Detection of parasites in food of animal origin - Echinococcus

Echinococcus is the larvae of Echinococcus granulosus and Echinococcus multilocularis, which can cause zoonotic parasitic diseases——— echinoclccosis, also known as hydatidiosis. Echinococcus parasitizes the internal organs of a variety of intermediate hosts, compressing the internal organs and causing the parasitic organs to atrophy, which in turn causes corresponding symptoms. Adults parasitize the small intestines of canines, and the larvae can infect a variety of mammals, including cattle, sheep, pigs and camels in livestock, cattle and sheep are the most affected, and humans can also be infected. The disease is widespread around the world, causing serious harm to human and animal health and causing serious economic losses in endemic areas.

1 Etiological features

Echinococcus fine-grained (single-chamber echinococcus) is a free-band encapsulated structure that contains liquid. The shape varies, and the shape often varies depending on the parasitic site, and the size often ranges from the size of a pea to the size of a human head. Generally nearly spherical, diameter 5 to 10 cm. Single-house echinococcus can be divided into three categories:

(1) Humanoid echinococcus hominis (echinococcus hominis) sac contains liquid, and the sac wall is composed of two layers: the outer layer is thicker, called the stratum corneum; the inner layer is very thin, called the hair growth layer. On the hair growth layer, there can be a birth capsule, and on the inner wall of the hair growth sac, an unequal number of protocephalic cymbs can grow, some hair growth sacs are separated from the hair growth layer, or some head segments are separated from the hair growth sac. Free in the sac fluid is called "echinococcosis sand", and the naked eye examines the echinococcosis sand as yellowish-white, such as the size of a needle needle nose. A second generation of cysts can also be grown on the sac wall hair layer, called ascosacs. Ascomycetes can grow into the cavity of the original sac (also known as the mother sac), called "endogenous ascomycetes", or can grow outside the mother sac, called "exophytic ascomycetes". Grandchild sacs can also grow on the hair growth layer of the ascomycetes, and the ascomycetes and grandchilds have the same structure as the mother sacs, and their hair growth layer grows on the hair sacs and forms cephalic segments. Thus, a echinococcal sac may contain many ascomycetes and grandsmal sacs. This type is most common in humans, and is only found in cattle in livestock.

(2) The echinococcus veterinarum is basically similar in structure to the humanoid, except that ascomycetes and grandisans no longer grow on the hair growth layer. This type is most common in sheep.

(3) Echinococcus acephalocysta (echinococcus acephalocysta) has completely different characteristics from the above types, because there is no hair layer in the sac, it cannot grow cephalic segments, ascomycetes and grandstic sacs. This type is most common in cattle.

Echinococcus multiloceris is characterized by vesicles consisting of many small sacs and are mulberry-shaped.

2 Epidemiological features

Echinococcosis parviflora (Cystic echinococcosis, CE) is global in its distribution area, covering five continents, with the largest number of pastoral areas. Africa is dominated by countries such as Kenya, Libya, Tunisia, Algeria and Morocco. The Middle East region of Asia is dominated by countries such as the United Arab Emirates, Kuwait, Saudi Arabia, Iraq and Iran, and echinococcosis is endemic in countries such as Jordan, Syria and Lebanon in the eastern Mediterranean. The outbreak is more severe in South America, where it is found in countries such as Argentina, Brazil, Chile, Uruguay and Peru. North America is found mainly in northwestern Canada and Alaska in the United States. Echinococcosis is present in most European countries, but is more prevalent in Spain, Sardinia, Italy, and Corsica, France. The early severe epidemic of echinococcosis along the island of Somania in Iceland, Cyprus, New Zealand and Australia has been largely controlled through active integrated control measures. Xinjiang is the most serious in China, with sheep infection rates of 50% to 80%, and some areas as high as 90.85%, followed by Qinghai, Ningxia, Gansu, Inner Mongolia, Sichuan and other provinces. There are 2,000 cases of human echinococcosis in China every year. In recent years, sporadic cases have been reported in Guizhou, Yunnan, the three northeastern provinces, Henan, Shandong, Hebei, Anhui, Guangxi, Hubei, Hunan and other provinces.

