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.

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.

Aspergillus awamori-fermented mung bean seed coats enhance the antioxidant and immune responses of weaned pigs.

The potential benefits of Aspergillus-fermented mung bean seed coats (FMSC) for weaned pigs remain unexplored. Both in vitro and in vivo experiments were employed to evaluate the potential of FMSC supplement on the growth, antioxidant and immune responses of weaned pigs. The total polyphenols and DPPH scavenging capability of ethanol extract of FMSC exhibited a greater (p < 0.01) increase than those of pre-fermentation. With the addition of the polyphenol of FMSC extract, an increase in phagocytosis by neutrophils and proliferation of peripheral blood mononuclear cells (PBMC) were found. However, these observations were significantly inhibited (p < 0.05) in those activated cells. Next, 96 weaned pigs were allotted with a randomized complete block design into four dietary treatments, including 0 (control), 600, 1200 or 1800 mg/kg FMSC in a corn-soya bean meal basal diet for a 35-day trial. The pigs were injected with swine enzootic pneumonia (SEP) vaccines at day 3 and day 21 respectively. The results showed that dietary treatment failed to affect growth performance or serum SEP titre. The diet supplemented with 600-1800 mg/kg FMSC decreased faecal lactoferrin on day 21 and increased plasma trolox equivalent antioxidant capacity (TEAC) and erythrocytes catalase activity, as well as decreased (p < 0.01) plasma malondialdehyde (MDA) concentration on day 35. Diet supplementation of 1800 mg/kg FMSC increased phagocytosis by neutrophils and PBMC proliferation induced by pokeweed mitogen (PWM). However, the polymorphonuclear leucocytes (PMN)-positive respiratory burst cells were decreased in the supplementation of 1200 or 1800 mg/kg FMSC respectively. In addition, the serum haptoglobin concentration was decreased in the supplementation with 1200 mg/kg FMSC. Taken together, FMSC enriches polyphenols with antioxidative and immune modulated properties. After feeding FMSC, an improvement in antioxidative capability and immunocompetence was found, implying that FMSC could provide as a feed additive at optimal level 1200 mg/kg for weaned pigs.

Erysipelothrix rhusiopathiae and Mycoplasma hyopneumoniae: the sensitivities of enzyme-linked immunosorbent assays for detecting vaccinated sows of unknown disease status using serum and colostrum, and the correlation of the results for sow serum, colostrum, and piglet serum.

Due to relatively high concentrations of immunoglobulins, colostrum has the potential to improve the sensitivity of diagnostic tests for diseases in pigs when compared with serum. It is possible that colostrum could improve the sensitivity of the antibody enzyme-linked immunosorbent assay (ELISA) compared with serum. Colostrum is also essential for piglets, providing protection against infections in the first few weeks and months of life. The sensitivity of 2 commercially available ELISAs, one for the detection of Erysipelothrix rhusiopathiae and the second for Mycoplasma hyopneumoniae antibodies, when used with sow colostrum in comparison with serum was investigated. The correlation of maternal E. rhusiopathiae- and M. hyopneumoniae-specific antibody levels with specific-antibody serum levels in the piglet was also determined. The sensitivity was defined as the proportion of vaccinated sows that were correctly identified as vaccinated at a given cutoff point. The true disease status of the sows with regard to the 2 infections was unknown. Blood and colostrum samples were collected from 20 sows, 10 primiparous and 10 multiparous, and blood samples were also collected from the piglets of each sow, 48-72 hr post-farrowing. The sensitivities of both ELISAs were significantly improved when using colostrum compared with serum. Sow serum and colostrum optical density (OD) values were significantly correlated. The mean sow OD values for serum for E. rhusiopathiae and M. hyopneumoniae and colostrum for E. rhusiopathiae were significantly correlated with piglet serum OD levels. If the improved sensitivity of colostrum can be demonstrated in infected animals, this will increase the ability of the test to identify infected animals using both individual and pooled colostrum. Testing serum and/or colostrum using ELISA can be useful predictors of piglet disease-specific OD values.

