Use of Nanostructured Silica SBA-15 as an Oral Vaccine Adjuvant to Control Mycoplasma hyopneumoniae in Swine Production.

Mycoplasma hyopneumoniae is a difficult-to-control bacterium since commercial vaccines do not prevent colonization and excretion. The present study aimed to evaluate the performance of an orally administered vaccine composed of antigens extracted from Mycoplasma hyopneumoniae and incorporated into mesoporous silica (SBA-15), which has an adjuvant-carrier function, aiming to potentiate the action of the commercial intramuscular vaccine. A total of 60 piglets were divided into four groups (n = 15) submitted to different vaccination protocols as follows, Group 1: oral SBA15 + commercial vaccine at 24 days after weaning, G2: oral vaccine on the third day of life + vaccine commercial vaccine at 24 days, G3: commercial vaccine at 24 days, and G4: commercial vaccine + oral vaccine at 24 days. On the first day, the piglets were weighed and, from the third day onwards, submitted to blood collections for the detection and quantification of anti-Mycoplasma hyopneumoniae IgG. Nasal swabs were collected to monitor IgA by ELISA, and oropharyngeal swabs were used to assess the bacterial load by qPCR. Biological samples were collected periodically from the third day of life until the 73rd day. At 41 days of life, 15 individuals of the same age, experimentally challenged with an inoculum containing M. hyopneumoniae, were co-housed with the animals from groups (1 to 4) in a single pen to increase the infection pressure during the nursery period. At 73 days, all piglets were euthanized, and lungs were evaluated by collecting samples for estimation of bacterial load by qPCR. Quantitative data obtained from physical parameters and laboratory investigation were analyzed by performing parametric or non-parametric statistical tests. Results indicate that animals from G2 showed smaller affected lung areas compared to G3. Animals from G2 and G4 had a low prevalence of animals shedding M. hyopneumoniae at 61 days of age. Additionally, no correlation was observed between lung lesions and M. hyopneumoniae load in lung and BALF samples in animals that received the oral vaccine, while a strong correlation was observed in other groups. In the present study, evidence points to the effectiveness of the oral vaccine developed for controlling M. hyopneumoniae in pig production under field conditions.

Efficacy evaluation of a novel oral silica-based vaccine in inducing mucosal immunity against Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae, the main etiological agent of Porcine Enzootic Pneumonia, is widely spread in swine production worldwide. Its prevention is of great interest for the productive system, since its colonization in the lung tissue leads to intense production losses. This study aimed to compare the M. hyopneumoniae shedding and acute-phase response in 30 pigs submitted to different vaccination protocols: an experimental oral vaccine using a nanostructured mesoporous silica (SBA-15) as adjuvant (n = 10); an intramuscular commercially available vaccine at 24 days of age (n = 10); and a control group (n = 10) following experimental challenge with M. hyopneumoniae. Laryngeal and nasal swabs were collected weekly and oral fluids were collected at 7, 10, 14, 17, 23, 28, 35, 42, and 49 days post-infection to monitor pathogen excretion by qPCR. Nasal swabs were also used to detect anti-M. hyopneumoniae IgA by ELISA. Blood samples were collected for monitoring acute phase proteins. The antibody response was observed in both immunized groups seven days after vaccination, while the control group became positive for this immunoglobulin at 4 weeks after challenge. Lung lesion score was similar in the immunized groups, and lower than that observed in the control. SBA-15-adjuvanted oral vaccine provided immunological response, decreased shedding of M. hyopneumoniae and led to mucosal protection confirmed by the reduced pulmonary lesions. This study provides useful data for future development of vaccines against M. hyopneumoniae.

