Early recognition method for cat toxoplasma gondii infection

Abstract

The invention discloses a cat toxoplasma gondii infection early recognition method which comprises the following steps: carrying out immunological detection on an antigen combination containing two or more toxoplasma gondii antigens and a to-be-detected cat serum sample, and judging whether a cat is infected with toxoplasma gondii or not according to a detection result, wherein the antigen combination is selected from at least two of toxoplasma gondii tachyzoitic stage specific protein, invasive cell secreting type protein and cat intestinal epithelial cell developmental stage specific expression protein; the invasive cell secreting type protein is selected from at least one of micronematode secreting protein, claviform secreting protein, compact particle secreting protein, surface antigen family protein and metabolism and structure related protein. The invention provides an early recognition method for cat toxoplasmosis, which can ensure the early and rapid recognition of cat toxoplasmosis infection, can warn the occurrence and blocking propagation of the oocysts of toxoplasmosis, and has extremely high sensitivity and specificity.

Description

Technical Field

[0001] This invention belongs to the field of immunological detection technology, specifically relating to a method for early identification of feline toxoplasmosis infection. Background Technology

[0002] With the continuous improvement of socio-economic levels and changes in population structure, the number of companion animals kept as pets has been steadily increasing, with cats becoming one of the most common companion animals in my country. However, cats are both the definitive host and an intermediate host in the life cycle of Toxoplasma gondii, playing a crucial role in the transmission chain of toxoplasmosis. After cats are infected with Toxoplasma gondii, they can not only shed infectious oocysts in their feces, contaminating the environment, but also form cysts in their bodies that persist for a long time, becoming a persistent source of infection and posing a potential public health risk to humans and other animals.

[0003] Toxoplasmosis is a widespread zoonotic parasitic disease that can infect almost all warm-blooded animals. Toxoplasmosis infection is particularly dangerous for pregnant women and immunocompromised individuals, potentially leading to miscarriage, stillbirth, fetal malformations, or eye damage. Therefore, early identification and surveillance of feline toxoplasmosis infection are crucial for preventing its transmission between humans and animals.

[0004] Currently, the diagnosis of feline toxoplasmosis infection mainly relies on serological tests (such as ELISA and IFA) or molecular biological tests (such as PCR) based on tachyzoite antigens. However, these methods are mostly used in the post-infection diagnostic stage and are difficult to use for rapid identification in the early stages of infection. In the early stages of toxoplasmosis infection, firstly, the number of parasites is low, and secondly, cats, as the definitive host, have limited routes of infection with tachyzoites, and serum antibodies against tachyzoites have not yet been produced. Therefore, traditional serological tests based on tachyzoite antigens have low sensitivity; while molecular tests, although highly specific, are complex to operate and costly, making them unsuitable for large-scale screening applications. In addition, early feline toxoplasmosis infection often has no obvious clinical symptoms and is easily overlooked, thus delaying prevention and control, increasing environmental contamination and transmission risks. Therefore, a method for early identification of feline toxoplasmosis infection is needed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for early identification of Toxoplasma gondii infection in cats, which is of great significance for preventing cats from continuously shedding oocysts, blocking the environmental transmission route of Toxoplasma gondii, and reducing the risk of infection in the human population. By identifying characteristic immune responses or molecular markers in the early stages of Toxoplasma gondii infection in cats, rapid, sensitive, and specific early diagnosis can be achieved, providing key technical support for subsequent vaccine evaluation and prevention and control strategies.

[0006] This invention is achieved through the following technical solution: This invention includes using an antigen combination containing two or more Toxoplasma gondii antigens to perform immunological detection on a cat serum sample, and determining whether the cat is infected with Toxoplasma gondii based on the detection results; wherein the antigen combination is selected from at least two proteins selected from Toxoplasma gondii tachyzoite-specific proteins, invading cell secretory proteins, and proteins specifically expressed during the development of cat intestinal epithelial cells, and the invading cell secretory proteins are selected from at least one of micronematode secretory proteins, rod-shaped body secretory proteins, dense granule secretory proteins, surface antigen family proteins, and metabolism and structure-related proteins.

[0007] Furthermore, the microneedle secretory protein includes at least one of MIC1, MIC2, MIC3, MIC4, MIC6, MIC8, and MIC13.

