Brazilian Clinical Strains of Actinobacillus pleuropneumoniae and Pasteurella multocida: Capsular Diversity, Antimicrobial Susceptibility (In Vitro) and Proof of Concept for Prevention of Natural Colonization by Multi-Doses Protocol of Tildipirosin.

One hundred Actinobacillus pleuropneumoniae (App) and sixty Pasteurella multocida subsp. multocida serogroup A (PmA) isolates were recovered from porcine pneumonic lungs collected from eight central or southern states of Brazil between 2014 and 2018 (App) or between 2017 and 2021 (PmA). A. pleuropneumoniae clinical isolates were typed by multiplex PCR and the most prevalent serovars were 8, 7 and 5 (43, 25% and 18%, respectively). In addition, three virulence genes were assessed in P. multocida isolates, all being positive to capA (PmA) and kmt1 genes, all negative to capD and toxA, and most of them (85%) negative to pfhA gene. The susceptibility of both pathogens to tildipirosin was investigated using a broth microdilution assay. The percentage of isolates susceptible to tildipirosin was 95% for App and 73.3% for PmA. The MIC50 values were 0.25 and 1 µg/mL and the MIC90 values were 4 and >64 µg/mL for App and PmA, respectively. Finally, a multiple-dose protocol of tildipirosin was tested in suckling piglets on a farm endemic for both pathogens. Tildipirosin was able to prevent the natural colonization of the tonsils by App and PmA and significantly (p < 0.0001) reduced the burden of Glaesserella parasuis in this tissue. In summary, our results demonstrate that: (i) tildipirosin can be included in the list of antibiotics to control outbreaks of lung disease caused by App regardless of the capsular type, and (ii) in the case of clinical strains of App and PmA that are sensitive to tildipirosin based on susceptibility testing, the use of this antibiotic in eradication programs for A. pleuropneumoniae and P. multocida can be strongly recommended.

Oncogenic viral DNA related to human breast cancer found on cattle milk and meat.

Bovine leukemia virus (BLV) is a major cause of lymphoma in cattle and has been recently correlated to breast cancer in humans. How and whether BLV might reach humans remains unknown but it could be through cattle-derived milk and meat. Here our aim was to investigate whether BLV DNA could be found in fresh milk and raw meat destined to human consumption and whether anti-BLV antibodies could be detected in human blood at the same geographical region. Milk (n = 36) and meat (n = 54) samples were collected from cows knowingly seropositive or negative to BLV and evaluated by nested PCR targeting BLV tax gene. Human serum samples (n = 900) were tested by ELISA to detect anti-BLV antibodies. BLV DNA was detected in 39 % of the milk samples and in 32 % of meat samples from BLV positive cows. Anti-BLV antibodies were found in 4.1 % of the human serum samples. Our data further supports the hypothesis that BLV might cause a zoonotic infection and indicate that milk and meat from BLV-infected cattle might be considered a potential source of infection to humans.

Development and validation of a flow cytometry antibody test for Lawsonia intracellularis.

Lawsonia intracellularis is the etiologic agent of porcine proliferative enteropathy (PPE), an inflammatory bowel disease with a major economic impact on the pig industry. The serological diagnosis of PPE can be performed using Blocking or Indirect ELISA, Immunoperoxidase Monolayer Assay (IPMA) and Indirect Fluorescence Antibody Test (IFAT). Here, we designed a most sophisticated immunological method for the detection of porcine anti-L. intracellularis IgGs, named Flow Cytometry Antibody Test - FCAT. This assay uses whole, live-attenuated L. intracellularis bacteria derived from a commercial vaccine. For the assay, we set up the optimal antigen concentration (106 bacterium/assay), primary antibody dilution (1:100), time of incubation (20 min), antigen stability (15 days), precision (coefficient of variation - CV < 10%), reproducibility (CV ≤ 13%) and Receiver Operating Characteristic (ROC). When using a cut-off of >15.15% for FCAT, we determined that it showed a sensitivity of 98.8% and specificity of 100%. The rate of agreement with IPMA was 84.09% with a kappa index of 0.66. FCAT was used to screen 1,000 sera from non-vaccinated pigs housed in 22 different farms and we found that 730 pigs (73%) from 16 farms (72.7%) had L. intracellularis IgG. This high prevalence confirms that L. intracellularis is endemic on Brazilian pig farms. Finally, we determined that FCAT is an easy to perform diagnostic assay and we would highly recommend it for: i) seroepidemiological studies; ii) evaluation of infection dynamics; and iii) characterization of the humoral response profile induced by vaccines.