Echinococcosis multicultura (Echinococcus vesicularis, AE) is more limited in distribution area, mainly prevalent throughout the northern hemisphere tundra, especially in alaska, Japan, Hokkaido and Siberia, Europe is found in France, Germany and other countries. Echinococcosis multi-house in China is mainly endemic in Gansu, Ningxia, Xinjiang and the Qinghai-Tibet Plateau, and there are sporadic case reports in northeast China and Inner Mongolia.

Echinococcosis is a disease transmitted between canines and intermediate hosts, and canines have important implications for the epidemic of the disease as the terminal host of adult echinococcus. The terminal hosts are mainly dogs, especially wild dogs and sheepdogs. Eggs contaminate grasslands and living environments, causing infections in livestock and people. The intermediate host of Echinococcus parviflora is widespread, and epidemiologically important sheep (adult sheep) have the highest rate of infection. The transmission route of echinococcosis is mainly the digestive tract route, and there are two ways of oral infection: one is that the human body is in direct contact with the terminal host animal infected with Echinococcus taenus, resulting in accidental swallowing of eggs and infection. Epidemiological investigations have shown that the prevalence of echinococcosis is related to the lifestyle habits of the population. Certain occupations have a high rate of infection, such as herders (especially Tibetans), reindeer keepers, children, students, dairy workers, slaughterhouse personnel, refrigeration plant personnel, livestock fur processing or tannery workers, wool spinning mill workers, etc., whether echinococcosis seropres or prevalence is relatively high. Long-term exposure of the human body to the environment infected with echinococcus can enhance the body's immunity, and it is beneficial to defend against echinococcal infection due to the increase of antibodies in the blood. Therefore, new patients infected with echinococcus infection shortly after first entering the endemic area are susceptible. Another way is indirect infection, where eggs infect the human body by contaminating water, vegetables, or fruits. Eggs can also be inhaled into the lungs by respiratory route, so in a few cases there are only lung infections and no liver infections.

3 Hazards

Echinococcus parasitizes the internal organs and muscles of mammals such as pigs, cattle and sheep, causing serious harm to humans and animals. The severity of the harm of echinococcus to animals depends mainly on the size, number and parasitic site of echinococcus. The main hazards are mechanical compression that causes the surrounding tissues to shrink and function. In addition, all kinds of animals can have severe allergic reactions due to vesicle rupture, and sudden death, which is particularly harmful to people. Because the echinococcal sand and broken hair sac in the echinococcal cyst fluid can grow into new echinococcal sacs in any part of the body, and the cyst fluid is an allogeneic protein to humans and animals, and even contains toxins, the bursting of the vesicles causes severe allergic reactions in humans and livestock, causing breathing difficulties, elevated body temperature, diarrhea, and is particularly sensitive to people. At present, there is no effective treatment for fine-grained echinococcosis, mainly through surgical removal to control the disease; the harm of multi-atrial echinococcus is far more serious than that of echinacea, and prevention and control are particularly difficult. The growth characteristics of polyamos echinococcus are diffuse infiltration, the formation of countless small vesicles, compressing the surrounding tissues, causing organ atrophy and dysfunction, just like malignant tumors; it can also be transferred to various organs throughout the body, so it is called "insect cancer", which poses a great threat to the health of the population.

4 Domestic and foreign health requirements

In the quarantine process, the animal organs detected by echinococcus should be destroyed; when echinococcus is found in the muscle, the affected part is cut off and destroyed, and the other parts are not restricted; but if the infection is serious and the vesicle ruptures and contaminates the flesh carcass, it should be treated at high temperature and used for industrial use.

5 Detection methods

5.1 Etiological testing

5.1.1 Live animal testing

Mild or initial infection in livestock is asymptomatic. Sheep are most susceptible to the disease, with poor fattening when they are seriously infected, being hairy and prone to hair removal, and those affected by the lungs cough continuously and cannot stand up on the ground. When the bovine liver is affected, it is malnourished, ruminated and weak, often puffy, thin and weak, and coughing when the lungs are affected. In severe infection, the animal is emaciated, coughing, and the right side of the abdomen is enlarged.

