Pasteurella multocida strain was obtained from the lungs of pigs showing clinical pneumonia. Samples were cultured on brain-heart infusion agar (Difco Laboratories, Detroit, MI, USA) and incubated aerobically overnight at 37˚C. Pasteurella multocida type A was identified by standard procedures [15] [16]. The biomass was inactive with formaldehyde 0.5% for 12 hours by washing the plates with PBS, and the bacterial concentration was standardized spectrophotometrically (660 nm) at 10⁸ CFU/ml. To obtain optimal growth conditions, the bacterial suspension (5%) was inoculated into flasks with 50 ml brain-heart infusion broth with three different pH values (6.0, 6.5 and 8.0) and incubated at 37˚C for 18 h with constant shaking at 50 rpm., at the same time dilutions are made of this sample and plated on agar, so the count of each dilution is made and the CFU/ml is obtained, it was repeated 3 times. The bacterial concentration in units of colony forming units per milliliter (CFU/ml) was determined at different times.

Pasteurella multocida was prepared using the traditional method of emulsification but without incorporating stabilizers [17]. The first step was to prepare a primary w/o emulsion by homogenizing the aqueous phase (bacteria inactivated in PBS) in the oil phase (mineral oil USP/NF 55, Cosmopolita S. A. de C.V. México) with vigorous propeller stirring (1500 rpm Caframo R2R2-64 Ontario, Canada) at 37˚C. Then, the w/o emulsion was blended with hydrophilic silica (Aerosil^Ò 200, Degussa, W. Germany) to obtain a turbid liquid. Finally, hydrophobic silica (Aerosil^Ò R972) was added, and a viscous fluid powder (dry emulsion) was obtained. One gram of hydrophilic silica for each 5.7 ml of aqueous medium and 1 g of hydrophobic silica for each 3 ml of oil medium were used. Twelve batches were prepared for the study. The distribution of bacteria in the system was determined by light microscopy and scanning electron microscopy (SEM). Pasteurella multocida viability was evaluated after preparation by determining the number of CFU/ml. To detect the presence of the dry emulsion in the in vivo test on mucosa, a marker consisting of 300 μl of colloidal silver flakes obtained from an electroconductive silver adhesive (SPI, high purity silver paint) was dispersed in the final product. The flakes were selected as markers due to their morphology and their ability to emit secondary electrons.

The bioadhesive properties of the dry emulsion were evaluated using an in situ method as proposed by Rao and Buri [18]. Briefly, longitudinal sections (30 cm long) from the duodenum, jejunum or ileum were removed from commercial Yorkshire pigs at the slaughterhouse immediately after death and were quickly transported to the laboratory in 0.9% saline solution. The intestine samples were clipped to a cylindrical transversal section of a polyvinyl chloride tube (diameter = 10 cm) and fixed in a platform at a 45˚ angle. The intestine sections were washed with Sorensen buffer (pH 6.0) using a peristaltic pump with a flow rate of 25 ml/min for 30 min. Then, 100 mg of dry emulsion was homogenously distributed on the tissue sections, allowing interaction of the powder with the mucosa for 10 min. Afterwards, the system was eluted with simulated intestinal medium at a flow rate of 25 ml/min for 60 minutes. During the entire process, the fluid covered the tissue area to determine the persistence of the dry emulsion or bacteria on the mucosa. Different mucosa sections were taken after treatment and examined with light microscopy and scanning electron microscopy.

The determination of adhesion strength to intestinal pig mucosa was evaluated following a previously described method using a modified Du Noüy tensiometer [13] [19]. The ring was replaced by a square metallic plate (4.00 cm²), and the original filament was replaced by a stainless-steel filament similar to piano strings of 0.22 gauge. The device was calibrated using reference weights and validated for this kind of measurement, the calibration and validation were performed following manufacturer instructions. Each section of mucosa was placed on the plate with the mucous surface exposed (3 cm²) and fastened by a plastic holder and 4 staples. A dry emulsion sample was attached to the tensiometer platform. It was allowed to be in contact with the membrane for 30 s, thus causing the dial to spin at a constant rate. The adhesion force reading is obtained when the gel disk is detached from the mucosa. Evaluations were made in triplicate. The in vitro dissolution assay was performed by using the USP 24 apparatus II (paddles) using 250 ml of simulated intestinal fluid at 37˚C and a stirring rate of 50 rpm. One gram of the disperse-adsorbed system was placed between filter paper (diameter of 2.5 cm) and mesh (U. S. Standard 60, 250 mm pore), and this system was immersed in the dissolution medium. Samples were taken at different intervals between 0 and 120 hours, and the bacteria released were correlated to the CFU/g.