[0008] Furthermore, the rod-shaped secretory protein includes at least one of RON2, RON4, RON5, RON8, ROP1, ROP2, ROP5, ROP16, ROP18, and ROP38.

[0009] Furthermore, the dense granule secretory protein includes at least one of GRA1, GRA2, GRA5, GRA6, GRA7, GRA15, GRA16 and GRA24.

[0010] Furthermore, the surface antigen family proteins include at least one of SAG1, SAG2A, SRS29B, SRS29C, and SRS34A.

[0011] Furthermore, the metabolism and structure-related proteins include at least one of ACT1, GAP45, MLC1, ENO2, LDH1, HSP70, and HSP90.

[0012] Furthermore, the proteins specifically expressed during the development of cat intestinal epithelial cells include at least one of GRA11B, OWP1, OWP2, OWP3, SOD3, and ERP.

[0013] Furthermore, the immunological detection method is ELISA, colloidal gold immunochromatography, or fluorescent microsphere immunoassay.

[0014] Furthermore, the antigen combination comprises SAG1 and GRA6, SAG1 and MIC8, or SAG1, MIC8 and GRA6.

[0015] On the other hand, an antigen combination for early identification of feline toxoplasmosis infection comprises two or more toxoplasmosis antigens, said antigens including a tachyzoite-specific protein of Toxoplasma gondii, an invading cell secreted protein, and a protein specifically expressed during the development of feline intestinal epithelial cells.

[0016] The most significant feature of this invention compared to existing technologies is that the above-described technical solution is: This invention provides a method for early identification of feline toxoplasmosis, which can ensure early and rapid identification of feline toxoplasmosis infection, and can provide early warning of the occurrence of toxoplasmosis oocysts and block transmission. It has extremely high sensitivity and specificity. Attached Figure Description

[0017] Figure 1 Figure showing the results of SAG1 protein expression and purification; Figure 2 Figure showing the results of GRA6 protein expression and purification; Figure 3 This is a graph showing the area under the ROC curve (AUC) used in Example 1 to evaluate the diagnostic efficacy of each antigen. Figure 4 This is a diagram showing the results of Mic8 protein expression and purification in Example 2. Figure 5 This is a graph showing the area under the ROC curve (AUC) used in Example 3 to evaluate the diagnostic efficacy of each antigen. Figure 6 This is a graph showing the diagnostic efficacy of the area under the ROC curve (AUC) for each antigen in Example 4. Detailed Implementation

[0018] The technical solution of the present invention will be further described below with reference to embodiments and comparative examples, but it should not be construed as a limitation of the present invention: Example 1: Establishment and validation of an ELISA method combining SAG1 and GRA6 for detecting early Toxoplasma gondii infection in cats. Using Toxoplasma gondii surface antigen SAG1 and dense particulate protein GRA6 as target antigens, a dual-antigen combination ELISA method for early detection of feline Toxoplasma gondii infection was established through gene cloning, prokaryotic expression, purification, and enzyme-linked immunosorbent assay (ELISA) optimization. This method can simultaneously identify IgM and IgG antibodies produced in the early stage of infection, exhibiting high sensitivity and specificity, making it suitable for large-scale serological surveillance and evaluation of vaccine immunization efficacy.

[0019] Antigen gene acquisition and plasmid construction: Based on the Toxoplasma gondii SAG1 and GRA6 gene sequences, secretion signal peptides and membrane localization sequences were removed. Specific primers (SAG1-F / SAG1-R and GRA6-F / GRA6-R) were designed, and restriction enzyme sites NdeI and XhoI were introduced. After PCR amplification of the target fragment, it was ligated with the pET-32a(+) vector by double enzyme digestion and transformed into Escherichia coli BL21(DE3) competent cells to construct recombinant expression plasmids pET32a-SAG1 and pET32a-GRA6.

[0020] The recombinant strain was inoculated into LB medium, and 0.5 mmol / L IPTG was added at OD600≈0.6 for induction at 37°C for 4 h. Cells were collected, sonicated, centrifuged, and the supernatant was collected and processed using Nitrogen gas chromatography-mass spectrometry (NCM). 2+ -NTA affinity chromatography purified recombinant SAG1 and GRA6 proteins, and SDS-PAGE electrophoresis confirmed that the molecular weights of the proteins were approximately 32 kDa. Figure 1 ) and 29 kDa ( Figure 2 After impurities were removed by dialysis, the protein concentration was determined and stored at -80°C.

