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.

Determination of Mycoplasma hyopneumoniae-Specific IgG, IgG1, and IgG2a Titers in BALB/c Mice Induced by Mineral Oil-Based Oil-in-Water Emulsion Adjuvants Prepared Using a Self-Emulsifying Drug Delivery System.

We prepared mineral oil-based emulsion adjuvants by employing simple self-emulsifying drug delivery system (SEDDS). Mineral oil emulsions (3%, 5%, and 7%) were prepared using deionized water and C-971P NF and C-940 grade carbomer solutions with concentrations 0.01% (w/v) and 0.02% (w/v). In total, 15 emulsions were prepared and mixed with a solution containing inactivated Mycoplasma hyopneumoniae (J101 strain) antigen and porcine circovirus type 2 antigen to prepare vaccines. Droplet sizes in the submicron range and zeta potential values between - 40 and 0 mV were maintained by most emulsion adjuvants for a period of 6 months. Emulsion adjuvants were regarded safe, and their M. hyopneumoniae-specific IgG, IgG1, and IgG2a titers were either better or comparable to those of aluminum gel.

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.

Innate and adaptive immune responses of snatch-farrowed porcine-colostrum-deprived pigs to Mycoplasma hyopneumoniae vaccination.

BACKGROUND: The snatch-farrowed porcine-colostrum-deprived (SF-pCD) pig model, in which neonates are raised on commercially available bovine colostrum, is an alternative model for porcine infectious disease research. It is not known if SF-pCD pigs possess growth performance and immunity comparable to conventional, farm-raised pigs. The current experiment compared growth performance and immune responses of SF-pCD pigs to their farm-raised siblings following Mycoplasma hyopneumoniae (Mhyo) vaccination. Twelve SF-pCD and 13 farm-raised siblings were vaccinated on day 7 (D7) and D26 of age. Body weights were measured once or twice weekly and average daily gain (ADG) was calculated. Peripheral blood mononuclear cells (PBMC) were isolated on D40. Cytokine secretion from PBMC stimulated with Mhyo antigen or phorbol myristate acetate plus ionomycin (PMA/Iono) was assessed using a multiplexed fluorescent microsphere immunoassay (FMIA). Additionally, interferon gamma (IFNγ) secretion from stimulated PBMC was assessed using ELISPOT. Mhyo IgG titers were measured by an ELISA in D40 sera. RESULTS: Growth performance did not differ between groups before weaning, but SF-pCD pigs had higher ADG after weaning. In response to Mhyo stimulation, numbers of IFNγ secreting PBMC and levels of interleukin 8 (IL8) and IL10 in PBMC supernatants were significantly higher in SF-pCD pigs, as were Mhyo antibody levels in sera, and levels of IL1ß, IL8 and IL12 in supernatants of PMA/Iono stimulated PBMC. CONCLUSIONS: Under the conditions of this experiment, SF-pCD pigs demonstrated superior growth performance and enhanced humoral and cell-mediated immunity following vaccination. Whether or not this reflects greater resistance or tolerance to infection is unknown but the ability to react positively to the vaccination provides evidence that SF-pCD pigs are a suitable alternative model for swine disease research.

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.

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.

Mycoplasma hyopneumoniae bacterins and porcine circovirus type 2 (PCV2) infection: induction of postweaning multisystemic wasting syndrome (PMWS) in the gnotobiotic swine model of PCV2-associated disease.

Groups (5 to 15 per group) of gnotobiotic swine were infected oronasally with porcine circovirus type 2 (PCV2) at 3 days of age and then given 1 of 6 different commercial Mycoplasma hyopneumoniae (M. hyopneumoniae) bacterins as either a single dose (7 d of age, 1 application products) or 2 doses (7 and 21 d of age, 2 application product). Control groups received PCV2 alone (n = 9) or were infected with PCV2 and immunized twice with keyhole limpet hemocyanin (KLH) emulsified in incomplete Freund's adjuvant (ICFA) (n = 7). Five of 7 (71%) PCV2-infected piglets immunized with KLH/ICFA developed mild or overt PMWS, whereas none of 9 piglets infected with PCV2 alone developed PMWS. Five of 12 (42%) piglets vaccinated with a commercial bacterin containing mineral oil adjuvant developed PMWS following vaccination. None of the PCV2-infected piglets in the other bacterin-vaccinated groups developed PMWS in this model of PCV2-associated disease. This difference in prevalence of PMWS in piglets given the mineral oil-adjuvanted M. hyopneumoniae bacterin and the other M. hyopneumoniae bacterin vaccination groups was statistically significant (P < 0.05).

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.