Use of nanostructured silica SBA-15 as an oral vaccine adjuvant to control mycoplasma hyopneumoniae in swine production

Mycoplasma hyopneumoniae is a difficult-to-control bacterium since commercial vaccines do not prevent colonization and excretion. The present study aimed to evaluate the performance of an orally administered vaccine composed of antigens extracted from Mycoplasma hyopneumoniae and incorporated into mesoporous silica (SBA-15), which has an adjuvant-carrier function, aiming to potentiate the action of the commercial intramuscular vaccine. A total of 60 piglets were divided into four groups (n = 15) submitted to different vaccination protocols as follows, Group 1: oral SBA15 + commercial vaccine at 24 days after weaning, G2: oral vaccine on the third day of life + vaccine commercial vaccine at 24 days, G3: commercial vaccine at 24 days, and G4: commercial vaccine + oral vaccine at 24 days. On the first day, the piglets were weighed and, from the third day onwards, submitted to blood collections for the detection and quantification of anti-Mycoplasma hyopneumoniae IgG. Nasal swabs were collected to monitor IgA by ELISA, and oropharyngeal swabs were used to assess the bacterial load by qPCR. Biological samples were collected periodically from the third day of life until the 73rd day. At 41 days of life, 15 individuals of the same age, experimentally challenged with an inoculum containing M. hyopneumoniae, were co-housed with the animals from groups (1 to 4) in a single pen to increase the infection pressure during the nursery period. At 73 days, all piglets were euthanized, and lungs were evaluated by collecting samples for estimation of bacterial load by qPCR. Quantitative data obtained from physical parameters and laboratory investigation were analyzed by performing parametric or non-parametric statistical tests. Results indicate that animals from G2 showed smaller affected lung areas compared to G3. Animals from G2 and G4 had a low prevalence of animals shedding M. hyopneumoniae at 61 days of age. Additionally, no correlation was observed between lung lesions and M. hyopneumoniae load in lung and BALF samples in animals that received the oral vaccine, while a strong correlation was observed in other groups. In the present study, evidence points to the effectiveness of the oral vaccine developed for controlling M. hyopneumoniae in pig production under field conditions.

Effect of testing protocol and within-pen prevalence on the detection of Mycoplasma hyopneumoniae DNA in oral fluid samples.

Combinations of 2 nucleic acid extractions and 3 Mycoplasma hyopneumoniae (MHP) PCRs (namely Protocol 1, 2, 3, and 4) were compared in terms of the probability of detecting DNA in pen-based oral fluid samples as a function of within-pen MHP prevalence. Oral fluid samples were created by randomly assigning 39 7-week old pigs to one of 5 pens, i.e., negative control pen (3 pigs) and 4 pens of 9 pigs each that differed in the proportion of MHP-inoculated pigs (1, 3, 6, or 9). Deep tracheal swabs were collected twice weekly to establish individual pig MHP infection status and derive within-pen prevalence estimation. On DPI 3, tracheal swabs from 15 of 19 inoculated pigs were MHP DNA positive. Oral fluids (n = 320) were collected daily from - 4 to 59 days post inoculation (DPI). Using a piecewise exponential model to account for within-pen transmission dynamics followed by a mixed-effect logistic regression, the probability of detecting MHP DNA in oral fluids was positively associated with within-pen prevalence (P < 0.0001) and differed among test protocols. MHP DNA was detected in 173 oral fluid samples with Protocol 3 versus 148, 134, and 101 with Protocols 4, 2, and 1, respectively. At 100% within-pen prevalence, the probability of detecting MHP DNA in oral fluids was highest using Protocol 3 (95.7%), followed by Protocols 4 (70.1%), 2 (60.1%), and 1 (34.0%). The fact that PCR protocols performed differently suggests that further improvements in extraction methods and MHP PCRs are possible. In the field, the dynamics of MHP infections should be taken into account if using oral fluid samples in surveillance.

Influence of Mycoplasma hyopneumoniae natural infection on the respiratory microbiome diversity of finishing pigs.