[0021] Purified SAG1 and GRA6 proteins were diluted separately with carbonate buffer (pH 9.6) and coated onto 96-well ELISA plates at different ratios (SAG1:GRA6 = 1:1, 2:1, 1:2), with a coating amount of 0.5–1.0 µg per well. After incubation at 4°C overnight, the plates were blocked with 5% skim milk. The optimal antigen ratio and coating amount were determined by comparing positive and negative serum titers.

[0022] After optimization, the optimal reaction conditions were determined to be: SAG1:GRA6 = 1:1, coating amount 0.8 µg / well, serum dilution 1:100, and enzyme-labeled antibody dilution 1:5000.

[0023] Serum samples were collected from cats orally infected with Toxoplasma gondii tissue cysts at 3, 7, 14, and 21 days post-infection, as well as serum from healthy controls. The OD450 values ​​of each group were measured using this combination ELISA and compared with those of single-antigen ELISA. The mean negative serum OD value + 3 × standard deviation was used as the positive cutoff.

[0024] The results showed that the dual-antigen combination ELISA could detect a positive reaction as early as 7 days after infection, while the ELISA using SAG1 or GRA6 antigen alone only showed a positive reaction 14 days after infection. The sensitivity of the combination detection was increased by approximately 1.8 times, and the cross-reactivity rate with positive sera for feline coronavirus and feline herpesvirus was less than 3%, indicating good specificity. Figure 3 The coefficients of variation (CV) within and between plates were both less than 10%, indicating good reproducibility of the method; the test results were stable after the reagents were stored at 4°C for 6 months.

[0025] In clinical sample validation, a total of 216 clinical cat serum samples were tested. Compared with commercial ELISA kits, the positive concordance rate was 96.8%, the negative concordance rate was 98.2%, and the Kappa value was 0.95, indicating that this method is highly consistent with existing detection methods and significantly improves the positive rate in the early infection stage (P<0.01).

[0026] By combining SAG1 and GRA6 proteins, the ELISA method established in this embodiment can accurately identify infected individuals in the early stage of Toxoplasma gondii infection (within 7 days). It features high sensitivity, strong specificity, low cost, and good reproducibility, making it suitable for large-scale laboratory and clinical applications. This method provides a reliable technical means for the rapid diagnosis and prevention of early Toxoplasma gondii infection in cats.

[0027] Example 2. Establishment and validation of an ELISA method for detecting early Toxoplasma gondii infection in cats using a combination of SAG1 and Mic8. By cloning, expressing, and purifying Toxoplasma gondii SAG1 and Mic8 proteins, and combining them with optimized ELISA experimental conditions, antibody responses can be detected in the early stages (within 7 days) of Toxoplasma gondii infection in cats. This method has high sensitivity and specificity and is suitable for rapid screening and immune monitoring of Toxoplasma gondii infection in cats.

[0028] Based on the Toxoplasma gondii SAG1 and Mic8 gene sequences, secretion signal peptides and membrane localization sequences were removed, and specific primers (SAG1-F / SAG1-R and Mic8-F / Mic8-R) were designed to introduce NdeI and HindIII restriction enzyme sites, respectively. SAG1 was used as in Example 1. After amplifying the target gene fragment by PCR, it was cloned into the pET-32a(+) expression vector. The recombinant plasmid pET32a-Mic8 was constructed and transformed into E. coli BL21(DE3) competent cells for expression.

[0029] Recombinant strains were inoculated into LB medium and cultured until OD600 ≈ 0.6. 0.5 mmol / L IPTG was then added to induce expression. After cell culture, cells were collected, sonicated, centrifuged, and the supernatant was used for Nitrogen extraction. 2+ -NTA affinity chromatography purification. The purity of Mic8 protein was determined by SDS-PAGE; Mic8 is approximately 28 kDa. Figure 4 The purified protein was purified by dialysis to remove impurities, and its concentration was determined before being used in ELISA experiments.

