, paralysis and even death; compression of the eyeball can cause vision loss, or even blindness. Among them, cerebroscysticercosis is the most serious and is the most common cause of secondary epilepsy in developing countries. There are about 50 million people with cerebral cysticercosis worldwide, more than 80% of whom live in low- and lower-middle-income countries, and more than 50,000 people die each year from the disease.

Cysticercosis and human taeniasis are parasitic diseases that form a mutually causal, circular infection between pigs and humans, and if the sanitary environmental control measures are lax, it will lead to a vicious circle. Patients with taeniasis in pigs exclude pregnancy and eggs from the outside world every day, and they can last for years or even more than 20 years, so that pigs are under threat for a long time. Humans are the only terminal host of cysticercosis, and infection with tapeworms depends mainly on dietary hygiene and cooking and carnivorous methods. People eat pork or contaminated food with live cysticerci and contract taeniasis. Residents of endemic areas prefer to eat raw or undercooked pork and beef, which plays a decisive role in the spread of taeniasis. Human cysticercosis is caused by people eating food contaminated with tapeworm eggs or autoinjuvation in patients with tapeworm disease, and 16% to 25% of patients with tapeworm swine tapeworm have concurrent cysticercosis. Therefore, cysticercosis is one of the parasitic diseases that seriously endanger the human body.

4 Domestic and foreign health requirements

According to the "Sanitary Quarantine Code for Livestock and Poultry Slaughter" (NY467-2001), the main test sites of cysticercosis slaughtered are masseter muscles, deep psoas muscles and diaphragm muscles, and other detectable sites are myocardium, lateral scapula muscles and medial femoral muscles. According to the relevant provisions of the national standard GB16548-2006, "If cysticercles and calcified insect bodies are found, the whole corpse is used for industrial use or destroyed." ”

5 Detection methods

5.1 Etiological testing

5.1.1 Live animal testing

The biopsy sites of porcupine cysticercosis are mainly the tongue muscles and extraocular muscles. Use special openers or wooden sticks and other tools to pry open the pig's mouth, one hand pad a coarse cloth to pull the tongue out for careful observation, and with your hand touch the bottom of the tongue, the side of the tongue, the root of the tongue with a cyst, when touching the bean nodule that feels elastic, it is a cyst, and its detection rate is about 30%. When examining the extraocular muscles, the conjunctiva and outer skin of the eye are pinched with the thumb and index finger, and the initial judgment of the cyst is when a protrusion is felt.

5.1.2 Meat testing after slaughter

When the pig is inspected after slaughter, in accordance with the requirements of the "Trial Procedures for Meat Hygiene Inspection", the masseter muscle, tongue muscle, heart muscle, deep psoas muscle, etc. are cut open, and the vesicles the size of beans or rice grains can be seen, oval, white and transparent, and the sac contains translucent liquid and white head segments with rice grains. Pick the subcutaneous nodule, peel off the outer fiber membrane, the worm body is directly observed under the microscope, the sac wall is divided into two layers, the outer cortex, the inner interstitial layer, the interstitial layer has a thickened into the capsule to form a head node that rolls inward and contracts, and its structure is similar to the head segment of the adult insect. Tableting, fixation, dehydration, transparency, and post-stain identification when necessary.

5.2 Intradermal allergic reactions

Take the cysticercosis head joint from fresh pork to develop a 1:100 suspension. Inject 0.1 to 0.2 mL of intradermal part of the outer fold of the ear to be tested, and the reaction begins to appear after 5 min. Judgment: Redness and swelling occur at the injection site, the diameter is more than 11 mm, and it begins to subside after 45 minutes, which is a positive reaction; if there is no above reaction or the reaction is not significant, it is negative.

5.3 Serological testing

The pathogenic diagnosis process of cysticercosis is complex, it is difficult to obtain accurate diagnostic results, and it is easy to miss tests, so serological and immunological tests are often used to assist in the diagnosis and identification of cysticercosis. Laboratory methods for diagnosing cysticercosis in pigs include complement binding reaction, precipitation reaction, indirect erythrocyte agglutination test and enzyme-linked immunosorbent assay. Complement binding reactions have long been used to diagnose cysticercosis, but have been gradually replaced by other methods due to the high false-positive rate. Precipitation reactions include liquid phase precipitation reactions and solid phase precipitation reactions, and the diagnosis of cysticercosis by this method has the disadvantage of too low detection rate. At present, the commonly used detection methods are indirect erythrocyte agglutination test and enzyme-linked immunosorbent assay.

