80 jorge lopez vaccine 2014

Vaccine 32 (2014) 6805–6811

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Oral immunization against porcine pleuropneumonia using the cubic phase of monoolein and purified toxins of Actinobacillus pleuropneumoniae Jorge Lopez-Bermudez a , David Quintanar-Guerrero b , Horacio Lara Puente e , d ´ ´ ¨ ´ Carrasco a , Jorge Tortora Perez c , Francisco Suarez Guemez , Abel Ciprian Susana Mendoza Elvira a,∗ a

Laboratorio de Virología-FES-Cuautitlán, UNAM, Mexico Laboratorio en Tecnología Farmacéutica FES-Cuautitlán, UNAM, Mexico c Laboratorio de Patología-FES-Cuautitlán, UNAM, Mexico d Facultad de Medicina Veterinaria y Zootecnia, UNAM, Mexico e Laboratorios Avi-Mex, S.A. de C.V., Mexico b

a r t i c l e

i n f o

Article history: Received 4 June 2014 Received in revised form 11 September 2014 Accepted 24 September 2014 Available online 19 October 2014 Keywords: Porcine pleuropneumonia Apx I Apx II Apx III Immunity Oral vaccine

a b s t r a c t The main goal of this work was to obtain an orally administered immunogen that would protect against infections by Actinobacillus pleuropneumoniae. The Apx I, II and III toxins were obtained from the supernatants of cultures of serotypes 1 and 3 of A. pleuropneumoniae. The capacity of monoolein gel to trap and protect the Apx toxins, and the effect of their incorporation on the stability of the cubic phase were evaluated. The gel was capable of trapping a 400-␮g/ml concentration of the antigen with no effects on its structure. Approximately 60% of the protein molecules were released from the gel within 4 h. Four experimental groups were formed, each one with four pigs. All challenges were conducted in a nebulization chamber. Group A: Control (−) not vaccinated and not challenged; Group B: Control (+) not vaccinated but challenged; Group C: vaccinated twice intramuscularly with ToxCom (a commercial toxoid) at an interval of 15 days and then challenged; and Group D: vaccinated orally twice a week for 4 weeks with ToxOral (an oral toxoid) and challenged on day 28 of the experiment with a same dose of 2.0 × 104 UFC of A. pleuropneumoniae serotypes 1 and 3. The lesions found in group B covered 27.7–43.1% of the lungs; the pigs in group C had lesions over 12.3–28%; and those in group D over 15.4–32.3%. No lesions were found in the Group A pigs. A. pleuropneumoniae induced macroscopic lesions characteristic of infection by and lesions microscopic detected by histopathology. The etiologic agent was recovered from the infected lungs, tonsils and spleen. The serotypes identified were 1 and 3. An indirect ELISA test identified the antibodies against the Apx toxins in the serum of the animals immunized orally. © 2014 Published by Elsevier Ltd.

1. Introduction Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the etiologic agent of contagious porcine pleuropneumonia (CPP), a highly transmissible respiratory illness responsible for large economic losses on pig farms [1]. To date, 15 serotypes of App have been identified, based on capsular antigens, all with different degrees of virulence [2,3]. CPP is characterized by

∗ Corresponding author at: Facultad de Estudios Superiores – Cuautitlán, UNAM, Laboratorio de Virología, Av. 1о , De Mayo S/N Campo I, Santa María las Torres, CP 54700 Cuautitlán Izcalli, Estado de México, Mexico. Tel.: +55 5623 2058; fax: +52 55 56232058.

E-mail address: seme@unam.mx (S. Mendoza Elvira). http://dx.doi.org/10.1016/j.vaccine.2014.09.056 0264-410X/© 2014 Published by Elsevier Ltd.

manifestations of variable clinical signs in the acute or chronic phases [4,5]. The bacteria present a series of actions coordinated with diverse virulence factors, such as capsular antigens [6,7], external membrane protein antigens [8,9], external membrane lipopolysaccharides [10,11], the Ca2+ -dependent Apx I, II, III and IV toxins, with hemolytic and/or cytotoxic activity. These toxins have been identified in all 15 serotypes of A. pleuropneumoniae [3,4,12–15]. The virulence of A. pleuropneumoniae is multifactorial, several studies have shown that virulence is strongly associated with production of the Apx toxins [7,11,16]. The various types of vaccines, commercially available combine bacterins, bacterins/toxoids and toxoids, but they continue to be applied by parenteral route. Thus, developing oral immunization methods with carrier and protection systems could be of great importance in controlling CPP [17–19].