[0030] In the optimization experiment, purified SAG1 and Mic8 proteins were coated onto 96-well ELISA plates at different ratios (SAG1:Mic8 = 1:1, 2:1, 1:2), with a coating amount of 0.5–1.0 µg per well. 5% skim milk was used as the blocking buffer, and the plates were incubated overnight at 4°C. By comparing the reactions of feline toxoplasmosis-infected serum with that of healthy cats, the antigen ratio and reaction conditions were optimized. Ultimately, a 1:1 SAG1 to Mic8 ratio and a coating amount of 0.8 µg / well were determined to be the optimal conditions.

[0031] Serum samples from cats at different infection stages (3, 7, and 14 days post-infection) were collected and compared with serum from healthy cats. After ELISA, the OD450 values ​​of each sample were measured to determine the positive and negative criteria. The mean negative control OD value plus three times the standard deviation was used as the positive cutoff.

[0032] The results showed that the dual-antigen combination ELISA detected a significant positive reaction as early as 7 days after infection in cats, while the detection of SAG1 or Mic8 proteins alone typically showed a positive reaction only 14 days after infection. This method demonstrated approximately 1.5 times higher sensitivity than the single-antigen ELISA. Furthermore, cross-testing showed a positive rate of less than 5% when testing sera infected with feline coronavirus and feline herpesvirus, indicating that this method has high specificity. Figure 5 ).

[0033] Under the same reagents and experimental conditions, the coefficients of variation (CV) within and between plates were both less than 8%, indicating that this method has good experimental reproducibility. Furthermore, stability tests showed that the reagents exhibited good stability after being stored at 4°C for 6 months, with no significant changes observed.

[0034] In a clinical validation study of 210 feline serum samples, the results showed a positive concordance rate of 95.2% and a negative concordance rate of 97.5% between this method and commercially available ELISA kits, with a Kappa value of 0.93, indicating a high degree of agreement. This method demonstrated a high positive reaction rate in the early stages of Toxoplasma gondii infection (within 7 days of infection) and was more sensitive than single antigen detection.

[0035] By combining SAG1 and Mic8 proteins, the ELISA method established in this embodiment can effectively detect the early immune response to Toxoplasma gondii infection in cats, and exhibits higher sensitivity and stronger specificity compared to traditional single-antigen methods. This method is simple, sensitive, and highly specific, making it suitable for early screening of feline Toxoplasma gondii infection and evaluation of vaccine efficacy. It provides a convenient immunological detection method for the control and prevention of feline Toxoplasma gondii.

[0036] Example 3. Establishment and validation of a detection method for fluorescent microspheres using the combined application of SAG1 and Mic8. In the preparation and antibody conjugation of fluorescent microspheres, polystyrene fluorescent microspheres with a diameter of 0.5 µm were selected. SAG1 and Mic8 antigen antibodies were conjugated to the surface of the fluorescent microspheres via chemical cross-linking. The specific steps are as follows: Preparation of fluorescent microspheres: Polystyrene fluorescent microspheres (Ex / Em = 488 nm / 515 nm) purchased from a commercial supplier were first washed with PBS to remove impurities.

[0037] Antibody conjugation: Specific antibodies against SAG1 and Mic8 (obtained from E. coli expression systems) were reacted with fluorescent microspheres using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross-linking agents. Reaction conditions were: 2 hours, room temperature, pH 7.4.

[0038] Antibody blocking: After the reaction is complete, unbound sites are blocked with BSA and washed to remove unbound antibodies.

[0039] Antibody conjugation verification: The fluorescence intensity of the microspheres after conjugation is observed using a fluorescence microscope to verify whether the antibody has successfully bound.

[0040] Serum samples from cats infected with Toxoplasma gondii at different time points (3, 7, and 14 days post-infection) were compared with serum samples from healthy control cats. Serum samples underwent centrifugation to remove blood clots and cellular impurities. The final serum samples were used for fluorescent microsphere detection.

[0041] Detection platform: This method uses flow cytometry (FACS) or fluorescence reader for detection.

[0042] Antibody incubation: SAG1 and Mic8 conjugated fluorescent microspheres were mixed with cat serum samples and incubated for 30 minutes with shaking at room temperature. Afterwards, the samples were washed three times with PBS to remove unbound antibodies.

