BR102021018518A2 - KIT FOR THE DETECTION OF IMMUNE RESPONSES AGAINST SALMONELLA ENTEICA BACTERIA (SALMONELLA) - Google Patents

Abstract

A kit for the detection of immune responses against the bacteria Salmonella enterica (salmonella) with lipopolysaccharide (LPS) – or subunits thereof – from salmonella immobilized on a solid substrate; and flagellin – or subunits thereof – from salmonella immobilized on a solid substrate. A kit for detecting immune responses against Salmonella enterica (salmonella) bacteria produced by several sequential steps. A kit for the detection of immune responses against the bacteria Salmonella enterica (salmonella) configured to differentiate between animals infected or vaccinated against salmonella for a period between 1 day and 21 days or for periods longer than this.

Description

KIT FOR THE DETECTION OF IMMUNE RESPONSES AGAINST SALMONELLA ENTEICA BACTERIA (SALMONELLA) LEGAL NOTICE

[001] The information below will soon be passed on to the public in the form of posters at a scientific event.

FIELD OF THE INVENTION

[002] The present invention relates to the process of developing a kit for detecting intestinal immune responses or other sites with mucosal tissue ("mucosal" immune responses) against Salmonella enterica enterica subspecies of the non-typhoid subgroup. The proposed kit is based on the Enzyme-Linked Immunosorbent Assay (ELISA) technique. The nontyphoidal salmonella subgroup includes serotypes such as S. enterica Heidelberg, S. enterica Enteritidis, S. enterica Tiphymurium, S. enterica Minesotta, among about two thousand other serotypes. Salmonella in this subgroup are flagellar and contain a wall containing lipopolysaccharides (LPS). Variations in flagella and LPS are used to define serotypes of Salmonella enterica subspecies enterica of the non-typhoid subgroup (also called paratyphus). Thus, in this invention, LPS and flagella of the serotypes of interest are immobilized on immunoadsorbent plates using specific solutions and conditions; plates are blocked with specific solution and then screened with specific dilutions of samples kept in specific preservation solution. Samples are diluted in specific blocking solution for use in the reaction. IgA antibodies associated with flagella and LPS are detected with enzyme-linked secondary antibody. The differential of the present invention in relation to the state of the art is in the combination of LPS and flagellin antigens to screen each sample; in the specific solutions and conditions used for sample transport, for immobilization of antigens on plates, for plate blocking and for sample dilution. In addition, the differential of the present invention lies in the analysis of samples from mucous membranes, in particular fecal content and cloacal mucosa swabs, to determine the animal immune response against paratyphic salmonella.

[003] The present invention also relates to the use of said ELISA kit for assessing the health status of poultry and swine flocks in relation to bacteria Salmonella enterica enterica subspecies of the non-typhoid subgroup.

FUNDAMENTALS OF THE INVENTION

[004] With the massive production of poultry and commercial pigs that moves the world market, it is presumable the increase in the occurrence of health disorders, with a significant number of animals carrying infectious agents. Such agents may be responsible for diseases caused by bacteria and viruses in different types of animals and in humans, consumers of products of animal origin [Vidic J. et al.; Advanced biosensors for detection of pathogens related to livestock and poultry; 2017; Wyk V.B. et al.; Induction of functional interferon alpha and gamma responses during acute infection of cattle with non-cytopathic bovine viral diarrhea virus; 2016; Okamoto, A.S et al.; Immunological evaluation of the intestinal mucosa of broiler chicks treated with Lactobacillus Spp. and challenged with Salmonella Enteritidis; 2007] .

[005] The assessment of the health conditions of animals in the field, in herds and farms, also guarantees the well-being of the human population, whether man is a worker in the aforementioned environment and/or a consumer of animals. From an economic point of view, the producer can still be very affected by the loss of his meat and egg production when sick animals are found. Therefore, there is a great need for monitoring animal health, which is also a requirement of the National Poultry Health Program, whose objective is to act in the prevention and/or treatment of diseases in production. This program requires strict control over the prevalence of non-typhoid salmonella [Programa Nacional de Sanidade Avícola, Ministry of Agriculture; 2017] .

[006] Among the reasons for the need for strict control of paratypic salmonella in poultry and pig production are demands from consumer markets. The presence of paratyphic salmonella reduces the value of animal products, as they are rejected by more demanding markets that pay more, such as Europe. Brazil's failure to meet these sanitary requirements regarding salmonella has resulted in trade sanctions and the return of containers loaded with meat products to Brazil [https://reporterbrasil.org.br/2019/07/vetado-na-europa- chicken-contaminatedby-salmonella-and-resold-in-brazil/; accessed on June 10, 2021] .