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Glyceryl monooleate, or monoolein, is a molecule characterized by low toxicity, biodegradability and biocompatibility [20]. Its molecular structure, presents both polar and hydrophobic characteristics [21–24]. In the presence of excess water, monoolein forms a viscous gel called the cubic phase, within which it is feasible to trap protein molecules [20,25,26]. These properties allow monoolein to function as a vehicle and protection system for the orally administered protein molecules [21,22,27,28].

The principle objective of this work was to study this glyceryl monooleate-based gel as a vehicle, protector and promoter of absorption of A. pleuropneumoniae toxins in order to assess its possible use as an oral immunogen to control CPP. 2. Materials and methods 2.1. Culture of the strains A. pleuropneumoniae serotypes 1 (Strain: 4074) and 3 (Strain: 1421), were used as reference strains and cultured in a medium of brain heart infusion (BHI, Becton Dickinson de México) with nicotinamide adenine dinucleotide (NAD) added at 0.5%, and incubated at 37 ◦ C for 18 h.

2.2. Production of the A. pleuropneumoniae toxins Colonies of serotypes 1 and 3 of A. pleuropneumoniae were taken independently, inoculated in BHI-NAD liquid medium with 5 mM of CaCl2 , and incubated at 37 ◦ C for 4–6 h. The systems were then concentrated by tangential ultrafiltration (Pellicon Millipore, USA), lyophilized (Labconco, Freezone 6, USA), and stored at −70 ◦ C in 1.0 ml aliquots [29,30].

2.3. Assays of the Apx toxins The protein concentrations in the culture supernatants were determined (400 ␮g of total protein) by the Bradford method [31].

Electrophoresis in SDS polyacrylamide gel was also conducted. The Apx toxins were inactivated with formaldehyde at 1.0%/PBS and 1 ml of the adjuvant (aluminum hydroxide). Hens (Rhode Island) were immunized following a program of three immunizations every 7 days. After 21 days the animals were bleed from the wings, the serum recuperated was evaluated by Western Blot [32], and isoelectric test in order to confirm the presence of the Apx toxins in the supernatants (data not shown).

2.4. Tests for activity of the Apx toxins The study used the non-inactivated supernatants of A. pleuropneumoniae serotypes 1 and 3 to determine the amount of antigen by assessing hemolytic activity in sheep erythrocytes, and cytotoxicity tests in cultures of Vero and PK-15 cells, respectively [30,33,34].

2.5. Preparing the ELISA assay The toxins obtained by ultrafiltration and concentrated by lyophilization were diluted to a concentration of 10 ␮g/ml using a carbonate-based buffer solution (0.1 M, pH 9.5). From this solution, 100 ␮l were placed in each well of a 96-well ELISA plate and incubated overnight at 4 ◦ C, then washed three times with 300 ␮l of the PBS/Tween-20 0.05% buffer solution and blocked for 1 h with skim milk at 5% [35,36]. The hemolytic activity of the supernatants of A. pleuropneumoniae was determined using ovine erythrocytes in TS buffer (Tris 10 mM, NaCl 0.8%, pH 7.5) as follows: 1.0 ml of filtered at an initial concentration of 1000 ␮g/ml, or dilutions in TS buffer to concentrations of 500, 250, 125, 62.5, 31.2, 15.6, 7.8 and 3.9 ␮g/ml, were mixed with 1.0 ml of ovine erythrocytes at

1%. The amount of hemolysis was then measured by absorbency at 540 nm. A microplate was prepared for each type of cell line up to a confluence of 90% in RPMI medium (ICN Biomedicals, Inc.), then they were inoculated with 100 ␮l of the different protein concentrations (100, 200, 300, 400, 500 and 600 ␮g/ml) of each bacteria serotype. A hemolytic filtrate was reported when the ovine erythrocytes were completely hemolyzed. A strongly cytotoxic filtrate was considered when the titer was >62.5, while a titer >31.5 was considered moderately cytotoxic [30,33,34].