Phylogenetic study and comparison of different TbpB obtained from Glaesserella parasuis present in Spanish clinical isolates.

Glaesserella parasuis (Gp) is the etiological agent of Glässer's disease (GD), which causes important economic losses for the pig intensive production worldwide. This organism uses a smart protein-based receptor to acquire specifically iron from the porcine transferrin. This surface receptor consists of transferrin-binding protein A (TbpA) and transferrin-binding protein B (TbpB). TbpB has been considered the most promising antigen to formulate a based-protein vaccine with broad-spectrum of protection against GD. The purpose of our study was to determine the capsular diversity of Gp clinical isolates collected in different Spanish regions between 2018 and 2021. A total of 68 Gp isolates were recovered from porcine respiratory or systemic samples. A species-specific PCR based on tbpA gene, followed by multiplex PCR for typing Gp isolates were performed. Serovars 5, 10, 2, 4 and 1 were the most prevalent and involved almost 84% of isolates. TbpB amino acid sequences from 59 of these isolates were analyzed, and a total of ten clades could be established. All of them showed a wide diversity with respect to capsular type, anatomical isolation site and geographical origin, with minor exceptions. Regardless of the serovars, the in silico analysis of TbpB sequences revealed that a vaccine based on a TbpB recombinant protein could potentially prevent Glässer's disease outbreaks in Spain.

TbpBY167A-based vaccine is safe in pregnant sows and induces high titers of maternal derived antibodies that reduce Glaesserella parasuis colonization in piglets.

Glässer's disease is one of the main diseases affecting young piglets, particularly during the nursery phase, that can significantly impact pork production. Vaccination of sows has the potential to prevent Glaesserella parasuis infection during the first weeks of life that is to a substantial degree due to the transfer of maternal derived antibodies (MDA) in colostrum. In this study we compare the antibody response to two vaccines administered to pregnant sows. A subunit vaccine containing the mutant transferrin-binding protein, TbpBY167A, and an autogenous vaccine formulated with the LM96/20 strain of G. parasuis (SV4) administered on days 65 and 86 of the gestational period were safe and induced high titers of antibodies in sows. The IgG peak was reached on day 100 of gestation, and the translocation of IgG to the mammary gland was confirmed in colostrum at the time of delivery. Piglets born from vaccinated sows maintained positive IgG titers against TbpBY167A or G. parasuis SV4 for the duration of the experiment (35 days of life). Piglets born from sows vaccinated with the TbpBY167A-based vaccine had a significantly (p = 0.001) lower load of G. parasuis in the respiratory tract compared to those born from sows vaccinated with the autogenous vaccine. Finally, we demonstrate that the LM96/20 (SV4) strain is highly virulent and a primary agent of Glässer's disease.

Molecular diagnosis of abdominal angiostrongyliasis by PCR using serum samples.