5.1.2 Meat testing

Echinococcus echinococcus is mainly parasitic in the liver, followed by the lungs. Echinococcus can be found in the liver and lungs in the liver and lungs with significantly increased volume and uneven surfaces; sometimes echinococcus can be found in other organs such as spleen, kidney, brain, subcutaneous, muscle, bone, spinal canal, etc. Incision of echinococcus may have liquid flowing out, forming a cavity cavity, precipitating the liquid, and many hair sacs and protocephalic larvae (i.e., echinococcus sand) can be seen with the naked eye or under the autopsyoscope; sometimes the ascomycetes or even grandchild sacs in the liquid can also be seen with the naked eye; occasionally calcified echinococcal larvae or purulent foci can be seen. The echinococcal protocephalic segment is stained with 0.1% eosin stained under the microscope, and the etrophic or inverted protocephalic segment is visible, and accessories such as apical process, small hooks and suction cups are visible. The sac wall of Echinococcus is composed of the inner capsule, the former being the hair-generating membrane of the single-cell nucleus layer and the stratum corneum of the cell-free layer of the plate-like structure, which is the fibrous membrane and inflammatory cell infiltration produced by the host's response to echinococcus. Echinococcus multilocereformis is observed under the microscope to form multi-atrial echinococcus nodules formed by infiltration of small vesicle groups and the fibro connective tissue around them by membrane and inflammatory cells, and the proliferation of polyadenococcus is endogenous and extrogenous, and the protocephalic segment is usually rare.

5.2 Serological testing

Echinococcus cystic fluid has been used as a major antigen source for primary immunological diagnosis of cystic echinococcosis in humans. However, this antigen lacks sensitivity, specificity, and is difficult to standardize, especially in the cross-reactivity of serum from other parasitic infected patients. Although recombinant proteins have been synthesized from these antigens, to date, there is no standard high sensitivity and specific test for immunodiagnosis of echinococcosis. Among the commonly used immunodiagnostic methods, most of them use dialysis echinococcal sac fluid as antigens, and there are also affinity chromatography and polyacrylamide gel electrophoresis methods to concentrate and separate antigens, and live or dead protocephalics can be used as effective antigens. Among them, animals and humans can be diagnosed by intradermal allergy (Kasonyl test), high sensitivity, but poor specificity, the general accuracy rate is about 70%, but its operation is simple, high sensitivity, can quickly measure the reaction results, especially suitable for large-area census of the initial screening, still has practical value. Complement binding test generally has a positive rate of 50% to 80%, and there are a variety of false positive reactions. Immunoserological tests for echinococcus are mainly indirect erythrocyte agglutination test (IHAT), the former is fast and easy, with a detection rate of 83.3%, and ELISA, the latter has high specificity and sensitivity. In addition, there are enzyme-linked immunosorbent assays (PPA-ELISA) labeled with enzyme-linked Staphylococcus aureus protein, dot-ELISA tests, and biotin-affinity enzyme-linked immunosorbent tests (ABC-ELISA). Zhang Jingyuan et al. compared 8 immunodiagnostic methods, and the results showed that ELISA and ABC-ELISA had the highest sensitivity, followed by IHAT, and agarose gel diffusion was the worst. Specificity is highest in enzyme-labeled convective immunoelectrophoresis. Since these tests have different levels of false positives and negatives, it is recommended that a positive reaction in 2 to 3 methods be used as a diagnostic indicator of the disease. The current laboratory diagnosis is based on the imaging structure identification technology of cysts [ultrasound, computed tomography (CT), X-ray] and immunological diagnostic methods, mainly ELISA and western blots, and X-ray and CT detection rates are high.

5.2.1 dot-ELISA

Dot-ELISA is a new development of ELISA technology in recent years, the selection of nitrocellulose thin (NC) membrane with strong adsorption capacity of protein as a solid phase carrier, the substrate is enzymatic reaction after the formation of colored precipitate to color the film, and then visually or quantitatively with an optical density scanner. Dot-ELISA can be used to detect antibodies and can also be used to detect antigens, because this method is easier to perform than other immunological tests, so it is currently used for antigen detection. Here's how:

The serum to be tested was diluted 1:(1~20), 1 μL of serum was dripped on the nitrocellulose membrane (NC) with a microsampler, placed at 70 °C and immersed in the NC membrane in 1% BSA-PBS for 1 h, shaken at room temperature for 1h, washed twice, added 1:1000 diluted monoclonal antibody (McAb) enzyme marker, shaken at room temperature for 2 h, washed 3 times, added substrate 3,3'-diaminobenzidine or 4chloro-1-ethanol, 15 min later, the flow of water to terminate the reaction, visually judging the result. Those who show brown spots are positive, otherwise they are negative. The highest dilution to produce the brown spot reaction is the antigen titer.