2.6. Elaboration of the monoolein gel In 300 ␮l of the culture medium containing an amount equivalent to 200 ␮g of Apx toxins was mixed with 700 mg of monoolein.

The dispersion thus obtained was centrifuged at 14,000 rpm for 10 min. The integrity of the cubic phase of the monoolein (CPM) and the effect of the inclusion of the Apx toxins in the gel were determined by polarized light microscopy and visual observation [21–23,27]. The release profile was determined in Franz diffusion cells (20 ml) using filter paper as the separation membrane [31].

2.7. Experimental design Used twenty 5-week-old pigs Yorkshire pigs that were free of antibodies against A. pleuropneumoniae, Mycoplasma hyopneumoniae, Pasteurella multocida, Aujeszky’s disease, and Porcine Reproductive and Respiratory Syndrome (PRRS). Throughout the experiment, the animals received antibiotic-free feed. Four groups of four pigs each were formed. Group A: Control (−) not vaccinated and not challenged; Group B: Control (+) not vaccinated but challenged; Group C: ToxCom (Commercial Toxoid), vaccinated intramuscularly twice at an interval of 15 days and challenged; and, Group D: ToxOral (Oral Toxoid), vaccinated orally twice a week for 4 weeks and challenged on day 28 of the experiment.

2.8. Immunization On day one of the experiment, the pigs in Group C received their first vaccination; each animal was inoculated intramuscularly with 2.0 ml of the immunogen, this immunization was repeated 15 days later using the same dose and route. The pigs in group D received two 400-␮g doses of total protein orally each week for 3 weeks (6 immunizations). Blood samples were taken from all pigs in all groups on day 1, 7, 14, 21, 28 and 32 of the experiment. These samples were used for determined Apx toxin by indirect modified ELISA assay [35–38].

2.9. Challenge system using nebulization Aerosolization was conducted in a nebulization chamber [39] with a holding capacity of 20 piglets. Nebulization continued for 45 min with 36 ml of the inoculum at a titer of each serotype of 2.0 × 104 UFC for A. pleuropneumoniae serotypes 1 and 3 [40].

2.10. Clinical evaluation of pigs The pigs’ body temperatures were monitored throughout the experiment, and the following signs of the presence of CPP identified: nasal secretion, sneezing, ocular secretion, conjunctivitis, cough, expectorations, dyspnea, and abdominal respiration [1].

2.11. Sacrificing the animals, necropsy and evaluation of macroscopic lesions Necropsies were performed on all the animals, both those that died during the experiment and those that were slaughtered

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Fig. 1. Effect of Apx toxins on the temporal development of the humoral immune response in A. pleuropneumoiae-infected pigs. Group A: Control (−) not vaccinated and not challenged; Group B: Control (+) not vaccinated but challenged; Group C: ToxCom, vaccinated intramuscularly twice at an interval of 15 days and challenged; and, Group D: ToxOral, vaccinated orally twice a week for 4 weeks and challenged on day 28 of the experiment. The blood samples were taken from each animal on days: 1 = day 1; 2 = day 7; 3 = day 15; 4 = day 21; 5 = day 28 and 6 = day 32: The day 28, the pigs in Groups B, C and D were infected with A. pleuropneumoniae. From day 21 the difference in the increase in optical density (OD at 480 nm) of immunoglobulin levels in Groups C and D was statistically significant differences between groups (p < 0.05).

upon its conclusion. The pigs were euthanized by sedation with 3 mg/kg of azaperone (Sural, Chinoin Productos Farmaceuticos, S.A. de C.V., Mexico) and deep anesthesia with 0.3 ml/kg of metomidate (Hypnomidate, Janssen-Cilag, S.A. de C.V., Mexico) followed by exsanguination [41]. The study was approved by the Ethics Committee on Animal Experiments at the Faculty of Veterinary Medicine, UNAM (Universidad Nacional Autónoma de México), and was conducted in compliance with Mexican Regulations for Animal Care and Maintenance [42]. The pathological lesions in all animals from the four groups were evaluated [8].

activity, determined by the hemolysin and cytotoxicity trials, showed titers of 62.5 and of 31.5, respectively.

2.12. Statistic study

3.3. Serology

The extent of pneumonic lesions was measured by planimetry on standard lung diagrams [43]. Statistically significant differences between groups (p < 0.05) were determined with a student’s t-test using SAS software [44].