5.3.1 Indirect erythrocyte agglutination test

Indirect erythrocyte agglutination test (IHAT) is a serological method in which red blood cells are used as vectors for immune ligands and red blood cell agglutination readings are used. The most commonly used red blood cells are sheep red blood cells or human (O-type) red blood cells, which are conveniently sourced. At present, aldehyde-ingriocytes are used, which can be stored for half a year without losing immunosorbent properties. The steps are as follows:

(1) Erythrocyte tanning and sensitization Take the aldehyded red blood cells with 0.15mol/L PBS (pH7.2) centrifugation wash twice, the precipitation is mixed with PBS into a 2.5% suspension; add an equal amount of 1:2000 tannic acid solution (different batches of tannic acid, the mass difference is large and must be pre-tested to determine the appropriate concentration) 37 °C incubation for 20min, shake frequently; centrifuge to remove the supernatant, wash once with PBS, and then use 0.15mol/ L's PBS (pH6.4) is formulated into a 10% suspension; each suspension is mixed with an equal amount of appropriately diluted antigenic solution, placed in a 37 °C water bath tank for 30 min (1 vibration every 5 min), centrifuged to remove the supernatant, washed twice with PBS (pH7.2), and then formulated with 5% cell suspension containing 1% normal rabbit serum (NRS) and 10% sucrose buffer. Add 0.1% sodium azide to prevent corrosion, store at 4 °C or freeze-dried under reduced pressure for later use, and each batch of sensitized cells needs to be titrated with known positive and negative serum sensitivity or specificity. Positive titers above 1:640, negative serum without reaction can be used.

(2) Micro-hemagglutination test On the U-shaped (or V-shaped) micro-hemagglutination plate, the serum to be tested is diluted with 1% NRS or bovine serum albumin (BSA) normal saline series, containing 0.05mL of diluted serum per well. Add 0.01 mL of sensitized red blood cell suspension per well (can be added dropwise with a calibrated syringe needle), shake well with full shaking, and cover at room temperature for 1 to 2 h to read the results.

Depends on the type of red blood cells deposited at the bottom of the well. "-", erythrocytes sink at the bottom of the tube, dot-shaped, and smooth peripheral; "±", erythrocytes sink at the bottom of the tube, surrounded by not smooth or have small white dots in the center; "+", the red blood cell deposition range is very small, showing a more pronounced ring; "++", the red blood cell deposition range is small, in which a faint ring can appear; "+++", red blood cells are covered with the bottom of the tube, appearing ground glass; "++++", red blood cells are flaky agglutination or margin curl. The highest dilution of a significantly positive response (+) is the titer or titer of the serum. Indirect erythrocyte agglutination test detects cerebrospinal fluid or serum antibody titers greater than the upper limit of normal reference values, that is, serum agglutination titers < 1:8 and cerebrospinal fluid < 1:2, which can be diagnosed as neurocysticercosis, with a positive rate of 78% to 91.4%.

5.3.2 Enzyme-linked immunosorbent assay

At present, the most reliable diagnostic method of tail ceratophyllosis is enzyme-linked immunosorbent technology. The early establishment of the enzyme-linked immunosorbent assay (ELISA) is widely used in the diagnosis of cysticercosis in pigs. On this basis, the spot enzyme-linked immunosorbent assay, the biotin-affinity enzyme-linked immunosorbent assay, and the inhibitory enzyme-linked immunosorbent assay have been developed. In addition, the gold label test strip technology for rapid diagnosis of cysticercosis prepared using the principle of enzyme-linked immunity and colloidal gold chromatography has been successfully developed. Enzyme-linked immunosorbent assay detection rate is high, specific, stable, has been included in China's animal quarantine regulations, the specific operation steps are as follows:

(1) The whole blood tablet of the detected pig marks the number of the pig to be tested at one end of the 10cm × 1cm ordinary filter strip, and the other end draws 1 to 2 drops of blood from any part of the detected pig, dries in the indoor shade, and places it in a 4 °C refrigerator (can be stored for 6 months).

(2) Wash each well of elisa reaction plate with antigen coating solution 3 times. Dilute the antigen to the working concentration of the ELISA plate according to the instructions for use with the antigen-coated solution, 0.1mL per well, and place it at room temperature (11~29 °C) overnight after capping.

(3) Forcefully shake off the antigen-coated liquid in the elisa reaction plate wells that are coated overnight, add washing liquid to each well, soak for 3min, shake off the washing liquid with force, and use filter paper to suck away the residual washing liquid and exorcise the bubbles in the wells, re-add the washing liquid, and wash 3 times according to the same method, that is, 3×3min rinse.

(4) The blood tablets, standard negative blood tablets and standard positive blood tablets will be cut into 1cm×1cm size, placed in penicillin bottles, and diluted solution 0.3mL is added to every 1cm × 1cm blood tablets, soaked for 20min, that is, after the blood tablets turn white.

(5) Add 2 wells to each part of the blood tablet infusion solution, 0.1mL per well. Standard negative and standard positive control wells plus 2 wells of corresponding blood tablet immersion, 0.1 mL per well. Blank control wells plus dilution 2 wells at 0.1 mL per well. After the sample is loaded, it is covered, and it is left at room temperature (11~29 °C) for 30 minutes and then washed with washing solution according to the (2) method.