Abdominal angiostrongyliasis (AA) is a zoonotic disease caused by the nematode Angiostrongylus costaricensis, which is endemic in southern Brazil. Humans become infected by ingesting third-stage (L3) larvae and are considered accidental hosts since neither eggs nor first-stage (L1) larvae are found in feces. The definitive diagnosis can be made by histopathologic examination of surgical specimens or intestinal biopsies. The present study assessed the use of PCR to carry out the molecular detection of AA from serum samples. A total of 62 human serum samples were divided into three groups: (i) 28 serum samples from human patients with presumptive histopathological diagnosis of AA; (ii) 23 serum samples from individuals with unknown serology for AA; (iii) 11 serum samples from patients that suffered from different parasitosis were included. The serum samples were initially tested by in-house indirect ELISA and then by PCR. A total of 14 samples were positive by ELISA, and 6 were positive by PCR. Six samples that were negative by ELISA were positive by PCR. Amplicons were sequenced, and Angiostrongylus DNA was confirmed. We conclude that PCR amplification can be used to confirm Angiostrongylus DNA in serum, which is especially important in cases where antibody levels are too low to be detected. It may also serve as a useful target for survey studies.

Modulation of expression of proinflammatory genes and humoral immune response following immunization or infection with Aeromonas hydrophila in silver catfish (Rhamdia quelen).

The early immune-related events arising from the interaction of antigen and innate immune cells are central to modulating the acquired immune response. Ideally, the immunizing antigen should elicit immunological changes similar to that observed after infection with the wild type pathogen. Here, we evaluated early changes on the expression of selected proinflammatory genes (TNF-α, IL-1ß, IRAK4 and myeloperoxidase) and innate immune parameters (serum myeloperoxidase, lysozyme and complement hemolytic activity) in silver catfish vaccinated or infected with Aeromonas hydrophila, the etiological agent of hemorrhagic septicemia. The humoral immune response and resistance to challenge were also evaluated in vaccinated and placebo inoculated fish. We found that the expression of TNF-α and IL-1ß genes was higher (p<0.05) in vaccinated or infected fish at 24 h post inoculation (p.i) compared to the control group but returned to basal levels at 72 h p.i. The expression of IRAK4 gene, however, was not altered by vaccination or infection. In addition, the natural hemolytic activity of complement was higher (p<0.05) at 24 h and 72 h p.i. in the vaccinated and infected groups; serum myeloperoxidase was higher (p<0.05) in these groups but only at 24 h p.i. and lysozyme activity was higher (p<0.05) only in the infected group at 72 h p.i. Furthermore, vaccination induced the production of IgM-like antibodies and protection to challenge with the A. hydrophila. Our results indicate that the vaccine formulation induces an immune response similar to that induced by the infecting pathogen and might be a valuable tool in the prophylaxis of hemorrhagic septicemia in silver catfish.

TbpBY167A-Based Vaccine Can Protect Pigs against Glässer's Disease Triggered by Glaesserella parasuis SV7 Expressing TbpB Cluster I.

Glaesserella parasuis is the etiological agent of Glässer's disease (GD), one of the most important diseases afflicting pigs in the nursery phase. We analyzed the genetic and immunological properties of the TbpB protein naturally expressed by 27 different clinical isolates of G. parasuis that were typed as serovar 7 and isolated from pigs suffering from GD. All the strains were classified as virulent by LS-PCR. The phylogenetic analyses demonstrated high similarity within the amino acid sequence of TbpB from 24 clinical strains all belonging to cluster III of TbpB, as does the protective antigen TbpBY167A. Three G. parasuis isolates expressed cluster I TbpBs, indicating antigenic diversity within the SV7 group of G. parasuis. The antigenic analysis demonstrated the presence of common epitopes on all variants of the TbpB protein, which could be recognized by an in vitro analysis using pig IgG induced by a TbpBY167A-based vaccine. The proof of concept of the complete cross-protection between clusters I and III was performed in SPF pigs immunized with the TbpBY167A-based vaccine (cluster III) and challenged with G. parasuis SV7, strains LM 360.18 (cluster I). Additionally, pigs immunized with a whole-cell inactivated vaccine based on G. parasuis SV5 (Nagasaki strain) did not survive the challenge performed with SV7 (strain 360.18), demonstrating the absence of cross-protection between these two serovars. Based on these results, we propose that a properly formulated TbpBY167A-based vaccine may elicit a protective antibody response against all strains of G. parasuis SV7, despite TbpB antigenic diversity, and this might be extrapolated to other serovars. This result highlights the promising use of the TbpBY167A antigen in a future commercial vaccine for GD prevention.