The serological response (IgG) detected differences between vaccinated groups C and D. While the sera of the animals in group B appeared to have only a minimal increase in their responses up to day 32, in groups C and D this appeared to increase gradually on day 21, and more markedly on day 28 post-vaccination with statistically significant differences (p < 0.05) (Fig. 1).

2.13. Evaluation of histopathological lesions Samples of several tissues – tonsils, lung, spleen, liver and kidney – were taken, fixed in formaldehyde at 10%, and embedded in paraffin. The sections obtained were stained with hematoxylin and eosin [1].

2.14. Recovery of the inoculated agents To isolate A. pleuropneumoniae, the samples of tonsils, lung, spleen, liver and kidney were cultured in blood agar medium and BHI agar with NAD at 0.05%. The colonies recovered were serotyped individually using specific anti-sera against serotypes 1 and 3 [45].

3. Results 3.1. Production and identification of A. pleuropneumoniae toxins The Apx toxins I, II and III were obtained from supernatants of 4-h cultures of serotypes 1 and 3 of A. pleuropneumoniae. Their presence in the supernatants was confirmed by means of the 100-to-120 kD bands to enrich the supernatants. The supernatant

3.2. Dose monoolein gel and Apx toxins The dose used for oral immunization was determined from the results of the cytotoxicity and the hemolytic test of the Apx toxins.

A concentration of 400-␮g was the optimal oral dose for studying these toxins.

3.4. Clinical observations None of the pigs in Groups C and D that were vaccinated with the Tox/Com or Tox/Oral immunogens showed any post-vaccination reaction, included hyperthermia. In contrast, the groups inoculated with A. pleuropneumoniae became feverish on day 30. The Group B animals had a higher average body temperature (40.1 ± 0.5 ◦ C) than the uninfected ones (39.3 ± 0.4 ◦ C); while in Groups C and D temperatures remained elevated when measured on day 30 post-infection (40.3 ± 0.4 ◦ C, and 40.5 ± 0.4 ◦ C, respectively) (Fig. 2).

From day 29, all the inoculated pigs in group B presented moderate coughing that lasted until day 31, and also displayed severe fever, coughing, dyspnea, and signs of death from A. pleuropneumoniae infection. The animals in Groups C and D began to manifest mild coughing on day 30, which increased to moderate on day 32. Dyspnea in pigs in Groups B, C and D began on day 29. Dyspnea at rest was more pronounced than under effort until day 30. In the Group B pigs, dyspnea lasted until slaughter. From day 32 until the end of the experiment, dyspnea under effort was more marked than dyspnea at rest in Group C (Table 1).

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Fig. 2. Average body temperatures for each group were determinate during 32 days of the different pig’s groups: Group A: control (−) not vaccinated and not challenged; Group B: control (+) not vaccinated but challenged; Group C: ToxCom, vaccinated intramuscularly twice at an interval of 15 days and challenged; and, Group D: ToxOral, vaccinated orally twice a week for 4 weeks and challenged on day 28 of the experiment.

3.5. Gross pathology and bacteriology

thrombi and dilatation. Three of the pigs in Group C did not present active BALT, and the presence of PMN was scarce. Only one of those pigs showed an abundant presence of PMN in the bronchioles and alveoli. While three pigs in Group D presented a milder signology, the symptoms of most of the animals in the oral-vaccination group were less pronounced than those in the other animals, with serous exudate in the alveoli and discrete vascular changes.

All animals in Groups B, C and D inoculated with A. pleuropneumoniae showed consolidated hemorrhagic pneumonic lesions in the right and left diaphragmatic lobes and accessory lobes, while the right and left mid-cranial and caudal lobes presented minimal lesions. There were lesions covering an area of 27.7–43.1% in the pigs in Group B, while those in Group C had lesions over 12.3–28%, and those in Group D 15.4–32.3% (Fig. 3). The distribution, extent and appearance of the macroscopic lung lesions are summarized in Table 2. The others organs doesn’t show microscopic effect.

