Contributions from the Autonomous University of Zacatecas in the Epidemiology, Diagnosis and Treatment of Trichinellosis, 1986-2021

Trichinellosis is worldwide distributed zoonosis, which affects humans and other living organisms including mammals, birds, and reptiles. The distribution is facilitated by the modifications of ecosystems, and the repercussions on nature, such as increase of greenhouse gases and the increase of temperature. Having economic, social, and health repercussions. The present work aims to present the research carried out at the Autonomous University of Zacatecas from the year 1986 to 2021, on Trichinellosis parasitosis caused by parasite Trichinella spiralis , working on the biology of the parasite, the life cycle, experimental models, diagnosis, and treatments with drugs and immunogens. Methodology:


Introduction
In Mexico, the first reports of Trichinellosis were by Dr. Olvera population is dedicated to agriculture, livestock, mining, services and industry [4]. The Autonomous University of Zacatecas (UAZ) is considered the civilizer of the north, it was founded in 1832, and its autonomy was in 1968, which currently has an approximate population of 40 thousand students and 6 thousand teachers and administrative personnel UAZ 2020. Trichinellosis was found to be more frequent in urban than rural areas, the age group from 15-44 years being more affected (49% of cases) and referring to sex 1: 1.8 male / female. Zacatecas currently has 58 municipalities and Zacatecas, Villanueva, Valparaíso, Panucho, Jerez, Jalpa and Guadalupe were affected at that time, and the transmission route was due to the consumption of poorly sewn chorizo, the diagnosis was by direct plate compression techniques, that received the name of trichinoscopy, and by the indirect technique of micro immuno-diffusion-double [5]. The objective of the present work is to make a report of the work carried out in this parasitosis during the period of 1986-2021 in the Autonomous University of Zacatecas.
b) Characterization of the life cycle of Trichinella spiralis [11,14,15]. c) Establishment of direct techniques (plate compression, artificial digestion, and hematoxylin / eosin) and indirect (double microimmunodiffusion, Dot-ELISA, IFI, Western Blot, intradermal reaction) for the diagnosis of Trichinella spiralis [16][17][18][19][20][21][22][23]. d) Evaluation of albendazole in a murine and pig experimental model, its evaluation with 3, 5, 7, 10, 14 days of treatment. In the initial phase of the infection, in the intestinal and muscular phase [11,23,25,26]. e) Evaluation of albendazole in pregnant rats, with the same treatment days [27]. f) Evaluation of the total antigen of Trichinella spiralis in murine and pig models, and of the 45 KDa band in murine models. In the intestinal, muscular phase and after 30, 60, 90 and 120 days of immunogen application. The purpose was to evaluate the modifications of the T. spiralis nurse cell in Long Evans rats immunized with total soluble antigen of T. spiralis and sacrificed at different times. We worked with 25 male rats of 2 and a half months of age, immunizing 20. Subsequently, the 25 rats were challenged with infected T. spiralis meat, sacrificing 5 rats every month, plus a control rat per 4 months, when sacrificing them, direct plate compression techniques, artificial digestion and the hematoxylin-eosin technique were performed; indirect MIDD and Western Blot techniques were performed on the sera [11,26,[28][29][30][31][32].
Another major finding is that the total extract of T. spiralis and the band of 45 KDa are excellent immunogens, and confirmatory technique of Trichinellosis by Wester-Blot, and the rest of the techniques are useful in epidemiological diagnoses and for research. The present study contributes to the understanding of Trichinellosis, as a silent disease present in the state of Zacatecas, Mexico, which unfortunately is not correctly diagnosed as in the other places of the Mexican Republic. Hence the importance of prevention. The mainly carriers are rats, pigs, and recently wild boar. Some of these animals are already consumed regularly in our region posing a risk for the spreading of the disease.