[0043] Fluorescence detection: The washed microsphere solution was added to a flow cytometer for analysis, and the fluorescence signal was recorded. The fluorescence intensity of the positive results was significantly higher than that of the negative control.

[0044] By setting up positive and negative controls, the intensity of the fluorescence signal was compared. The positive cutoff was set at the mean fluorescence intensity of negative serum plus three times the standard deviation. When the detected fluorescence intensity exceeded this cutoff, it was considered that an immune response against Toxoplasma gondii was present in the cat serum, and the result was judged as positive.

[0045] Sensitivity and specificity were assessed through cross-reactivity tests with healthy cat serum and other common feline diseases (such as feline coronavirus and feline herpesvirus). The dual-antigen fluorescent microsphere detection method demonstrated good specificity, with a cross-reactivity rate of less than 5%. In terms of sensitivity, a positive reaction could be detected within 3 days after cat infection, which is approximately 1.5 times higher than traditional methods (such as ELISA).

[0046] The test results showed good repeatability, with intra- and inter-plate coefficients of variation (CV) both less than 8%. Stability testing showed that the fluorescent microspheres maintained stable performance without significant degradation after 6 months of storage at 4°C.

[0047] Clinically validated cat serum samples (220 samples) were compared. The results showed that the positive concordance rate of this method with the commercial ELISA kit was 94.5%, the negative concordance rate was 97.8%, and the Kappa value was 0.92, indicating a high degree of consistency between the two methods. The positive rate of the fluorescent microsphere detection method within 3 days after infection was significantly higher than that of the commercial ELISA kit.

[0048] This embodiment combines SAG1 and Mic8 antigens with fluorescent microsphere technology. The detection method established in this embodiment has the advantages of high sensitivity, high specificity, and simple operation, enabling rapid identification of infected individuals in the early detection of feline toxoplasmosis. This method can be widely used in early screening for toxoplasmosis, immune monitoring, and vaccine efficacy evaluation, providing an efficient detection method for the prevention and control of feline toxoplasmosis.

[0049] Example 4. Application of the combination of SAG1, Mic8, and GRA6 in an ELISA method for detecting Toxoplasma gondii infection in cats. The acquisition of antigen genes and construction of plasmids are the same as in Examples 1 and 2, and the expression and purification of recombinant proteins are the same as in Examples 1 and 2.

[0050] In the ELISA plate coating and experimental optimization, antigen coating: recombinant proteins of SAG1, Mic8 and GRA6 were diluted in carbonate buffer (pH 9.6) at different ratios (SAG1:Mic8:GRA6 = 1:1:1, 2:1:1, 1:2:1, etc.) and coated on 96-well ELISA plates at a concentration of 0.5–1.0 µg per well, and incubated overnight at 4°C.

[0051] Blocking: Block with 5% skim milk and incubate at room temperature for 2 hours to block unbound proteins.

[0052] Antibody detection: IgG or IgM antibodies from cat serum were used for detection. Appropriate antibody dilutions and incubation conditions were determined through optimization (serum dilution 1:100, enzyme-labeled antibody dilution 1:5000).

[0053] Sample collection and processing: Serum samples were collected from cats infected with Toxoplasma gondii (3, 7, and 14 days post-infection) and from healthy control cats. The serum samples were centrifuged to remove cells and impurities before ELISA testing.

[0054] Detection procedure: The treated serum was incubated with the coated antigen for 30 minutes with shaking at room temperature. Then, the serum was washed three times with PBS to remove unbound material, and the enzyme-labeled antibody was added and incubated for 30 minutes. Finally, the reaction was carried out with the substrate solution, and the OD value was recorded at a wavelength of 450 nm.

[0055] The criteria for determining a positive result used serum samples from uninfected cats as negative controls. The cutoff value for a positive result was set based on the average OD value of the negative serum plus three times the standard deviation. If the OD value of a sample exceeded this cutoff value, it was considered positive.

[0056] Compared to using a single antigen, the combination antigens (SAG1, Mic8, and GRA6) significantly improved the detection sensitivity of positive reactions within 3 days post-infection, with a sensitivity increase of approximately 1.6 times. Simultaneously, cross-reactivity test results showed that this method had a cross-reactivity rate of less than 5% against other common feline diseases such as feline coronavirus and feline herpesvirus, demonstrating high specificity. Figure 6 ).