4. Discussion CPP causes enormous economic losses worldwide, so it is necessary to develop more efficacious methods of control. Today, many commercial bacterins include the complete microorganism, with the serotype (or serotypes) predominant in each area or predetermined region [46,47]. Attenuated vaccines have produced such mutations as: unencapsulated; attenuated; and auxotrophic, which have been used to induce protective immunity, but decreases in mortality and lesions under experimental conditions in which the vaccinated pigs resisted the intratracheal challenge with homologous and heterologous serotypes [48–50].

At present, the commercially available vaccine is based on subunits with three toxoids of Apx I, Apx II and Apx III, and an OMP with an oleaginous adjuvant [51–53,56–59]. Therefore, developing oral immunization methods, since stimulating the immune response at the level of the mucosa may well provide protection against the disease. This study developed an immunogen with these characteristics using the Apx I, II and III toxins of A. pleuropneumoniae and the CPM as the system for delivering, promoting and

3.6. Recovery of the inoculated agents The inoculated agent was not isolated or identified in group A, but A. pleuropneumoniae was isolated and identified in the diaphragmatic lobes of all pigs in groups B, C and D. 3.7. Histopathological lesions Lungs: The pigs in Group A showed no pathological changes attributable to A. pleuropneumoniae infection. The animals in the positive control Group B presented typical lesions of the disease with hyperplasia of the bronchiolar epithelium, BALT proliferation, and PMN in the bronchioles, alveoli and interlobular septa.

Animals in Groups C and D had pulmonary lesions characteristic of CPP, consisting in hyperemia with hemorrhaging in the alveoli and the interlobular and pleural septa, and the presence of venous

Table 1 Clinical signs observed during 32 days in the pigs of the different groups. Group A: control (−) not vaccinated and not challenged; Group B: control (+) not vaccinated but challenged; Group C: ToxCom, vaccinated intramuscularly twice at an interval of 15 days and challenged; and, Group D: ToxOral, vaccinated orally twice a week for 4 weeks and challenged on day 28 of the experiment.

Group N=4

A B C D a

Pathological presentation

Nasal secretion %

Ocular secretion/conjunctivitis %

Cough %

Abdominal respiration %

Dyspnea at rest %

Dyspnea under effort %

Rales and post-challenge deatha %

0 25 0 0

0 0 25 25

0 0 25 25

0 100 50 50

0 100 25 25

0 100 50 50

0 100 0 0

When the pigs in Group A died 24-to-72 hours post-heterologous challenge with A. pleuropneumoniae serotypes 1 and 3, the pigs in Groups B, C and D were sacrificed.

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Fig. 3. Percentage of extension of lesions (%) in the lungs of pigs experimentally infected with A. pleuropneumoniae after 32 days for the different groups. Group A: control (−) not vaccinated and not challenged; Group B: control (+) not vaccinated but challenged; Group C: ToxCom, vaccinated intramuscularly twice at an interval of 15 days and challenged; and, Group D: ToxOral, vaccinated orally twice a week for 4 weeks. All pigs were euthanized and the percentages of the areas covered by lesions in their lungs were recorded. The averages for Groups B (a) and C (b) were statistically different by least significant difference (p < 0.05) and were determined with the t-test.

protecting an orally administered vaccine which protects animals that are susceptible to the disease [18,34,54,55].

The presence of the Apx I, II and III toxins in the supernatants obtained from the cultures of serotypes 1 and 3 of A. pleuropneumoniae was demonstrated by in the polyacrylamide gels, Western Blot tests, and isoelectric focusing. The filtrates showed a pronounced hemolytic activity, while cytotoxic activity [30,33,34].

CPM, an amphiphilic lipid carrier that gels in water excess, it was capable to protect molecules; it releases the incorporated toxin in a controlled manner; and it promotes interaction/adhesion with the surface of the mucosa where absorption, occurs, through its surfactant qualities [24,26,28,56–58].

Serological monitoring to identify the antibodies against the Apx’s toxins was conducted using the modified indirect ELISA assay

[35,36]. During the first week, the antibodies were similar in groups C and D. The serological behavior of ToxCom and ToxOral in the subsequent samples was typical of a secondary response of greater magnitude (Fig. 1).