[007] Innovative strategies have been eagerly sought to control paratyphal salmonella in the production chain of animal products, given the importance of these bacteria for public health and the international trade of these products. Methods for differentiating infected animals are essential for controlling the bacteria in herds, and the ELISA method is recognized as important in this context. However, available methods only check for the presence of serum antibodies, although many paratyphal salmonella infections are restricted to the mucosa. Even when it comes to isolates with invasive capabilities, paratyphus salmonella may not induce the production of antibodies in serum, generating false-negative results in serology [OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Mammals, Birds and Bees). Volumes 1 & 2, Chapter 3.9.8. World Organization for Animal Health; 2009] [ Chappell, L., Kaiser, P., Barrow, P., Jones, M. A., Johnston, C., & Wigley, P. (2009). The immunobiology of avian systemic salmonellosis. Veterinary immunology and immunopathology, 128(1-3), 53-59].

[008] Intestinal immune responses are very important in the defense against paratyphic salmonella. The infection invariably occurs through the digestive system, which highlights the immune importance of antibody production at these sites [Moor, K., Diard, M., Sellin, M. E., Felmy, B., Wotzka, S. Y., Toska, A., . .. & Slack, E. (2017). High-avidity IgA protects the intestine by enchaining growing bacteria. Nature, 544(7651), 498-502]. The most relevant antibodies for intestinal protection against S. enterica enterica are those called immunoglobulin A (IgA) [Richards, A.F., Doering, J.E., Lozito, S.A., Varrone, J.J., Willsey, G.G., Pauly, M., .. & Mantis, N.J. (2020). Inhibition of invasive salmonella by orally administered IgA and IgG monoclonal antibodies. PLoS neglected tropical diseases, 14(3), e0007803]. Although the immune response in the mucous membranes is of special relevance, the immune tests for the evaluation of salmonella infection are exclusively based on the detection of serum antibodies, whose immune response against paratyphic salmonella in animals can be flawed, as previously described in this text [Kuhn, K.G., Falkenhorst, G., Ceper, T.H., Dalby, T., Ethelberg, S., Mølbak, K., & Krogfelt, K.A. (2012). Detecting non-typhoid Salmonella in humans by ELISAs: a literature review. Journal of medical microbiology, 61(1), 1-7].

[009] There are, in the scientific literature, parallel methods for the detection of IgA in samples of commercial birds. However, these techniques were performed in house, that is, these are not commercially available kits. Crucially, these in-house techniques have not gone through validation processes regarding reagent stability, sensitivity and specificity, relevance in detecting batches of positive animals in field (non-experimental) situations, comparison with other methods, evaluation of immune responses in different age groups or in animals subjected to different conditions, such as during the application of antibiotics. In particular, these in-house methods are also based on the use of a single salmonella antigen, although the immune responses against LPS and flagellin are extremely different, something that is not contemplated in the analysis of the results [Revolledo, L. (2005). Study of the immune response, colonization and invasion by Salmonella enterica subsp enterica serotype Typhimurium Nalr in broilers treated with glucan, probiotics and competitive exclusion products. Doctoral Thesis, Faculty of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo. doi:10.11606/T.10.2006.tde-07022007-152110. Retrieved on 2021-06-10, from www.teses.usp.br] [ Okamoto, A. S., Andreatti Filho, R. L., Lima, E. T., Pereira, R. E. P., Menconi, A. I. I. I., Rocha, T. S., & MariettoGonçalves, G. A. (2007) . Immunological evaluation of the intestinal mucosa of broiler chicks treated with Lactobacillus spp. and challenged with Salmonella Enteritidis. Brazilian Journal of Poultry Science, 9(4), 259-262] [ Chappell, L., Kaiser, P., Barrow, P., Jones, M. A., Johnston, C., & Wigley, P. (2009). The immunobiology of avian systemic salmonellosis. Veterinary immunology and immunopathology, 128(1-3), 53-59]. In short, it is not possible to assess, based on the data available in the literature or commercial datasheets, the feasibility of using mucosal IgA to determine the health status of lots of animals with regard to paratyphic salmonella.

SUMMARY OF THE INVENTION

[010] The inventors developed and tested an ELISA kit through which it is possible to verify the health status of animal herds regarding paratyphal salmonella, through the detection of IgA in samples from mucous membranes, such as tears, cloacal swab or fecal content. The invention includes the use of said kit in commercial flocks. The applicability of the kit depends on knowing the dilution ranges of the samples, the age of the animals to have samples collected, other factors associated with the immune capacity of the animals, determining the status of the lot against paratyphic salmonella from the interpretation of the percentage of positive samples, the sample number to be obtained from each animal group, the interpretation of the results regarding the data obtained for the different antigens used in the kit (LPS and flagellin).