Effects of dietary Centella asiatica (L.) Urban on growth performance, nutrient digestibility, blood composition in piglets vaccinated with Mycoplasma hyopneumoniae.

To investigate the effects of Centella asiatica (L.) on growth performance, nutrient digestibility and blood composition in piglets, 32 nursery pigs were fed 0.0, 0.5, 1.0 and 2.0% dietary C. asiatica (L.) from 15 to 90 kg BW. At 30 kg BW, nutrient digestibility was measured and at 35 kg BW piglets were vaccinated with Mycoplasma hyopneumoniae. Hematological parameters were checked at 40 and 80 kg BW. Compared with the control, growth performance was not affected. The ether extract, ash and calcium digestibility were lower at 0.5%, and dry matter, crude protein, crude fat, phosphorus and energy digestibility were lower at 1.0% (P<0.05). On hematological values, at 40 kg hematocrit, total white blood cells, neutrophils, eosinophils, basophils, monocytes and lymphocytes were higher at the 2.0% level (P<0.05). Most of these values except basophils and monocytes continued until at 80 kg, at which total white blood cells, neutrophils, eosinophils and lymphocytes were higher even at 1.0% (P<0.05); neutrophil-to-lymphocyte ratio tended to be higher at 2.0% (P<0.03). Cholesterol, triglycerides and antibody levels against M. hyopneumoniae did not differ except that at 40 kg the cholesterol of 0.5% was lower (P<0.05) and M. hyopneumoniae-specific antibodies tended to be higher with increasing levels of C. asiatica (L.) (P<0.07). The result that C. asiatica (L.) could not improve growth performance but increased values of serum hematocrit and white blood cells, and mycoplasma immunity to M. hyopneumoniae might suggest that C. asiatica (L.) has no function to elevate body weight but has the potential to enhance innate immunity.

Florfenicol feed supplemented decrease the clinical effects of Mycoplasma hyopneumoniae experimental infection in swine in México.

The therapeutic value of Florfenicol feed supplemented was evaluated in conventional pigs to eliminate consequences of chronic infection of Mycoplasma hyopneumoniae. The experimental animals were pigs with an average of 16 kg, after intratracheally inoculation with M. hyopneumoniae they were divided in two experimental groups: (a) the non-medicated; and (b) the feed supplemented group (20 g Florfenicol/ton of feed) during the ensuing 35 days. The average daily weight gain of the Florfenicol-treated pigs (0.33±0.14 kg/day) was significantly higher than that of the non-treated ones (0.21±0.10 kg/day). In medicated animals was still impaired relative to that of the uninfected ones control group (0.39±0.02 kg/day). The average percentage of pneumonic gross lesions extensions' of the pigs groups was: 13.99% for M. hyopneumoniae infected non-medicated group; 1.79% M. hyopneumoniae infected, Florfenicol-treated group and, 0.56% of the uninfected control group. M. hyopneumoniae; colonization was detected at these levels in 7 and 9 members of the respective infected groups. The extent of the pneumonic lesions and M. hyopneumoniae generally was greater in the non-medicated pigs. Therefore, oral administration of Florfenicol via feed ingestion seemed to be somewhat effective in ameliorating the clinical effects of M. hyopneumoniae infection of swine.

Effect of cross-fostering on transfer of maternal immunity to Mycoplasma hyopneumoniae to piglets.