The temperature measurements in the different groups B, C and D showed the appearance of hyperthermia on days 29–32 was due to A. pleuropneumoniae infection. The pigs in the positive control Group B that were inoculated with App presented greater respiratory difficulty and a tendency towards greater severity, which eventually caused death. Turning to the pigs in Groups C and D that were immunized with ToxCom and ToxOral respectively presented milder clinical and respiratory signs in general and a tendency to recover. The pigs in Group B presented lesions characteristic of acute pleuropneumonia [60] with consolidated areas that covered

Table 2 Location and appearance of macroscopic lesions in the lungs of A. pleuropneumoniae-uninfected, -infected, and vaccinated/infected pigs.a Groups N=4

Group A Uninfected

Location of lesionsb

Right apical

Right cardiac

Right diaphragmatic

Left apical

Left cardiac

Left diaphragmatic

Accessory

Appearance of lesions: Reddish, pleural hemorrhagic adhesions

0

0

0

0

0

0

0

0

0

3

4

0

1

4

4

4

4

2

4

1

1

4

3

4

4

2

4

1

1

4

3

4

4

Group Ba A. 3 pleuropneumoniaeinfected Group Ca Tox/Com 1 vaccine and A. pleuropneumoniaeinfected Group Da Tox/oral 1 vaccine and A. pleuropneumoniaeinfected

Group A: control (−) not vaccinated and not challenged; Group B: control (+) not vaccinated but challenged; Group C: ToxCom, vaccinated intramuscularly twice at an interval of 15 days and challenged; and, Group D: ToxOral, vaccinated orally twice a week for 4 weeks. All pigs were euthanized and the percentages of the areas covered by lesions in their lungs were recorded. a Number of animals with lung lesions. b Sites within the consolidated lobes of the lungs.

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from 27.7% to 43.1% of the lungs. The average area affected was 33.62%. The index of pneumonic lesions seen in this group represented 100% of the lesions caused by the experimental infection at the dose applied [61,62].

The pigs in the ToxCom Group presented chronic pleuropneumonic lesions with consolidated areas covering 12.3–28.0% and an average of 21.34%. In contrast, those in the ToxOral Group suffered chronic pleuropneumonic lesions with consolidated areas covering 15.4–32.3% and an average of 26.9%. The index of pneumonic lesions found in this group represented 63.5% of the lesions caused by the experimental infection at the dose utilized, which means a reduction of just 36.5%. The index of pneumonic lesions seen in this group represented 80% of the lesions caused by the experimental infection at the dose applied, indicating a reduction of 20% [61,62].

Recovery from A. pleuropneumoniae was achieved only in the groups B, C and D inoculated with the agent. The infectious agents recovered corresponded to the serotypes 1 and 3 of A. pleuropneumoniae with which the animals had been inoculated.

The integral immune system of the mucosa has been established in various studies utilizing oral administration [63]. Using a suspension of inactivated bacteria administered orally, some authors, such as Liao et al. [64] created a novel co-spray drying process capable of entrapping A. pleuropneumoniae immunogens in microspheres using aqueous dispersion polymers, and Wei et al. [19] have demonstrated production of high antibody titers in the sera of the animals tested, utilized a system of microspheres to protect and carry A. pleuropneumoniae cells inactivated with formaldehyde to show the effectiveness of oral immunization systems in preventing infection by this agent. They found not only high antibody titers against the antigen, but also a good percentage of protection against the disease after the challenge. The injection vaccine of a commercial whole-cell bacterin of A. pleuropneumoniae sometimes induced muscular lesions at the vaccination site, and caused severe inflammation, fever as asthenia, especially in piglets [19].

The antibodies in the serum of the animals immunized with CPM that contained the Apx toxins in titers similar to those of existing commercial preparations suggests the feasibility of developing an immunogen that is both easy to prepare and administer, and that will provide protection against CPP by oral application. Based on the test performed, the monoolein in the cubic phase showed to be a very good carrier and protective vehicle for Apx toxins, able to trap protein molecules in its water channels systems. Fortunately, the oral vaccine toxoid provides a good protection in to weaned piglets as growing pig stage decreasing the manipulation and animal stress produced by parenteral treatments. The data presented in this study demonstrated that it is possible to have a good immunization for CPP with a potential use in farms. Furthermore, this immunogen does not need special storage conditions.

Acknowledgments Grant: PIAPIC12 and PASPA DGAPA-UNAM. 2013 sabbatical stay of Dr. Abel Ciprián in Centre de Recerca en Sanitat Animal (CReSA), Campus Universitat Autònoma de Barcelona (UAB).

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