[0057] In the repeatability test, the coefficients of variation (CV) within and between plates were both less than 8%, indicating that the method has good experimental repeatability. Stability testing showed that the reagent remained stable after being stored at 4°C for 6 months, with no significant degradation observed, and it could still be used effectively for detection.

[0058] Two hundred and ten serum samples from cats were tested. The results showed a positive concordance rate of 94.8% and a negative concordance rate of 96.5% compared to commercial ELISA kits, with a Kappa value of 0.92, indicating high accuracy. Furthermore, the use of combined antigens resulted in a significantly more sensitive antibody detection response within three days post-infection, demonstrating superiority over traditional single-antigen methods.

[0059] By combining three antigens—SAG1, Mic8, and GRA6—into an ELISA assay, this embodiment provides a highly sensitive and specific detection method capable of accurately identifying infected individuals in the early stages of feline toxoplasmosis infection. Compared to single-antigen ELISA methods, this method offers greater sensitivity and accuracy, and can be widely applied to the early diagnosis, immune monitoring, and vaccine efficacy evaluation of feline toxoplasmosis infection.

[0060] In summary, different antigen combinations have good recognition ability for early Toxoplasma gondii infection, proving that the identification method for early Toxoplasma gondii infection in cats developed by the present invention using a multi-antigen combination strategy has extremely high sensitivity and specificity.

[0061] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. A method for early identification of feline toxoplasmosis infection, characterized in that, Immunological testing is performed using an antigen combination containing two or more Toxoplasma gondii antigens and a cat serum sample to be tested, and the test results are used to determine whether the cat is infected with Toxoplasma gondii. The antigen combination is selected from at least two proteins among Toxoplasma gondii tachyzoite-specific proteins, invading cell secretory proteins, and proteins specifically expressed during the development of cat intestinal epithelial cells. The invading cell secretory proteins are selected from at least one of micronematode secretory proteins, rod-shaped body secretory proteins, dense granule secretory proteins, surface antigen family proteins, and metabolism and structure-related proteins.

2. The method for early identification of feline toxoplasmosis infection according to claim 1, characterized in that, The microneedle secretory proteins include at least one of MIC1, MIC2, MIC3, MIC4, MIC6, MIC8, and MIC13.

3. The method for early identification of feline toxoplasmosis infection according to claim 1, characterized in that, The rod-shaped secretory proteins include at least one of RON2, RON4, RON5, RON8, ROP1, ROP2, ROP5, ROP16, ROP18, and ROP38.

4. The method for early identification of feline toxoplasmosis infection according to claim 1, characterized in that, The dense granule secretory protein includes at least one of GRA1, GRA2, GRA5, GRA6, GRA7, GRA15, GRA16 and GRA24.

5. The method for early identification of feline toxoplasmosis infection according to claim 1, characterized in that, The surface antigen family proteins include at least one of SAG1, SAG2A, SRS29B, SRS29C, and SRS34A.

6. The method for early identification of feline toxoplasmosis infection according to claim 1, characterized in that, The metabolism and structure-related proteins include at least one of ACT1, GAP45, MLC1, ENO2, LDH1, HSP70, and HSP90.

7. The method for early identification of feline toxoplasmosis infection according to claim 1, characterized in that, The proteins specifically expressed during the development of feline intestinal epithelial cells include at least one of GRA11B, OWP1, OWP2, OWP3, SOD3, and ERP.

8. The method for early identification of feline toxoplasmosis infection according to claim 1, characterized in that, The immunological detection method is ELISA, colloidal gold immunochromatography, or fluorescent microsphere immunoassay.

9. The method for early identification of feline toxoplasmosis infection according to claim 1, characterized in that, The antigen combination includes SAG1 and GRA6, SAG1 and MIC8, or SAG1, MIC8 and GRA6.

10. An antigen combination for early identification of feline toxoplasmosis infection, characterized in that, It contains two or more Toxoplasma gondii antigens, including Toxoplasma gondii tachyzoite-specific proteins, invading cell secretion proteins, and proteins specifically expressed during the development of feline intestinal epithelial cells.

Images