(6) Horseradish perhydrogenase labeled Staphylococcus aureus protein A (HRP-SPA) standard diluted to the working concentration with dilution according to the instructions for use. Tested, standard negative and standard positive wells, plus HRP-SPA marker 0.1 mL per well. Blank control wells are also added 0.1 mL. After adding, add the lid, leave it at room temperature (11 ~ 29 ° C) for 30 min and wash with the washing solution according to the (2) method.

(7) Add 0.1 mL of the prepared substrate solution per well and leave it at room temperature (11~29 °C) for 10 min.

(8) Add 2 drops of stop solution to each well to stop the reaction.

(9) Under the premise that the control hole is established, that is, the standard positive hole is dark yellow, the standard negative hole is colorless or light yellow, and the blank hole is colorless, and the test result is determined. First visual determination: compared with the standard negative pore, the color is darker than the standard negative pore, that is, the elisa-positive disease pig (+). It can also be determined with an enzyme-linked immunoassay (490 nm). Zero the blank control wells to determine the OD value of the wells to be detected. When the OD ≤ 0.22, it is determined that elisa is negative (-); the OD ≥ 0.26 is determined to be positive for ELISA (+); when 0.23≤od ≤ 0.25, it is determined that ELISA is suspected (±), and the suspect is retested once, and if it is still suspected, it is judged positive.

Cysticercosis antibody testing only responds to the presence of parasites, while antigen testing can indicate that the parasites are alive in vivo. Ideally, a combination of the two is tested. The various natural antigens used in serological tests are derived from the cyst fluid and can also be used as useful serological screening tools. Different methods use different soluble antigens to make test results difficult to compare. Most of the antigens detected by cysticercosis are crude antigens or chromatography purified antigens in the fluid of cysticercosis, mainly including the following categories: (1) purified antigen components of lentil lectins (cysticercosis 13ku, 14ku, 18ku, 21ku, 24ku, 39~42ku or 50ku glycoprotein) ;(2) antigen components of cysticer cyst fluid; (3) antigen components of the cyst wall of the cephalic segment.

After cysticercosis infects humans or animals, antibodies can be detected in serum, cerebrospinal fluid, saliva and even tears. Radioimmunology, hemagglutination tests, colloidal gold, complement binding, enzyme-linked immune responses, and immunoblotting techniques have been used to detect cysticercosis antibodies in infected people or animals. The initial antigens are derived from sac fluid, excreta G secretion (ES) products or crude homogens, these unrefined antigens have poor sensitivity and specificity, and are prone to false positives, false negatives and cross-reactions; therefore, purified antigens are developed or specific antigens are sought in the sac fluid. The best method of specificity is currently enzyme-linked immunoelectriportional transfer blotting (EITB) technology, which initially reported 100% specificity and 98% sensitivity. However, this approach is significantly less sensitive in the diagnosis of cerebral monocyst disease. Prabhakaran et al. report a sensitive immunodiagnostic approach (tertiary conformation of lentil lectins induced with urea). This method was used to diagnose 60 patients with cysticercosis in pigs, and antibodies were detected in the serum of 46% of them, while negative with standard western blot diagnostic methods. This commercialized EITB kit can be purchased directly and has been widely used to diagnose human and porcine cysticercosis, but the method also has certain drawbacks, such as antigen sources can not be guaranteed, antigen preparation is difficult, there are differences between antigens, probe hybridization technology is expensive, etc. To solve these problems, these antigens are identified, analyzed and synthetically synthesized, and the diagnostic test for cysticercosis in pigs is performed with LISA. Through ELISA detection, it was found that the specificity and sensitivity of these synthesized proteins were much lower than those of natural glycoprotein antigen (LLGP) proteins. Prasad reports a novel immunodiagnostic technique for detecting cystic antigen-induced lymphocyte proliferation. This technique is based on cyst fluid antigens, which measure the cellular immune response produced by cysticercosis stimulating the body by lymphocyte proliferation assays. The researchers reported that the specificity and sensitivity of 48 test groups and 79 control groups were 93.8% and 96.2%, respectively.

Another way to detect cysticercosis is to test for antigens. At present, researchers have established several tests for detecting cysticercosis antigens in serum. Two monoclonal antibodies, B158/B60 and HP10, have been validated and are routinely used for the detection of cysticercosis antigens, with a reported very high sensitivity, with up to 80% of serum samples and 90% of cerebrospinal fluid. In the epidemiological survey of cysticercosis in pigs, the researchers counted the sensitivity and specificity of B158/B60 and HP10, with the sensitivity of B158/B60 and HP10 being 84% and 76%, respectively, and the specificity of 55% and 83%, respectively. B158/B60 also has good diagnostic performance in human urine antigen testing, with a parasite sensitivity of 92% (compared to 62.5% in cases of single cysticercosis infection). Only the majority of patients with calcified cysticercosis (83%) tested negative. The diagnostic results of serum and urine are very consistent, so urine or saliva can be used as a diagnostic sample instead of cerebrospinal fluid and serum samples during the testing process, which can greatly simplify the screening process of cysticercosis.