Mycoplasma (M.) hyopneumoniae interacts with the respiratory microbiota and facilitates colonization of other pathogens. The present study investigated the pulmonary and nasal microbiota of M. hyopneumoniae-infected and M. hyopneumoniae-free pigs. Sixty-six pigs from three commercial herds were selected at the end of the finishing phase: 44 originated from two M. hyopneumoniae-positive herds and 22 from a M. hyopneumoniae-negative farm. At the slaughterhouse, samples of nasal turbinate (NT) and bronchus-alveolar lavage fluid (BALF) were collected. DNA was extracted with a commercial kit and the infection status was confirmed by qPCR. All samples from the same herd were pooled, and next-generation sequencing based on the hypervariable region V3-V4 of the 16 s bacterial rDNA was performed. Data analysis included the taxonomic analysis, Alpha diversity indexes, and Principal coordinates analysis (Pcoa) using Jaccard, Bray-Curtis, Weighted Unifrac, and Unweighted Unifrac distances. All pigs from the infected herds tested PCR positive for M. hyopneumoniae, whereas all pigs from the negative farm were negative. There was a greater diversity of microorganisms in BALF when compared to NT samples in all the farms. BALF samples from infected animals showed higher abundance of M. hyopneumoniae than NT samples and a predominance of Pasteurella multocida among the main species identified, which was also abundant in the M. hyopneumoniae-free herd. PCoa diagrams indicated that for most of the samples, dissimilarity on bacterial composition was observed, regardless of infection status and sample type. Therefore, the lung microbiota was modulated by M. hyopneumoniae infection, which could play a role in the pathogenesis of M. hyopneumoniae-disease.

Cough associated with the detection of Mycoplasma hyopneumoniae DNA in clinical and environmental specimens under controlled conditions.

BACKGROUND: The association of cough with Mycoplasma hyopneumoniae (MHP) DNA detection in specimens was evaluated under conditions in which the MHP status of inoculated and contact-infected pen mates was closely monitored for 59 days post-inoculation (DPI). METHODS: Seven-week-old pigs (n = 39) were allocated to five rooms (with one pen). Rooms contained 9 pigs each, with 1, 3, 6, or 9 MHP-inoculated pigs, respectively, except Room 5 (three sham-inoculated pigs). Cough data (2 × week) and specimens, tracheal swabs (2 × week), oral fluids (daily), drinker wipes (~ 1 × week), and air samples (3 × week) were collected. At 59 DPI, pigs were euthanized, and lung and trachea were evaluated for gross and microscopic lesions. Predictive cough value to MHP DNA detection in drinker and oral fluid samples were estimated using mixed logistic regression. RESULTS: Following inoculation, MHP DNA was first detected in tracheal swabs from inoculated pigs (DPI 3), then oral fluids (DPI 8), air samples (DPI 10), and drinker wipes (21 DPI). MHP DNA was detected in oral fluids in 17 of 59 (Room 1) to 43 of 59 (Room 3) samples, drinker wipes in 4 of 8 (Rooms 2 and 3) to 5 of 8 (Rooms 1 and 4) samples, and air samples in 5 of 26 (Room 2) or 3 of 26 (Room 4) samples. Logistic regression showed that the frequency of coughing pigs in a pen was associated with the probability of MHP DNA detection in oral fluids (P < 0.01) and nearly associated with drinker wipes (P = 0.08). Pathology data revealed an association between the period when infection was first detected and the severity of gross lung lesions. CONCLUSIONS: Dry, non-productive coughs suggest the presence of MHP, but laboratory testing and MHP DNA detection is required for confirmation. Based on the data from this study, oral fluids and drinker wipes may provide a convenient alternative for MHP DNA detection at the pen level when cough is present. This information may help practitioners in specimen selection for MHP surveillance.

Oral vaccination of piglets against Mycoplasma hyopneumoniae using silica SBA-15 as an adjuvant effectively reduced consolidation lung lesions at slaughter.