[011] The steps related to the production and use of the present invention are described in detail below.

[012] COMPONENTS OF THE PRESERVATIVE SOLUTION FOR COLLECTING MUCOSAL SWAB AND PREPARATION METHOD

[013] When it is necessary to collect mucosal swabs for IgA detection, the collected material is kept in a specific solution for preservation purposes, consisting of 0.1% casein, a mixture of: 5-chloro-2-methyl-2h 0.1% -isothiazol-3-one (3:1) and 2-methyl-2h-isothiazol-3-one (3:1), 0.5 mM phenylmethylsulfonyl fluoride, Ethyl(2-mercaptobenzoate-(2 -)-O,S) 0.1% sodium (1-) mercurate, 40% glucose and 0.01% sodium azide.

[014] Initially, the amount of glucose to be used for the desired volume of preservative solution is weighed. Then, deionized water corresponding to 40% of the final volume of the preservative solution is added in a glass beaker. With the aid of a magnetic stirrer, the glucose is added little by little. The solution is kept stirring until a homogeneous mixture is obtained. Then, the other components of the solution are added and the volume is completed with deionized water until reaching the final volume. Finally, the solution is fractionated into 5 mL aliquots in 15 mL polypropylene tubes and identified with the manufacturer's brand, produced batch number and expiration date.

[015] PREPARATION OF INACTIVATED CULTURE OF ESCHERICHIA COLI FOR ELISA

[016] The performance of this ELISA technique depends on an inactivated Escherichia coli solution that acts as an inhibitor of specific antibodies to bacterial antigens, especially in samples of feces and cloacal swab. Binding with Escherichia coli antigens reduces the occurrence of non-specific binding during assay performance. The preparation of the inactivated Escherichia coli solution begins with the cultivation of the bacteria in 50 mL of peptone water in an Erlenmeyer or Schott-type flask, which must remain at 175 rpm in a flask shaker, at a temperature of 37oC for 18 hours. After carrying out the cultivation, the quantification of the bacteria present in the medium used is performed. The culture should have an OD600 of 0.76 and about 150 CFU/100 µL (1.5x1012 CFU/mL) at the 10-9 dilution. After cultivation and quantification, the solution containing Escherichia coli must be inactivated in an oven at 65oC for 3 hours, being shaken every 15 minutes during this process. It is possible to attest the inactivation by plating an aliquot of the solution.

[017] PREPARATION OF ANTIGENS USED IN SENSITIVE PLATES

[018] For the production of flagellin and lipopolysaccharides (LPS) used in the sensitization of the plates used in the ELISA tests, a previous cultivation of Salmonella Enteritidis and Salmonella Typhimurium is carried out, each in 1L of LB medium at 37oC and 220 rpm for 16 hours . After preparing the bacterial cultures, an aliquot of 500 mL is collected for each antigen, which is centrifuged.

[019] For the production of flagellin, the culture is centrifuged at 11320xg at room temperature for 30 minutes. Then, the bacterial pellet is resuspended with 8 mL of 25 mM TrisHCl buffer at pH 7.0 with 150 mM NaCl. The resuspension is transferred to a 30 mL centrifuge flask and incubated on a shaker at 150 rpm and 37oC for 30 minutes. After incubation, 3 beads are added to up to 15mL of solution for homogenization in a vortex-type shaker at maximum speed for 1 minute. Then, the solution is centrifuged at 5000xg for 30 minutes at room temperature. At the end of centrifugation, flagellin will be present in the supernatant, which must be transferred to a 50 mL polypropylene bottle. The quantification of flagellin is carried out in a 25 mM TrisHCl buffer at pH 7.0 with 150 mM NaCl, in a high-resolution spectrophotometer.

[020] For the production of LPS, the culture must be centrifuged at 5000xg at room temperature for 30 minutes. After centrifugation, the bacterial pellet is resuspended in 40 mL of ultrapure water. The resuspension should be centrifuged at 9000xg for 10 minutes and resuspended again in 20 mL of ultrapure water, which will be mixed in 180 mL of ice-cold acetone (-18oC). The resuspension in acetone is maintained at -20oC for 96 hours and then centrifuged at 8000xg for 12 minutes and the pellet is resuspended in 50 mL of acetone. The procedures of centrifugation at 8000xg for 12 minutes and resuspension in 50 mL of acetone are performed a second time, and then the supernatant is discarded so that the bacterial pellet can dry in an oven at 37oC for 16 hours, and then macerated in clean and sterile pestle and mortar. The macerated pellet is weighed and an equivalent volume of 50 mM TrisHCl, pH 7.4, is added to obtain a 6% solution. Next, the pellet solution macerated in Tris-HCl must be heated to 65oC and, for the same volume of this solution, buffered phenol is added at the same temperature. The mixture of pellet solution and buffered phenol is then incubated at 65oC for 5 minutes, homogenizing in a vortex-type shaker every minute. At the end of the incubation, the mixture is centrifuged at 8000xg at 4oC for 20 minutes and then the supernatant is dialyzed on a 12kDa porosity membrane for 4 days, 2 days in running water and 2 days in distilled water. The dialysis product must be frozen and lyophilized.