5.2.2 Western blotting

Immuno blotting (IB) is a new type of immune probe technology combined with three technologies of sodium lauryl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrophoresis transfer and labeling immunization test, which is an effective method for analyzing protein antigens and identifying biologically active antigen components, and in recent years has been applied to detect the corresponding circulating antibody components or spectra of a molecular weight antigen in the body fluid of parasite infection. It has the characteristics of high sensitivity and high specificity. The western blotting technique used for diagnostics employs enzyme-labeled probes (i.e., secondary antibodies and their labeled conjugates), called enzyme-linked immunoelectriportunic transfer blots (EITBs). The specific steps are as follows:

(1) Sample separation Take fresh echinococcus in an appropriate proportion with buffer, homogenize, boiling water bath for 2min, centrifuge 10000g for 30min, take the supernatant for later use. Antigen samples are separated by single-comb SDS-PAGE electrophoresis. The left comb hole is added to the standard molecular weight protein, and the right side of the comb hole is added to the antigen solution, and the voltage is controlled at 160~180V.

(2) Electrophoretic transfer Remove the gel pieces of the completed electrophoresis from the electrophoresis plate and soak them in an enamel dish containing transfer buffer (TB). Form a transfer sandwich plate layer within the TB: take the corresponding size of the NC film, slowly soak in the TB, and press the gel sheet against the glossy surface of the NC film. Then place two layers of soaked filter paper on each side of the outer side of the gel and NC membrane and then a layer of sponge pad (thickness 0.5 to 1 cm), make a direction marker, and finally sandwich between the two layers of perforated plastic liner, absolutely avoid leaving bubbles between each layer. Invert the TB into the transfer slot and insert it into the transfer plate so that the gel sheet is on the cathode side and the NC membrane is on the anode side. Place the transfer slot in a 4 °C refrigerator, power on the transfer for several hours or overnight, the current is controlled at about 250mA (40 ~ 50V).

(3) Probe detection Remove the transferred NC membrane, put it horizontally into the quencher, shake at room temperature for 1h to block the area of unsorbed protein, and then wash 2 to 3 times with wash buffer, each time for 30min to remove the denaturant, so that the natural state and biological characteristics of the protein can be restored. Lay the NC membrane flat on filter paper soaked with Tris-buffer (TBS), divide the NC membrane into straight strips about 0.5 cm wide according to the direction of electrophoresis with a blade, and mark the upper end with a pencil. Take one of the thin strips and do an amino black stain with the standard protein band (also can be coomassie bright blue staining or silver staining), test the separation effect and determine the molecular weight position. The rest of the thin strips are left to dry at 4 °C for blotting test backup (antigen activity can be maintained for more than 3 months).

(4) Blotting test The above antigen strips are placed in the reaction tank of the compartment reaction plate, frontally upward, one per tank, and pre-soaked with 0.05% TBS-Tween (TBS-T) liquid; the serum to be tested is diluted with TBS-T liquid (1:150), added to the reaction tank, limited to the submerged membrane strip. It usually takes 0.5 to 1.5 mL, which is equivalent to 10 μL of blood sample (the same amount of liquid added per tank); oscillate for 60 min at room temperature (20 to 25 °C), wash 6 times with TBS-T for 3 min each time; add diluted sheep anti-human enzyme label secondary antibody conjugate, incubate for 1.5 h, wash as above; add freshly formulated substrate solution; rinse with distilled water several times after 15 min to terminate the reaction, the film strip is removed and placed on the glass plate to dry naturally; positive reactions can be seen blue-black (4-chloro-1-naphthalene phenol substrate) or tan diaminobenzidine (DAB) bands.