Mycoplasma (M.) hyopneumoniae is the main pathogen of porcine enzootic pneumonia (PEP). Its controlling is challenging, and requires alternative strategies. This study aimed to develop an oral vaccine against M. hyopneumoniae using a nanostructured mesoporous silica (SBA-15) as an adjuvant, and compare its effect with an intramuscular (IM) commercial vaccine (CV). Fifty 24 day-old M. hyopneumoniae-free piglets composed five equal groups for different immunization protocols, consisting of a CV and/or oral immunization (OI). Control piglets did not receive any form of immunization. All piglets were challenged with M. hyopneumoniae strain 232 on D49 by tracheal route. IgA antibody response in the respiratory tract, bacterial shedding and serum IgG were evaluated. The piglets were euthanized on 28 (D77) and 56 (D105) days post-infection. Lung lesions were macroscopically evaluated; lung fragments and bronchoalveolar fluid (BALF) were collected for estimation of bacterial loads by qPCR and/or histopathology examination. All immunization protocols induced reduction on Mycoplasma-like macroscopic lung lesions. IgA Ab responses anti-M. hyopneumoniae, the expression of IL-4 cytokine and a lower expression of IL-8 were induced by CV and OI vaccines, while IgG was induced only by CV. Oral immunization using silica as a carrier-adjuvant can be viable in controlling M. hyopneumoniae infection.

Artificial insemination of gilts with bovine viral diarrhea virus-contaminated semen.

Bovine viral diarrhea virus (BVDV) is a pestivirus that infects swine and other species and has genetic and antigenic similarity to classical swine fever virus. The objective of this study was to mimic the infection of swine by contaminated semen and evaluate the effects on their reproductive tracts and litters. Six gilts were artificially inseminated with semen containing BVDV-2 ncp (LVB 16557/15) and 2 were inseminated with BVDV-free semen. Blood samples from all gilts were collected for polymerase chain reaction and virus neutralization tests. No viremia or neutralizing antibodies were detected, and all the litters were born healthy.

Insémination artificielle de cochettes avec du sperme contaminé par le virus de la diarrhée virale bovine. Le virus de la diarrhée virale bovine (BVDV) est un pestivirus qui infecte les porcs et d'autres espèces et qui présente des similitudes génétiques et antigéniques avec le virus de la peste porcine classique. L'objectif de ce travail était de reproduire expérimentalement l'infection des femelles porcines par insémination avec du sperme contaminé, d'évaluer les effets sur l'appareil reproducteur de la truie et sur sa portée. Six cochettes primipares ont été inséminées artificiellement avec du sperme contenant du BVDV-2 ncp (LVB 16557/15) et deux femelles ont été inséminées avec du sperme sans BVDV. Des échantillons de sang de toutes les femelles ont été prélevés pour des tests de réaction d'amplification en chaîne par la polymérase et de neutralisation virale. Aucune virémie ni aucun anticorps neutralisant n'ont été détectés et toutes les portées sont nées saines.(Traduit par les auteurs).

Oral vaccination of piglets against Mycoplasma hyopneumoniae using silica SBA-15 as an adjuvant effectively reduced consolidation lung lesions at slaughter

Mycoplasma (M.) hyopneumoniae is the main pathogen of porcine enzootic pneumonia (PEP). Its controlling is challenging, and requires alternative strategies. This study aimed to develop an oral vaccine against M. hyopneumoniae using a nanostructured mesoporous silica (SBA-15) as an adjuvant, and compare its effect with an intramuscular (IM) commercial vaccine (CV). Fifty 24 day-old M. hyopneumoniae-free piglets composed five equal groups for different immunization protocols, consisting of a CV and/or oral immunization (OI). Control piglets did not receive any form of immunization. All piglets were challenged with M. hyopneumoniae strain 232 on D49 by tracheal route. IgA antibody response in the respiratory tract, bacterial shedding and serum IgG were evaluated. The piglets were euthanized on 28 (D77) and 56 (D105) days post-infection. Lung lesions were macroscopically evaluated; lung fragments and bronchoalveolar fluid (BALF) were collected for estimation of bacterial loads by qPCR and/or histopathology examination. All immunization protocols induced reduction on Mycoplasma-like macroscopic lung lesions. IgA Ab responses anti-M. hyopneumoniae, the expression of IL-4 cytokine and a lower expression of IL-8 were induced by CV and OI vaccines, while IgG was induced only by CV. Oral immunization using silica as a carrier-adjuvant can be viable in controlling M. hyopneumoniae infection.