[021] It is possible to perform confirmatory tests to verify the purity of the purification products. An aliquot of the contents of the purification products can be applied to a gel for electrophoresis in an SDS-PAGE or western blot assay.

[022] SENSITIZATION OF PLATES FOR ELISA

[023] 96-well polystyrene plates are sensitized with two different antigens (flagellin and LPS) from strains of Salmonella Enteritidis and Salmonella Typhimurium. Two plates are used to carry out the ELISA assay, one of the plates for flagellins, and the other for LPS. To prepare the sensitization solution, a 100 mM carbonate buffer solution is used to dilute the stock antigens. To sensitize the flagellin plate, add 5 ng/mL of purified flagellin solution from Salmonella Enteritidis and 5 ng/mL of purified flagellin solution from Salmonella Typhimurium, totaling 10 ng/mL of solution for sensitization. The volume added to each well of the 96-well plate is 50µL. To prepare the LPS plate sensitization solution, 1.5 ng/mL of purified Salmonella Enteritidis LPS solution and 1.5 ng/mL of purified Salmonella Typhimurium LPS solution are added, totaling 3 ng/mL awareness solution. The volume added to each well of the 96-well plate is 50 µl. The plate sensitization stage lasts 96 hours. After the end of the sensitization step, the contents of the antigen solution used in the sensitization are removed and the wells are washed with Tween 20 (0.1%) solution in phosphate buffer saline (PBS) 1x for three times, where each wash lasts 5 minutes. After washing, the plate is blocked with 1x PBS and 1% casein solution (200 µL per well) and incubated for 1 hour at room temperature. At the end of the blocking step, the volume of 1% casein present in the plates is discarded and the plate is stored in a cold environment (4oC).

[024] PERFORMING THE TEST

[025] To perform the ELISA, every sample must be previously diluted. Fecal samples are diluted 1:5 in 1x PBS and 0.1% casein solution and then diluted 1:10 in 1x PBS and 0.1% casein solution and 10% inactivated Escherichia coli to inhibit the binding of less specific antibodies that may interfere with the interpretation of results. After performing the initial dilution, stool samples can be serially diluted in the dilution plate in 1x PBS and 0.1% casein solution. Swab samples are diluted 1:30 in 1x PBS and 0.1% casein solution with 10% inactivated Escherichia coli. At the end of the initial dilution, the swab samples can be serially diluted in the dilution plate in a solution of 1x PBS and 0.1% casein with 10% inactivated Escherichia coli. It is also necessary to prepare the positive and negative controls. Positive controls are diluted 1:25, while negative controls are diluted 1:150, both in 1x PBS and 0.1% casein solution. There is no need to serially dilute the controls. At the end of the preparation of samples and positive and negative controls, 50 µL of the volume of each well of the dilution plate is transferred to the plate sensitized with flagellin and 50 µL of the volume of each well of the dilution plate to the plate sensitized with LPS. It is necessary to adjust the volumes of sample and reagents in the dilution plate so that there is sufficient volume at the time of transferring the contents to the flagellin-sensitized plate and the LPS-sensitized plate. The sensitized plates containing the samples are incubated for 2 hours at room temperature, so that the interaction between the antigens of the sensitized plates and the IgA antibodies present in the samples is possible. At the end of the incubation step, the sample content of the sensitized plates is removed and the wells are washed 5 times with a Tween 20 solution (0.1%) in 1x PBS. At the end of the washes, the content of the Tween 20 solution (0.1%) in 1x PBS remaining in the wells is removed and 50 µL of specific antibody solution to the IgA binding of the species from which the sample was collected are added. sample conjugated to horseradish peroxidase (HPR) in 1:30000 dilution in each well, diluted in 1x PBS and 0.1% casein solution. Plates with conjugated antibody solution are incubated for 1 hour at room temperature and then washed with Tween 20 solution (0.1%) in PBS 1x for 5 times. At the end of the washes, the solution of Tween 20 (0.1%) in 1x PBS remaining in the wells is removed and 50 µL of 3,3',5,5'-tetramethylbenzidine (TMB) solution is added to each well , at room temperature. The incubation of the plates with TMB must be carried out in a dark environment at room temperature to prevent the occurrence of nonspecific reactions. At the end of the incubation with TMB, 50 µL of sulfuric acid solution (H2SO4) at 1 M is added to each well containing TMB so that the reaction between HRP and TMB is stopped. After adding the H2SO4 solution, the absorbance of each well of the plate is read using a microplate reader unit, at a wavelength of 450 nm.