Short-term intestinal effects of water intake in fibre supplementation in healthy, low-habitual fibre consumers: a phase 2 clinical trial.

A randomised clinical trial was conducted on 20 healthy, low-habitual fibre consumers to assess the short-term effects of water intake (2 l/day) on fibre supplementation with wheat bran, pectin, and green banana flour. During the 14-days trial, fibre intake doubled in both fibre (n = 10) and fibre/water (n = 10) interventions (p < 0.001), whereas daily water intake increased from 538 to 1990 ml in the fibre/water group (p < 0.001). Weekly bowel movements increased similarly in both interventions (fibre: 6.8-8.8; fibre/water: 8.6-10; p < 0.01), while faecal weight (71-126 g; p = 0.009) increased in the fibre/water group. This group showed higher counts of faecal Bacteroides and Prevotella, Faecalibacterium prausnitzii, and Bifidobacterium, whereas both interventions decreased the count of Desulfovibrio. Transient abdominal symptoms occurred less frequently in the fibre/water than in the fibre group (3 vs. 9 participants; p = 0.020). In healthy, low-habitual fibre consumers, short-term water intake helps the intestinal adaptation to fibre supplementation.CLINICAL TRIAL REGISTRATION NUMBER: NCT02838849.

Unusual high prevalence of antibodies to hepatitis E virus in South Brazil.

Hepatitis E virus (HEV) is worldwide distributed and might cause acute or chronic hepatitis mainly in immunocompromised individuals. In previous studies we found a high prevalence of antibodies to HEV within blood donors in south Brazil and also within backyard-raised pigs. Here, we aimed to investigate the prevalence of anti-HEV antibody and HEV RNA within the general population from three major municipalities (Caxias do Sul, Passo Fundo and Santa Maria) in south Brazil. A total of 3000 blood samples were randomly obtained from clinical laboratories at each of the three municipality (n = 1000 each) to determine the presence of anti-HEV antibodies and HEV RNA. Overall, anti-HEV antibodies were detected in 574/1000 (57,4%) samples in Caxias do Sul, 655/1000 (65.5%) samples in Passo Fundo and 554/1000 (55.4%) samples in Santa Maria. The prevalence of HEV-positive samples increased steadily and significantly (P < 0,001) with age and was unusually higher within individual over 40 years. Despite of this, none of the pooled serum samples had detectable levels of HEV RNA. The high anti-HEV antibody prevalence suggests that the virus might be present on the environment and/or foodstuff and poses a permanent threat to immune-compromised individuals.

Orally administered ß-glucan improves the hemolytic activity of the complement system in horses.

BACKGROUND AND AIM: Immune-modulating molecules mainly act on innate immune cells, which are central to early defense against invading pathogens and contribute to developing adaptive immunity. Yeast-extracted ß-glucan, a model immune-modulating molecule, is widely used in several animal species; however, its effect on horse immune parameters has not been thoroughly investigated yet. This study aimed to evaluate the effects of orally administered ß-glucan on selected innate immune parameters in horses. MATERIALS AND METHODS: Eighteen thoroughbred horses were assigned equally into three groups as follows: One control group (no ß-glucan) and two ß-glucan experimental groups (one received 125 mg and the other 2 g of ß-glucan per day for 28 days). Blood samples were collected before and at the end of the experiment for hematological analysis, whole blood phagocytosis, respiratory burst assays, and to assess the serum lysozyme and complement hemolytic activities. RESULTS: At the end of the experiment, significant decreases (p<0.05) in monocyte numbers were observed in the control horses (258.8±45.9 vs. 115.3±41.5) and in those fed 125 mg/day of ß-glucan (208.8±72.3 vs. 99.2±60.7), whereas a significant increase in numbers was noted in the horses that were fed 2 g/day of ß-glucan (303.5±45.8 vs. 429.8±86.0; p<0.05). The natural hemolytic activity of the complement was higher only in horses fed 2 g/day of ß-glucan (p=0.018) compared to the other groups. The hemolytic activity in the classical pathway was higher in those fed 125 mg/day (p=0.0035) and 2 g/day of ß-glucan (p=0.0001). CONCLUSION: ß-glucan improves important innate immune parameters and might be fed to horses before stressful events.