The aim of this study was to assess the effect of cross-fostering on transfer of maternal Mycoplasma hyopneumoniae-specific humoral and cell-mediated immunity (CMI) from gilts to piglets. Cross-fostering, carried out within gilt pairs, was based on the gilts' M hyopneumoniae vaccination status in accordance with the following scheme: six pairs of vaccinated gilt × non-vaccinated gilt (V × N); five pairs of non-vaccinated gilt × vaccinated gilt (N × V); and five pairs of vaccinated gilt × vaccinated gilt (V × V). The piglets were cross-fostered at 0, six, 12 or 20 hours after birth. Two piglets per gilt per time point were cross-fostered (that is, eight piglets per gilt were moved) and the remaining piglets served as non-cross-fostered controls. In addition, four litters served as non-cross-fostered controls. A maximum of 10 piglets per gilt were sampled. The piglets' M hyopneumoniae-specific humoral immunity was assessed by ELISA and their CMI was assessed by delayed-type hypersensitivity testing. M hyopneumoniae-specific antibodies were detected in non-cross-fostered piglets from vaccinated dams and from piglets cross-fostered within the V × N gilt pair at six hours or more, and within the V × V gilt pair at all time points. Piglets cross-fostered within the N × V gilt pair had detectable M hyopneumoniae-specific antibodies only if they had been moved within six hours of birth. The transfer of M hyopneumoniae-specific CMI to piglets appeared to be source-dependent, and was detected only in piglets maintained on their vaccinated dams for at least 12 hours after birth.

Passive transfer of maternal Mycoplasma hyopneumoniae-specific cellular immunity to piglets.

Immunity in the neonatal animal is primarily maternally derived, either by lymphocytes that pass into the newborn across the placenta or following colostrum ingestion. However, the effect of this passively transferred cellular maternal immunity on the newborn's immune repertoire is not clearly understood. Various studies have shown that colostral lymphocytes are activated and possess functional abilities; however, no studies have shown the transfer of colostral antigen-specific T-cell-specific responses in a newborn. In this study we examined the transfer of vaccine-induced Mycoplasma hyopneumoniae cellular immunity from immune dams to newborn piglets. Newborn piglets from vaccinated and nonvaccinated dams were assessed in two ways for cellular immune responses specific to M. hyopneumoniae: (i) delayed-type hypersensitivity (DTH) testing and (ii) in vitro lymphocyte proliferation, assayed on piglet blood lymphocytes and sow colostral lymphocytes. DTH responses to M. hyopneumoniae were detected only for offspring of vaccinated sows, whereas DTH responses to the nonspecific mitogen phytohemagglutinin were seen for all piglets. M. hyopneumoniae-specific proliferation was seen for colostral lymphocytes from vaccinated sows and for blood lymphocytes from neonatal piglets of vaccinated dams but not for blood lymphocytes from piglets of nonvaccinated sows. Functional antigen-specific T cells were transferred to offspring from vaccinated sows and participated in the neonatal immune response upon stimulation. These data have implications for defining disease intervention strategies.

Antibody response to Mycoplasma hyopneumoniae infection in vaccinated pigs with or without maternal antibodies induced by sow vaccination.

Vaccination with bacterins is an important tool for the control of Mycoplasma hyopneumoniae infection of pigs. Because such vaccination often involves piglets that have suckled M. hyopneumoniae antibody-positive dams it is important to understand the effect of pre-existing (passively acquired) antibody on vaccine-induced immunity. To investigate this issue experimentally, 20 sows that were seronegative for M. hyopneumoniae were selected from a M. hyopneumoniae-infected herd and then randomly allocated to one of four treatment groups (five sows/group): Group A, vaccinated sows/vaccinated piglets; Group B, vaccinated sows/non-vaccinated piglets; Group C, non-vaccinated sows/vaccinated piglets; Group D, non-vaccinated sows/non-vaccinated piglets. Sows (Groups A and B) were vaccinated 14 days before farrowing and seroconverted within the next 14 days. Conversely, none of the non-vaccinated sows was seropositive at farrowing. Piglets (Groups A and C) were vaccinated when they were 7 days of age. Regardless of treatments none of the piglets had any evidence of an active immune response until many of those of Groups A and C and a few of those of Groups B and D seroconverted after it had been shown that at least some pigs of all groups had been naturally infected with a field strain of M. hyopneumoniae. This pattern of immune responsiveness (i.e. the collective results of Groups A, B, C and D) suggested that vaccination of pigs had primed their immune system for subsequent exposure to M. hyopneumoniae, and that passively acquired antibody had little or no effect on either a vaccine-induced priming or a subsequent anamnestic response. According to the statistical analysis sow serological status did not interfere with the antibody response in early vaccinated piglets. In conclusion, the results pointed out that early vaccination of piglets may assist M. hyopneumoniae control independently from the serological status of sows.