Ethical Approval
This study was reviewed and approved by the Bioethics Committee of the Biology Faculty of the Autonomous University of

Plate Compression Technique
For the plate compression technique, approximately 5 mg of tissue were used (diaphragm, masseter, tongue, intercostals, leg), each sample was placed between 2 lamellae and compressed, occupying an area of 1 x 5 mm, it was observed to the optical microscope, with the 10x and 40x lens [10,11,33,36,37].

Artificial Digestion Technique
30 g samples were used of homogenized tissue, and they were incubated at 37 °C, in a sack-shaped tulle sieve, suspended in a 0.3% solution of pepsin (10,000 U) and 37% HCl (0.2M) in 500 ml of distilled water, inside a separating funnel; 24 hours later, the larval package was separated with the ILs, which were deposited at the bottom of the funnel, observed in a newbawer camera under an optical microscope with a 10X lens, and the larval package was quantified [7,25,36,38].

Hematoxylin-Eosin Staining
Hematoxylin-Eosin (H-E) stain. Leg, tongue, and diaphragm samples were taken from each experimental model. The tissues were fixed in 10% buffered formalin for 24-48 hours. Subsequently, they were transferred to 70% ethyl alcohol for automatic processing for approximately 12 hours in two steps of 70% OH, 80% OH, 96% OH, 100% OH and xylol. From this last step, the samples were removed and embedded in paraffin, forming support blocks for making 5-8 µm thick histological sections on a Leica Model 820 microtome. The sections were placed in a float bath at 50 °C and they were lifted on a slide to dry, deparaffinize for an hour in an oven and then go on to the staining process with the Hematoxylin and Eosin technique according to the criteria of Viloria [28].

Double Microimmunodiffusion Technique (MIDD)
For double immunodiffusion, a 1% agar gel was made in distilled water with sodium azide, to avoid contamination; It was placed in an amount of 4.5 mL at 55 °C on a glass slide, once in solid form, the rosette was formed with a hole-hole, ensuring an equidistance of 0.5 cm between well and well; the comparison was carried out by always placing the antigen in an amount of 10 µL (10 µg) in the center and, around it, a serum of known reactivity, (in the same proportion by undiluted volume), leaving at room temperature environment in a humid chamber for 24 to 48 hours, until precipitation lines are observed between the positive serum and the antigen; then the gel was stained with Coomassie brilliant blue G 250, 25% by volume [10].

Dot-ELISA
Several nitrocellulose papers were squared, depending on the number of samples to be used, each 1 cm 2 square a 10x5 cm paper. The antigen was placed on the undiluted paper placing the equivalent of 10 µg / µL, then it was allowed to dry at room temperature and once dry, it was proceeded to block with 3% fat-free milk in PBS this for 18 hours. Once the blocking time had concluded, it was washed once with PBS 0.5% Tween 20 for 10 minutes and 2 consecutive times with PBS for 10 minutes each time, then, 10 µL of each test serum was placed in each square and incubated again with 3% fat-free milk in PBS for an hour and a half, at the end of this time we proceeded to wash again with PBS 0.5% Tween 20 for 10 minutes and 2 consecutive times with PBS for 10 minutes, then the second antibody, Anti-IgG, Anti-IgM or Anti-IgA at a concentration of 1: 2000 in PBS conjugated with peroxidase, 10 µL of the second antibody and allowed to dry at room temperature, once dry it was incubated once more with Milk 3% fat free in PBS for an hour and a half and the container containing the paper was completely covered with aluminum foil. At the end of the hour and a half, the paper was washed with PBS Tween 20 at 0.5% for 10 minutes and 2 consecutive times with PBS for 10 minutes. The paper was then developed with 3,3-diaminobenzidine DAB, using 37% hydrogen peroxide as substrate [11,19,27].

Indirect Immunofluoresence (IFI)
The infective larvae (IL) were obtained from the rat muscle with resin, they were observed under a confocal microscope [38].