Proof of Concept for Prevention of Natural Colonization by Oral Needle-Free Administration of a Microparticle Vaccine.

There has been substantial interest in the development of needle-free vaccine administration that has led to a variety of approaches for delivery through the skin for induction of a systemic immune response. The mucosal administration of vaccines has inherently been needle-free, but the simple application of vaccines on the mucosal surface by itself does not lead to mucosal immunity. Since many important bacterial infections develop after initial colonization of the upper respiratory tract of the host, prevention of colonization could not only prevent infection but also eliminate the reservoir of pathogens that reside exclusively in that ecologic niche. This study was designed to provide proof of concept for a needle-free immunization approach that would reduce or eliminate colonization and prevent infection. In order to accomplish this a microparticle vaccine preparation was delivered just below the oral mucosal epithelial cell layer where it would lead to a robust immune response. A vaccine antigen (mutant transferrin binding protein B) shown to be capable of preventing infection in pigs was incorporated into a polyphosphazene microparticle preparation and delivered by a needle-free device to the oral sub-epithelial space of pigs. This vaccination regimen not only provided complete protection from infection after intranasal challenge by Glaesserella parasuis but also eliminated natural colonization by this bacterium. Notably, the complete prevention of natural colonization was dependent upon delivery of the microparticle preparation below the epithelial layer in the oral mucosa as intradermal or intramuscular delivery was not as effective at preventing natural colonization. This study also demonstrated that a primary immunization in the presence of maternal antibody limited the resulting antibody response but a robust antibody response after the second immunization indicated that maternal antibody did not prevent induction of B-cell memory.

Tildipirosin: An effective antibiotic against Glaesserella parasuis from an in vitro analysis.

Tildipirosin is a latest generation macrolide that is used to battle infection diseases caused by Gram-negative bacteria. Recent studies have shown the effectiveness of this antimicrobial agent against Actinobacillus pleuropneumoniae; however, little information is available about Glaesserella parasuis, the etiological agent of Glässer's disease. In this study, the Tildipirosin activity to 100 Brazilian clinical isolates of G. parasuis was assessed using a broth microdilution assay. A total of 90% of G. parasuis isolates were sensitive at concentrations ≤ 4 µg/mL Tildipirosin, thus showing to be efficiently controlled by the therapeutic concentration recommended for pigs. On the other hand, a total of ten isolates have shown resistance to this antibiotic, with a minimal inhibitory concentration (MIC) ≥ 8 and ≤ 16 µg/ml. Notably, our findings highly support the use of Tildipirosin for treating Glässer's disease outbreaks, and it also advises the using of MIC approach to monitor the evolution of sensitivity or resistance exhibited by G. parasuis to this molecule, as well as to adjust therapeutic doses when necessary.

New Pathological Lesions Developed in Pigs by a "Non-virulent" Strain of Glaesserella parasuis.

Glaesserella parasuis is a Gram-negative bacterium that causes Glässer's disease, a common pathology found in young pigs characterized by polyarthritis, polyserositis, and meningitis. The bacterium has 15 known serovars that have been classified by virulence. Serovars 1, 4, 5, and 12 are considered highly virulent and used in most studies. Serovars 3, 6, 7, 9, and 11 are considered avirulent. Recent reports that serovar 7 is an emerging problem in the pig industry indicate that the association of virulence and serovar may not always be reliable. This led us to infect colostrum-deprived piglets with the reference serovar 7 strain (SV7 strain 174) that had been passaged through pigs and characterize the clinical and pathological signs. We observed that SV7 strain 174 caused clinical signs consistent with Glässer's disease in all infected piglets that succumbed to infection for up to day 5 post-infection. Macroscopic and microscopic lesions were consistent with those found in piglets infected with conventional virulent serovars. In addition, we describe novel microscopic lesions associated with Glässer's disease such as endophthalmitis and thymic depletion. Thus, our findings indicate that SV7 strain 174 causes classical signs of Glässer's disease in colostrum-deprived piglets and some caution should be used in employing vaccine strategies based on association between capsular serovar and virulence.