[Vaccination of sows against Mycoplasma hyopneumoniae with Hyoresp]. / Impfung von Sauen gegen Mycoplasma hyopneumoniae mit Hyoresp.

The aim of the study was to investigate the serological reactions of pregnant sows to vaccination with Hyoresp. Further investigations were performed in the offspring of these sows to follow the dynamics of maternal antibodies and the reaction to vaccination at different points in time. The study was conducted in three farrow-to-finish herds endemically infected with M. hyopneumoniae. A total of 30 gilts and 31 sows were vaccinated 8 and 4 weeks ante partum with Hyoresp (Merial GmbH) or given phys. saline solution as a placebo. The offspring was divided into three groups receiving Hyoresp at 1 and 4 or at 4 and 8 weeks of age. The control group was treated with phys. saline solution at 1 and 4 weeks of age. Before vaccination, antibodies against M. hyopneumoniae were detected in 85% of the gilts and 68% of the sows, confirming the endemic infection of the herds. Vaccination of the sows induced a significant increase in the antibody concentration in serum within four weeks and enhanced the concentration of antibodies in the colostrum. As expected, significantly enhanced levels of antibodies were also detected during the first four weeks of life of the offspring of vaccinated sows. The piglets' serological reaction to vaccination at 1 and 4 weeks of age showed marked interferences with maternal antibodies, so that a reaction could be demonstrated only at 8 weeks of age. The serological reaction of piglets vaccinated at 4 and 8 weeks of age was much stronger than that of piglets vaccinated earlier. Surprisingly, the vaccination status of the sow had no effect on the serological response of the piglets in either vaccination scheme. Maternal antibodies are known to reduce the risk of M. hyopneumoniae infections in piglets. Vaccinating the sows against M. hyopneumoniae may thus be an option for farrowing-to-finish herds with an enhanced risk for infections due to ineffective separation of different age groups, poor gilt acclimatisation or high gilt replacement rates.

Aspects of the transmission of protection against Mycoplasma hyopneumoniae from sow to offspring.

The aims of this study were to describe the variation in concentration of antibodies to Mycoplasma hyopneumoniae in the serum and colostrum of sows, and to compare the amount of antibodies in colostrum with that obtained in the serum of the smallest piglets in a litter. In addition, the efficacy of the passive immunity in natural conditions was studied. The study was performed in a sow pool herd (600 sows) that was endemically infected with M. hyopneumoniae. Blood samples were collected from sows 19 days (n = 25) before and 3 days (n = 15) after farrowing, and a colostrum sample (n = 25) was collected on the day of farrowing. All samples were analysed for antibodies to M. hyopneumoniae with a monoclonal blocking enzyme-linked immunosorbent assay (ELISA). Twelve sows (48%) were high-responders with respect to antibody concentration in colostrum. The amount of blocking decreased in serum during the last weeks of pregnancy and 3 days post-farrowing it was only 53% of the level found in colostrum. At the age of 14 days, 30 of the smallest piglets were weaned. They were divided into three experimental groups, being the offspring of high-responding sows, low-responding sows, or a mix of high- and low-responding sows. The groups were transported to three separated isolation units and were followed until slaughter. At slaughter, lung lesions were not found. Nor could M. hyopneumoniae be demonstrated either by cultivation or by polymerase chain reaction. However, a significant increase in absorbance values, assessed by an indirect-ELISA, was demonstrated in groups established from low-responding sows. It was concluded that a high antibody level in colostrum appeared to protect piglets from M. hyopneumoniae.