Cytokine expression and Mycoplasma hyopneumoniae burden in the development of lung lesions in experimentally inoculated pigs.

This study aimed to assess immunopathological factors and M. hyopneumoniae (M. hyo) load in macroscopic lesion formation at four timepoints after experimental infection of swine. To do this, 24 M. hyo-free pigs were divided into two groups: non-inoculated control (n = 8) and inoculated (n = 16). At day 0 post-infection (dpi), animals of infected group were intratracheally inoculated with 5 mL of lung inoculum containing 107 CCU (Color Changing Units) ∕mL of M. hyo strain 232, while control group was mock infected with 5 mL of sterilized Friis medium. At 14, 28, 42 and 56 dpi, four animals from the infected group and two from the control group were euthanized and necropsied. The extent of macroscopic lung lobe lesions was visually assessed, scored and lesion samples (qPCR, histopathology and gene expression) were collected. The macroscopic lesion score and estimated M. hyo load (in copies/µL) at the different timepoints were: 14 dpi: 18.5 %-1.55 × 103 copies∕µL; 28dpi: 15.8 %-8.4 × 103 copies∕µL; 42 dpi: 7.0 %-3.2 × 104 copies∕µL and 56 dpi: 6.3 %-1.11 × 105 copies∕µL; Significant and positive correlations between macroscopic lung lesion and the pathogen load were found (coefficient range: 0.77-0.99). The cytokine's IL-6 (0.73) and INF-γ (-0.69) gene expression were significantly (p < 0.05) correlated to macroscopic lung lesion score while IL-8, TNF- α, IL-1α and IL-1ß were associated to other pathological effects such as losses in average daily weight gain and microscopic lesion score. The results provide a better understanding about the pathogenicity of M. hyo strain 232 and the host-pathogen interactions, which may be helpful for the development of new treatments or control measures.

Congenital persistent infection with bovine viral diarrhea virus not observed in piglets.

This study was designed to determine whether congenital persistent infection occurs in piglets from gilts experimentally inoculated with bovine viral diarrhea virus type 2 (BVDV-2). Six pregnant gilts were divided into 2 groups, infected (n = 4), and control (n = 2). The gilts were inoculated at 45 days gestation. Piglets were assessed for 35 days following birth with nasal swab and blood sample collections every 72 hours. Reverse transcriptase-polymerase chain reaction (RT-PCR) tests were performed for direct diagnosis of virus in blood and nasal swabs, and virus neutralization was used for antibody detection. Transplacental transmission of BVDV-2 did not occur. Piglets were born free of the virus and did not shed BVDV during the experimental period.

Infection congénitale persistante par le virus de la diarrhée virale bovine non observée chez les porcelets. Cette étude a évalué l'occurrence d'infection congénitale persistante chez des porcelets nés de cochettes inoculées expérimentalement avec le virus BVDV-2. Six cochettes gestantes ont été divisées en deux groupes, infectées (n = 4) et témoins (n = 2). L'inoculation a eu lieu à 45 jours de gestation. Les porcelets ont été évalués pendant 35 jours par prélèvement d'écouvillons nasaux et d'échantillons de sang toutes les 72 heures. Des tests d'amplification en chaîne par la polymérase avec la transcriptase réverse (RT-PCR) ont été effectués pour le diagnostic direct dans le sang et les écouvillons, et la neutralisation virale pour l'évaluation sérologique. La transmission transplacentaire de BVDV-2 n'a pas été mise en évidence car les porcelets sont nés sans virus et n'ont pas éliminé le BVDV au cours de la période expérimentale.(Traduit par les auteurs).