Western Blot (WB)
The product obtained from the polyacrylamide gel run was transferred to NC paper [17], using the Transblot-Cell camera (Bio-

Intradermal Reaction (IDR)
Intradermal reaction was applied with Total Soluble Antigen of T. spiralis which were 10 units (10 µg of protein), the area of application of the AST was observed at 2, 24 and 48 hours [17,11].

Obtaining Immunogens
The antigen of T. spiralis was obtained, by means of extraction with liquid nitrogen, the infecting larvae were obtained by the artificial digestion technique, liquid nitrogen was added in sufficient quantity to cover the IL and by bursting the exit of antigenic components were centrifuged at 3500 rpm for 1.5 hrs.
The supernatant was the soluble antigen of T. spiralis, whose protein concentration was determined and used in the different immunological tests, and as an immunogen (secretion / excretion antigens) in protection studies [6,7,18,19,11,36].To determine that the antigen had the adequate protein concentration, a standard curve was obtained using bovine serum albumin, according to the methodology of Bradford, 1976, adjusting the concentration of proteins obtained to an optical density of 610 nm using Coomassie blue at 0 .06 % prepared in 2.2% HCl. The value of the optical density of the antigen was interpolated, to that of the standard curve of albumin, the concentration of proteins contained in the two types of antigenic extract was obtained [18].To obtain the immunogenic protein of 45kDa, the AST was subjected to polyacrylamide gel electrophoresis to separate by molecular weight (MW) the proteins that were identified by a PM marker of the 45kDa protein and by elution of bands the required protein was obtained.

Results
The experimental models of infection were implemented in a murine model, Balb / C mouse, Long Evans rat, domestic dog, eyelid edema, joint injury, increased temperature. At sacrifice, IL was detected in muscle and brain tissue (Figures 2 and 3).

Direct Diagnose Techniques
In all the experimental models by plaque compression (Figure   7), artificial digestion ( Figure 8) and the Hematoxylin-Eosin technique, IL from Trichinella spiralis (Figure 9) were observed.

Indirect
Techniques: Indirect techniques, double microimmunodiffusion (MIDD, Figure  9.), Dot-ELISA, Immunofluorescence (IFI, Figure 10), Wester Blot (WB, Figure 11) and Intradermoreaction were positive, in WB a triplet of 42,45 and infection with T spiralis, which is a very timely diagnosis for the pig without the need for a specialized infrastructure, as well as being inexpensive and can easily be carried out to the field with simple animal handling and not very aggressive, with a presumptive diagnosis after 2 hours of application of the intradermal reaction and confirmatory after 24 and 48 hours, which can prevent infection in man and even other animals in a timely manner.

Evaluation of the Total Soluble Antigen of Trichinella spiralis in Murine and Pig Models
A statistically significant protection effect was observed in muscle phase, by analysis of variance p <0.0001. (Figure 14

Evaluation of the Soluble Antigen of T. Spiralis, VITS, Bacterial Vaccine and Lactobacillus Casei
The

Epidemiological Studies
From Resulting a non-significant variation with a value of P = 0.5382.

Discussion
The present study is an analysis of the work carried out in the

Conclusions
The Autonomous University of Zacatecas has a research line in Trichinella spiralis, which has allowed to carry substantive functions, including teaching, research, extension, dissemination, and having an impact on the training of human resources. The findings reported in this research include: a reproducible experimental model for the study of Trichinellosis, reliable diagnostic techniques and confirmatory WB, an effective treatment using albendazole (which must be used under medical prescription), and the study of products such as the bacterial vaccine, the VITS immodulator, the Lactobacillus casei strain, which decrease the parasite load as well as the immunogen of the total soluble antigen of Trichinella spiralis.
The contribution of this study has been to provide deeper insights in a disease which is present in the state of Zacatecas, Mexico and is commonly not properly diagnosed or confused with other diseases, we consider very important to continue promoting having a proper diagnosis and prevention of the disease.