[026] REFERENCES
1. Baptista AAS, Donato TC, Souza EE, Gonçalves GAM, Garcia KCOD, Rodrigues JCZ, et al. Pesqui Vet Bras. 2013, 33:1215–1221. https://doi.org/10.1590/S0100-736X2013001000007.
2. Davis MR, Goldberg JB. J Vis Exp. 2012. https://doi.org/10.3791/3916.
3. Hassan JO, Barrow PA, Mockett AP, Mcleod S. Vet Rec. 1990, 126: 519-522. PMID: 2195754.
4. Okamoto AS, Andreatti Filho RL, Lima ET, Pereira REP, Menconi A, Rocha TS. Rev Bras Cienc Avic. 2007, 9:259–262. https://doi.org/10.1590/S1516- 635X2007000400009.
5. Oliveira BH, Silva MR, Braga CJM, Massis LM, Ferreira LCS, Sbrogio-Almeida ME, et al. 2011, 28:575–584. https://doi.org/10.1590/S0104- 66322011000400003.

ADVANTAGES OF THE INVENTION

[027] The advantages of the invention over the state of the art include: standardized methods and reagents that allow transnational shipment of samples at room temperature in a stable and safe way; reagents that allow the preservation of the kit at refrigeration temperature (4 to 8oC) for at least 1 year; detection of IgA from samples obtained from mucous membranes, such as fecal content and tears, which allows the real assessment of the immune status of animals exposed to paratyphic salmonella; possibility of certifying the health status of the herd as “positive” or “negative” for paratyphic salmonella based on standardized statistical analysis and participating in the invention based on the results obtained with the kit.

BRIEF DESCRIPTION OF THE FIGURES

[028] Figure 1 represents IgA titers to LPS flagellin before and after vaccine administration (via spray; graphs 1 and 2, and drinking water; graphs 3 and 4, respectively). There is a statistical difference between the times evaluated for the geometric mean of the titles (Mann-Whitney test, P < 0.05)

[029] Figure 2 represents the effect of antimicrobial use on the vaccine response against Salmonella (test with Ceftiofur; graphs 1 and 2, test with Gentamicin; graphs 3 and 4).

ACHIEVEMENTS OF THE INVENTION

[030] We report below two experiments in which the present invention was applied, with particular emphasis on the possibility of animal health assessment regarding paratyphus salmonella under “field” conditions, that is, non-experimental. Under these real conditions, the variations obtained with ELISA analyzes generally make the use of the technique unfeasible, which is only applicable after extensive standardization. In addition, in the state of the art, such a methodology has never been used to assess the health status of batches of animals under production conditions regarding paratyphic salmonella, nor has the possibility of using samples from mucous membranes (such as fecal content and tears) to the monitoring of batches, showing the inventive activity necessary for the proposal to be achievable. In fact, it was not possible to intuit from the literature about the stability of fecal content samples in relation to the IgA present there, since they are obviously contaminated samples and subject to rapid degradation, which prevented the previous development of similar kits

[031] COMPARISON OF INTESTINAL IgA IN BIRDS VACCINATED AGAINST SALMONELLA VIA SPRAY AND DRINK WATER

[032] Salmonella enterica infection first occurs in the intestine and then spreads to various compartments and organs systemically. It is important to remember that most of the local immunity on the surface of the intestinal mucosa is provided by IgA. This antibody is fundamental to prevent or reduce Salmonella penetration, representing a very efficient protection mechanism and contributing to the maintenance of healthy microbiota homeostasis by immune exclusion of enteropathogens (1).

[033] The vaccination process is a critical point in the protection of birds against Salmonella enterica. As a matter of practicality, most vaccinations with live Salmonella vaccines are carried out via drinking water. On the other hand, the spray route is also indicated and can be a more practical and safer alternative for the producer.

[034] The purpose of this work was to measure and compare the amount of intestinal IgA in birds vaccinated against Salmonella via spray and drinking water.

[035] To carry out this comparative study, chicks of the same origin were housed in 02 aviaries on the same rural property with identical structure and the same nutrition and management guidance. All chicks received live Salmonella Typhimurium vaccine (Poulvac ST) by coarse spray in the hatchery on the first day of life. When the birds reached 14 days of age, a booster dose was performed, with one of the aviaries being vaccinated via spray and the other via drinking water.