Synthetic gene as target to assess the sensitivity of PCR to detect Trichinella spp. larvae in meat from a non-endemic region.

Trichinellosis is a zoonotic disease exotic in Brazil but commonly found worldwide including South American countries like Argentina. International trading of swine meat needs an official Trichinella-free diagnosis commonly carried out by pepsin-HCl digestion of diaphragm tissue fragments followed by microscopic examination for the presence or absence of Trichinella larvae. The easiness of this diagnostic method allows it to be performed at slaughtering plants but, in contrast, it lacks sensitivity and does not allow species differentiation, which is fundamental for determining geographical and species distribution of different genotypes. In our study, we aimed to evaluate a highly sensitive diagnostic method based on the polymerase chain reaction (PCR) that would allow us to detect and classify different species of Trichinella. Thus, we designed a synthetic gene and selected five sets of primers targeting specific regions of the Trichinella genome. The synthetic gene was cloned into a plasmid and then used to optimize PCR conditions. Using our PCR, we were able to detect 0.001 pg of the synthetic gene, which corresponded to 0.01 larvae. Then, we collected 175 samples of Suidae (domestic and wild boars) diaphragm fragments that were pooled into groups, digested with pepsin-HCl, and had the DNA extracted for analysis by PCR. The clinical samples evaluated were negative by PCR. Our results indicate that the PCR-based method might be a useful diagnostic method complementary to the pepsin-HCl digestion method currently in use, mostly in non-endemic areas.

ß-Glucan improves wound healing in silver catfish (Rhamdia quelen).

The immune modulating activity of ß-glucan on aquatic species has been a matter of intense investigation. Here, we aimed to investigate the effect of ß-glucan on wound healing of silver catfish, a Neotropical South American scale-free fish. Small sections of skin and muscle (3 mm in diameter) were removed and fish were bathed daily with ß-glucan (0.1% and 0.5%) up to 28 days when cicatrization was complete. A group of fish similarly injured and non-exposed to ß-glucan was used as control. Wound closure and healing was monitored visually and by histopathological analysis. In fish bathed with 0.5% ß-glucan we found reduced blood cortisol levels at day one post-wounding and, by day 7 post wounding, the deposition of granulation tissue was higher compared to non-exposed fish. In addition, from day 7 forward, wound size was significantly lower in fish bathed with 0.5% ß-glucan. Histopathological analysis of the wounded site indicated a thin layer of immature epidermal cells at day one post wounding. A discrete inflammation with mixed inflammatory cell infiltrate was observed on wounded muscle and was lower by day 7 post wounding on fish bathed with 0.5% ß-glucan. By day 14 post wounding, the deposition of collagen fibers and the presence of fibroblast and new muscle fibers were higher in fish exposed to 0.5% ß-glucan, and dermis restoration was complete. Thus, our results indicate that in silver catfish wound healing occurs rapidly and improves greatly by daily bathing with ß-glucan.

Molecular serotyping of clinical strains of Haemophilus (Glaesserella) parasuis brings new insights regarding Glässer's disease outbreaks in Brazil.