Nine intact male Yorkshire weanling pigs (7 weeks old) were obtained from an animal house. The animals were kept in isolation units and fed standard pig feed and water ad libitum throughout the study. All experiments were conducted in compliance with the Mexican Regulations for Animal Care and Maintenance (NOM-062-200-1999). The pigs were randomly divided into three groups (n = 3): group A, control animals (untreated); group B, treated with the dry emulsion containing the marker at 0.5 ml; and group C, pigs treated once with a dry emulsion of Pasteurella multocida 10⁸ CFU/g. The pigs were fed for fifteen days. Serum samples were collected weekly over 2 weeks (1, 7 and 15 days), and pig antibody responses were monitored using ELISA [20] [21].

The pigs were euthanized (NOM-062-200-1999), necropsied and small fractions of duodenum, jejunum and ileum were collected for evaluation. The samples were fixed by perfusion with Karnovsky’s fixative containing 1% tannic acid [22] and were then dehydrated in absolute alcohol, dried at critical point in a Tousimis sample dryer (Samdri-780A, Tousimis Research Corporation, Rockville, USA) and gold coated in a Finecoat Ion Sputter (JFC-1100, JEOL LTD, Tokyo, Japan). Samples were then examined using a scanning electron microscope (JEOL JSM/25 S II. Tokyo, Japan). For the in vivo study, a low vacuum (SEM JEOL JSM5410LV) was used since the ion sputter device could not produce a continuous electroconductive coat to avoid charging.

The pig antibody responses were monitored using ELISA. Immunoplates (Nunc VWR Scientific, Bridgeport, NJ) were coated at 4˚C overnight with 100 ng of the following antigens, in house monovalent enzyme-linked immunosorbent assay (ELISA, the plate antigens were prepared by sonicating the Pasteurella multocida type A strain. Whole cell lysates were placed in borate buffer (pH 9.5) 1:1. The plates were washed with 50 μl of 0.01 M PBS containing 0.05% Tween 20 (pH 7.2), followed by the addition of 200 μl of blocking buffer (PBS containing 1% bovine serum albumin). Samples (50 μl) were then incubated at temperature ~20C for 30 min. After the plates were washed, 50 μl of antisera serially diluted with blocking buffer was added, and the plates were incubated at ~20C for 30 min. After a further washing, 50 μl of 1:5000 diluted rabbit anti-pig immunoglobulin G conjugated to horseradish peroxidase (Sigma Aldrich Chemistry Mexico: catalogue: A5670) was added, and the plates were incubated ~20C for 1 h. For color development, 100 ml of 3, 39, 5, 59-tetramethylbenzidine substrate was added, and the plates were incubated for 30 min. Then, 100 ml of a 0.25% solution of hydrofluoric acid was added to stop the reaction. Absorbance was read at a wavelength of 630 nm with an ELISA reader (Multiskan EX type 355; Thermo Fisher Scientific (Shanghai) Instrument Co. Ltd). The positive/negative ratio method of analysis was used to determine the antibody titer of treated pigs. After a log transformation of the ELISA titer, a linear relationship was confirmed between the ELISA titer and the positive/negative ratio [20] [21] [22].

The manuscript does not contain clinical studies or patient data; the samples used in this study were voluntarily provided by veterinarians and pig farmers.

Bacterial Strain. The viability of Pasteurella multocida in the dry emulsion was confirmed by the CFU/ml. In only two cases, the CFU/ml decreased from 10⁸ to 10⁶. There was no significant difference among the batches and bacterial suspensions with the same concentration. The dry emulsion and the preparation process do not affect the viability of Pasteurella multocida.

Disperse-Adsorbed System. The values obtained for bioadhesive force were 5045 ± 85 dynes/cm² and 4780 ± 76 dynes/cm² for duodenal and jejunal mucosa, respectively, the release profile of Pasteurella multocida from the dry emulsion where CFU/g correspond to the accumulative bacteria released by gram of the system. Two steps can be observed, first a burst release until 5 h followed by a slow constant release until 120 h.

Animals. After the experiment, the animals showed neither temperature nor clinical signs.

Some samples like examples of the SEM revealed that the system is an organized association of particles, and water globules of sizes between 2.5 - 5.0 mm. The bacteria were apparently intact and localized adjacent to the particles in the aqueous domain. The rheological behavior of the dispersion is similar to that observed for cosmetic and pharmaceutical pastes. The bioadhesion study revealed areas of intestine completely covered by the emulsion. The marker was located mainly on the duodenum and jejunum. Apparently, the structure of the villi in the ileum did not allow for the adherence of the dry emulsion. On the duodenum, the system was found as bridges between villi (Figure 1) and at the bottom of the villi (Figure 2). In the case of the jejunum, the emulsion adopted the form of a sheet that covered important areas of the intestine. In this region, the marker can be clearly distinguished together with bacterial bodies (Figure 3). This interaction between the dry emulsion and mucosa was confirmed by the measurements of bioadhesive force.

The ELISA technique was used to monitor the mean antibody titer of treated pigs over the 2-week period, and a classic primary response curve occurred when the titer was plotted against time. The sorbent alone did not detect an antibody response, in the sorbent and Pasteurella multocida group, antibodies were detected at 7 days and a double response at 15 days (Table 1).