[036] Before vaccination, 08 pools of fresh feces were collected from each aviary to analyze the presence of intestinal IgA. After 4 days of vaccination, the collection was also carried out in the two aviaries with the same sampling. Feces were serially diluted and each dilution was tested by specific ELISA (GIgA Kit – object intended for this patent application) containing LPS or Flagellin from Salmonella Typhimurium according to the manufacturer's guidelines. The highest dilution with a positive signal was used as a reference for the IgA titer in the feces.

[037] In the first collection, performed before the application of the second dose of live vaccine, the presence of anti-LPS and anti-flagellin IgA of S. Typhimurium was already detected due to the first vaccination of the hatchery. In the second collection, a significant increase in anti-LPS and anti-flagellin IgA of S. Typhimurium was observed in the spray-vaccinated poultry. This increase is expected and constitutes the main objective of the second massive dose carried out in the field. Mucosal antibodies have a tendency to decline rapidly and the booster dose significantly improves local mucosal protection. The results are compiled in FIGURE 1.

[038] The results of the second collection in the avian vaccinated via drinking water showed maintenance of the anti-LPS titer and a reduction in the anti-flagellin titer. Comparison between titre results after the second dose indicates that spray vaccination resulted in a response as good or even better than that of birds vaccinated with drinking water. This result is important from a practical point of view, as in certain farms the producer may prefer to perform spray vaccination given the ease and safety of this method. An advantage of the spray application is the speed and the use of a smaller volume of water compared to the drinking water method. In addition, spray vaccination depends exclusively on the applicator's skill and the quality of the equipment, whereas vaccination via drinking water depends on the farm's hydraulic system (biofilm, presence of chlorine, maintenance, dosage, water fasting, equipment quality) .

[039] In the group treated via spray, it was possible to verify an increase in the IgA titer for both anti-LPS and anti-flagellin during the test period. For the water treatment, the titers for LPS were not changed while the titer for flagellin was statistically reduced in the same period.

[040] REFERENCES
1. Corthésy B. Autoimmun Rev 2012, 12:661-665. 2012; https://doi.org:10.1016/j.autrev.2012.10.012.
2. Corthésy B. Future Microbiol 2010, 5:817-829. https://doi.org/10.2217/fmb.10.39.
3. Forbes SJ, Eschmann M, Mantis NJ. Infect Immun 1992, 80:2454-2463. https://doi.org/10.1128/IAL00018-12.
4. Gutzei C, Magri G, Cerutti A. Immunol See 2014. 260:76-85. https://doi.org/10.1111/imr.12189.
5. Mantis NJ, Rol N, Corthesy B. Mucosal Immunol 2011, 4:603-611. https://doi.org/10.1038/mi.2011.41.
6. Michetti P, Porta N, Mahan MJ, Slauch JM, Mekalanos JJ, Blum AL, Kraehenbuhl JP, Neutra MR. Gastroenterology 1994, 107:915-923. https://doi.org/10.1016/0016-5085(94)90214-3.

[041] MEASUREMENT OF INTESTINAL IgA IN BIRDS VACCINATED AGAINST SALMONELLA WITH AND WITHOUT INJECTIONAL MEDICATION IN THE HATCHERY

[042] The tropism of Salmonella enterica for the gastrointestinal tract suggests that the application of mucosal vaccines should be favored in order to reach specialized sites of the immune system such as Peyer's patches, resulting in a robust formation of local immunity by activating lymphocytes T and by antibody response (mainly IgA) in gut-associated lymphoid tissue. The prevention or reduction of penetration of the epithelial barrier by enteropathogens, such as Salmonella, by specific IgA has been identified as a crucial mechanism for protection and maintenance of microbiota homeostasis. On the other hand, the injectable medication performed in the hatchery has been used to reduce the initial mortality of broiler flocks.

[043] The purpose of this work was to measure and compare intestinal igA in birds vaccinated against Salmonella with and without injectable medication in the hatchery.

[044] To carry out this comparative study, chicks of the same origin were housed in 04 aviaries with identical structure and the same nutrition and management guidance. All chicks received live Salmonella Typhimurium vaccine (Poulvac ST) by coarse spray in the hatchery on the first day of life. The antimicrobials Ceftiofur and Gentamicin in ovo were used on the 18th day of embryonic development together with Marek's vaccine. For each medicated house, there was one house without medication (negative control group).

[045] When the birds were 4 days old, 07 pools of fresh feces were collected from each aviary to analyze the presence of intestinal IgA. IgA was measured by specific ELISA (GIgA Kit) containing LPS or ST Flagellin according to the manufacturer's instructions. The highest dilution with a positive signal was used as a reference for the IgA titer in the feces.

[046] The use of Ceftiofur raised the average of antibodies in the birds of the medicated aviary. The geometric mean of S. Typhimurium anti-LPS IgA passed the cut-off threshold (> 8.22) with the use of the antibiotic. There was also an increase (not statistically significant) in the amount of S. Typhimurium anti-flagellin IgA.