Glässer's disease (GD) is an important infectious disease of swine caused by Haemophilus (Glaesserella) parasuis. Vaccination with inactivated whole cell vaccines is the major approach for prevention of H. parasuis infection worldwide, but the immunity induced is predominantly against the specific polysaccharide capsule. As a consequence, the available vaccines may not induce adequate protection against the field strains, when the capsules present in the vaccine strains are different from those in strains isolated from the farms. Therefore, it is crucial to map H. parasuis serovars associated with regional outbreaks so that appropriate bacterin vaccines can be developed and distributed for prevention of infection. In this study, 459 H. parasuis field strains isolated from different Glässer's disease outbreaks that occurred in 10 different Brazilian States were analyzed for serotype using PCR-based approaches. Surprisingly, non-typeable (NT) strains were the second most prevalent group of field strains and along with serovars 4, 5 and 1 comprised more than 70% of the isolates. A PCR-based approach designed to amplify the entire polysaccharide capsule locus revealed 9 different band patterns in the NT strains, and 75% of the NT strains belonged to three clusters, suggesting that a number of new serovars are responsible for a substantial proportion of disease. These results indicate that commercially available vaccines in Brazil do not cover the most prevalent H. parasuis serovars associated with GD.

Bovine leukemia virus DNA associated with breast cancer in women from South Brazil.

Breast cancer is a neoplastic condition with a high morbidity and mortality amongst women worldwide. Recent data linking bovine leukemia virus (BLV) with breast cancer has been contested already. Our study investigated the presence of BLV genome in healthy (n = 72) and cancerous (n = 72) paraffin-embedded samples of breast tissues from women in south Brazil. BLV DNA was found most frequently (30.5%) in breast cancer tissue than in healthy breast (13.9%) (Odds ratio = 2.73; confidence interval = 1.18-6.29; p = 0.027). In contrast, antibodies to BLV were found in a very small percentage of healthy blood donors. There was no association between BLV DNA and other tumor prognostic biological markers such as hormonal receptors, HER2 oncoprotein, proliferation index, metastasis in sentinels lymph nodes, and tumor grade and size. Our findings suggest that BLV should be considered a potential predisposing factor to breast cancer in women.

Immunogenic characterization of vaccines based on Haemophilus parasuis Nagasaki strain, OmpP2, OmpP5 and OmpD15, in colostrum-deprived pigs experimentally challenged with the same strain.

Three recombinant outer membrane proteins (rOmps) from the Haemophilus parasuis Nagasaki strain (serovar 5 reference strain), rOmpP2, rOmpP5 and rOmpD15, which have previously shown protection against H. parasuis infection in mice, were cloned, expressed and evaluated as vaccine antigens in colostrum-deprived pigs. When these animals were immunized with these rOmps and were later challenged intratracheally with 108 CFUs of the Nagasaki strain, no protection was seen in terms of survival, clinical signs, pathological results and recovery of H. parasuis. We hypothesized that a possible explanation for this lack of protection could be the low number of epitopes accessible to the immune system as a consequence of their poor exposure on the bacterial surface so that the immune response would not be able to protect against experimental infection by H. parasuis when a fully susceptible animal model, such as pigs, was used.

Development of an indirect ELISA based on recombinant capsid protein to detect antibodies to bovine leukemia virus.

Serological testing and culling infected animals are key management practices aiming eradication of bovine leukemia virus infection. Here, we report the development of an indirect ELISA based on BLV recombinant capsid protein (BLVp24r) to detect anti-BLV antibodies in cattle serum. The BLVp24r was expressed in Escherichia coli and purified by affinity chromatography, and then used to set up the ELISA parameters. The Polysorp® plate coated with 50ng of antigen/well and bovine serum diluted 1:100 gave the best results during standardization. Using sera from infected and non-infected cattle we set up the cutoff point at 0.320 (OD450nm) with a sensitivity of 98.5% and specificity of 100.0%. Then, we tested 1.187 serum samples from dairy (736 samples) and beef cattle (451 samples) with unknown status to BLV. We found that 31.1% (229/736) and 9.5% (43/451) of samples amongst dairy and beef cattle, respectively, had IgGs to BLV. The rate of agreement with a commercial competitive ELISA was 84.3% with a κ value of 0.68. Thus, our BLVp24r iELISA is suitable to detect BLV infected animals and should be a useful tool to control BLV infection in cattle.