[047] The use of gentamicin reduced the amount of anti-LPS IgA, but both treated and untreated groups were below the cut-off. However, the use of gentamicin increased the amount of salmonella anti-flagellin IgA (FIGURE 2).

[048] The results indicate that there could be a synergy between the use of injectable antimicrobial with the live vaccine against Salmonella Typhimurium, especially in the case of Ceftiofur. This could be due to control over the amount of intestinal microbiota at the time of vaccine application, since the antimicrobial was administered in ovo. The active principle of Ceftiofur is rapidly metabolized and would act mainly before chick hatching, controlling bacterial contamination. This possible synergism can be used to make the vaccine more effective in inducing an immune response through intestinal IgA. Any intervention that increases the production of specific IgA against Salmonella is highly favorable given the importance of this immunoglobulin in the local intestinal response.

[049] Anti-LPS and anti-flagellin results are interpreted together. In short, the use of antibiotics increased the anti-salmonella IgA response in the analyzed samples. This effect was best observed in the case of Ceftiofur. Other studies should be carried out to broaden the understanding of this synergistic effect

DEFINITIONS

[050] The term "Antibody" encompasses the terms "IgA", "serum antibodies", "immunoglobulin" and "immunoglobulin A" contained in this document. The term refers to a type of glycoprotein belonging to the gamma globulin class. Antibodies act in defense in the animal organism against different pathogens (Alberts et al., 2002).

[051] The term "Antigen" encompasses the terms "bacterial antigens" and "Escherichia coli antigens" and "salmonella antigen". "Antigen" is defined as molecules captured, processed and presented to cells of the immune system, triggering a response (Alberts et al., 2002).

[052] The term “biofilm” refers to sessile communities of bacteria in an extracellular matrix adhered to a surface (Costerton, 1999).

[053] The term "control" applies to the use of samples with the presence (positive control) or absence (negative control) of IgA antibodies in the context of the diagnostic kit detailed in this document, in order to obtain parameters that may indicate positivity or negativity for the evaluated samples (Johnson and Besselsen, 2002).

[054] The term “enzyme” refers to proteins with catalytic activity for specific chemical reactions (Berg, Tymoczko and Stryer, 2002).

[055] The term “ELISA” refers to enzyme-linked immunosorbent assay (ELISA). This test evaluates the binding of an antigen to a specific antibody, whose binding triggers a chemical reaction that generates a color change in a liquid medium, which is measured by means of the absorbance of this medium at a specific electromagnetic wave length (Crowther, 2002).

[056] The term “enteropathogens” refers to potentially pathogenic microorganisms whose main access to the host animal organism is through the gastrointestinal tract (Pickering and Shane, 2012).

[057] The term “Escherichia coli” refers to a species of bacteria belonging to the Enterobacteriaceae family, commonly found in the environment and in the gastrointestinal tract of animals (Kaper et al., 2004).

[058] The term “flagellin” refers to a constituent protein of the flagellum of bacteria, used for cell motility (Gómez-Gómez and Boller, 2002).

[059] The term “homeostasis” refers to the characteristic and constant condition of the physical-chemical environment of a living system for optimal functioning (Davies, 2016).

[060] The term “hatchery” refers to the place of storage and maintenance of fertile eggs in optimal conditions for later hatching (Ribeiro et al., 2010).

[061] The term “cutoff threshold” refers to the threshold parameter that defines the presence or absence of the analyte in the analyzed sample (Greiner et al., 1995).

[062] The term “lymphocytes” encompasses the terms “T lymphocytes” and “B lymphocytes”. This term refers to a group of cells that make up the immune system of animals (Alberts et al., 2002).

[063] The term “lipopolysaccharide” encompasses the acronym “LPS”, and refers to an important constituent of the outer membrane of Gram-negative bacteria (Schumann et al., 1990).

[064] The term “microbiota” refers to a community of commensal, symbiotic or pathogenic microorganisms that inhabit multicellular organisms (Sekirov et al., 2010).

[065] The term “Peyer's patches” refers to a type of lymphoid tissue aggregate associated with the intestinal mucosa (Sato and Iwasaki, 2005).

[066] The term “SDS-PAGE” stands for “sodium dodecyl sulphate–polyacrylamide gel electrophoresis”. The term refers to a type of assay done with the objective of separating and marking proteins based only on their molecular weight (Blancher and Jones, 2001).

[067] The term “Western blot” refers to a technique for separating proteins by electrophoresis and marking with specific antibodies to an antigen of interest on a nitrocellulose membrane (Blancher and Jones, 2001).

[068] REFERENCES
1. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. https://www.ncbi.nlm.nih.gov/books/NBK26921/
2. Blancher C, Jones A. Metastasis Res Protoc. 2003, 57:145–162. https://doi.org/10.0.5.105/1-59259-136-1:145
3. Costerton JW. Int J Antimicrob Agents. 1999, 11:217–221. https://doi.org/10.0.3.248/S0924-8579(99)00018-7
4. Crowther JR. The ELISA guidebook. Vol. 149, Methods in molecular biology (Clifton, NJ). 2000. https://doi.org/10.0.5.105/1592590497
5. Davies KJA. Mol Aspects Med. 2016, 49:1–7. https://dx.doi.org/10.1016/j.mam.2016.04.007
6. Gómez-Gómez L, Boller T. Trends Plant Sci. 2002, 7:251–256. https://doi.org/10.0.3.248/S1360-1385(02)02261-6
7. Greiner M, Sohr D, Göbel P. J Immunol Methods 1995, 185:123–132. https://doi.org/10.0.3.248/0022-1759(95)00121-P
8. Johnson PD, Besselsen DG. ILAR J. 2002, 43:202–206. https://doi.org/10.0.4.69/ilar.43.4.202
9. Kaper JB, Nataro JP, Mobley HLT. Nat Rev Microbiol. 2004, 2:123–140. https://doi.org/10.0.4.14/nrmicro818
10. Ribeiro R, Alcazar A, Silva A, Pires J, Rosa P. The management of processes, activities and tasks through the use of performance indicators: a case study in an egg hatchery. XVII Brazilian Congress on Costs. 2010;
11. Sato A, Iwasaki A. Cell Mol Life Sci. 2005, 62:1333–8. https://doi.org/10.0.3.239/s00018-005-5037-z
12. Schumann RR, Leong SR, Flaggs GW, Gray PW, Wright SD, Mathison JC, et al. Sci. Rep. 1990, 249:1429–1432. https://doi.org/10.1126/science.2402637
13. Sekirov I, Russell SL, Caetano M Antunes L, Finlay BB. Physiol Rev. 2010, 90:859–904. https://doi.org/10.1152/physrev.00045.2009
14. Stryer L, Berg JM, Tymoczko JL (2002). Biochemistry (5th ed.). San Francisco: WH Freeman. ISBN 0-7167-4955-6.
15. Yi J, Shane AL. Princ Pract Pediatr Infect Dis 2017, 376–383. https://doi.org/10.1016/B978-0-323-40181-4.00055-4

Claims (10)

A KIT FOR THE DETECTION OF IMMUNE RESPONSES AGAINST THE BACTERIA Salmonella enterica (SALMONELLA) characterized by containing lipopolysaccharide (LPS) – or subunits thereof – from salmonella immobilized on a solid substrate; and flagellin – or subunits thereof – from salmonella immobilized on a solid substrate. A kit, according to claim 1, characterized in that it consists of a solid substrate that is a synthetic or natural polymer. A kit, according to claim 1, characterized in that it consists of a solid substrate that is a conductive metal. A kit, according to claim 1, characterized by detecting and quantifying an immune response that encompasses the production of antibodies of the IgA class. A kit, according to claim 1, characterized by detecting immune responses that include immune responses from animal species of veterinary interest. A kit, according to claim 1, characterized in that the immune responses to be detected are from samples of fluids, excreta or body waste, encompassing immune responses arising from the mucosal surfaces of the organism, including feces, poultry litter, swab cloacal, urine, saliva, milk, colostrum and tears. A kit, according to claim 1, characterized in that it consists of independent solid substrates or not that have immobilized LPS or salmonella flagellin - or subunits thereof, encompassing the possibility that the solid substrates have the same characteristics or no. A kit for the detection of immune responses against the bacteria Salmonella enterica (salmonella) characterized by the fact that the product will be produced by the following sequential steps: 1) extraction of salmonella flagellin by removing these proteins from bacteria that contain them by means of mechanical or physical-chemical rupture of them; 2) extraction of salmonella lipopolysaccharides by removing these molecules from bacteria that contain them by mechanically or physicochemically breaking them; 3) immobilization of flagellin and salmonella LPS on solid substrates independent or not through physical adsorption on polymer surfaces or through self-assembled layers or through cysteine bonds on conductive metal surfaces. A kit for detecting immune responses against the bacteria Salmonella enterica (salmonella) characterized by the fact that the product is configured to differentiate between animals infected or vaccinated against salmonella for a period between 1 day and 21 days or for periods longer than this. A kit, according to claim 10, characterized by the fact that the product is configured to differentiate between animals infected or vaccinated against salmonella for a period between 1 day and 15 days or for periods longer than this.

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