A protein-based subunit vaccine with biological adjuvants provides effective protection against Pasteurella multocida in pigs.

Streptococcus suis (S. suis) and Pasteurella multocida (P. multocida) are pathogens that can cause zoonotic diseases. P. multocida toxin (PMT) is an important virulence factor that causes atrophic rhinitis in pigs. Suilysin (Sly) is an extracellular protein of S. suis and has been shown to be a potential adjuvant. Previous studies have indicated that subunit vaccines containing several fragments of PMT as antigens are safer than traditional inactivated or live-attenuated vaccines. However, protein-based vaccines need strong adjuvants to enhance their immunogenicity. In this study, recombinant PMT-NC (rPMT-NC) protein antigen was formulated with either recombinant Sly (rSly) or CpG oligodeoxynucleotides (CpG) as the adjuvant. The immune responses elicited by these vaccines and the protective efficacy after challenge with live P. multocida were evaluated in piglets. In the dose-dependent test, piglets immunized with the low dose (100 µg) of rSly had increased antigen-specific total IgG, interferon (IFN)-γ gene expression, and CD4+ and CD8+ T-cell populations. Compared to piglets in the commercial (Al-gel) adjuvant and the control groups (p < 0.05), piglets in the biological adjuvant groups showed significantly reduced turbinate atrophy, nasal distortion, and lung lesion scores after challenge with P. multocida serotype A. Vaccines containing rSly or CpG adjuvant enhanced humoral and cellular immune responses and protection against P. multocida. This combination of a protein-based antigen formulated with a biological adjuvant showed synergistic and protective effects against atrophic rhinitis and has potential to be developed as part of a bivalent vaccine.

Effects of Aspergillus-meal prebiotic diet on the growth performance, health status and gut microbiota of Asian seabass, Lates calcarifer.

In this study, the growth performance, health status and intestinal microbiota of juvenile Asian seabass, Lates calcarifer, were assessed after dietary administration of a prebiotic product obtained from fermented Aspergillus orizae, Fermacto®. Asian seabass were fed three diets; control (without Aspergillus-meal prebiotic), 0.2% and 0.3% Aspergillus-meal prebiotic for 56 days. Fish were raised in freshwater with acceptable water quality. No significant differences were found in the growth performance and composition of dorsal fish muscle among all groups. Fish fed diets supplemented with 0.3% of Aspergillus-meal prebiotic had a significantly higher survival rate after being challenged with V. alginolyticus than fish fed with the control diet. Supplementation of the Aspergillus-meal prebiotic significantly improved immune responses by inducing higher respiratory burst, superoxide dismutase, phagocytic and lysozyme activity compared to the control group. In addition, prebiotic doses significantly induced an up-regulation of heat shock cognate 70 kDa protein (hsp70) in the liver compared to the control group. Signaling pathways were also affected with significantly higher gene expression of complement c-3 (c3), mechanistic target of rapamycin (mtor), and mammalian lethal with SEC13 protein 8 (mlst-8) in the liver of fish fed 0.3% Aspergillus prebiotic. The pro-inflammatory gene, tumor necrosis factor (tnf) and anti-inflammatory gene, transforming growth factor beta-1 (tfg-ß1) were significantly higher in the head kidney of fish offered prebiotic diets. Fish receiving Aspergillus-meal prebiotic revealed significantly higher expression of Mx gene 24 h post nervous necrosis virus injection compared to the control. Additionally, the α-diversity of gut microbiota, including genus, Pielou's evenness, Shannon diversity index, and Margalef's species richness were significantly higher in fish fed 0.3% Aspergillus-meal prebiotic than the control group. The principal component analysis eigenvector plots showed that a high abundance of beneficial bacteria, such as Entercoccus faecium, Lactococcus lactis, Macrococcus caseolyticus and Vagococcus fluvialis, along with potentially pathogenic bacteria, such as Staphylococcus sciuri and L. garvieae subsp. garvieae were present in fish treated with Aspergillus-meal prebiotic. Although dietary Aspergillus-meal prebiotic did not improve the growth performance of Asian seabass, 0.3% of Aspergillus-meal prebiotic is recommended to elevate the immunological status of fish.

Dietary administration of a postbiotic, heat-killed Pediococcus pentosaceus PP4012 enhances growth performance, immune response and modulates intestinal microbiota of white shrimp, Penaeus vannamei.

The efficacy of postbiotics on the immune-related gene expression and gut microbiota of white shrimp, Penaeus vannamei remains unexplored. A commercial heat-killed postbiotic Pediococcus pentosaceus PP4012 was used to evaluate the growth performance, intestinal morphology, immunological status, and microbial community of white shrimp after dietary administration in this study. White shrimp (0.040 ± 0.003 g) were divided into three treatments; a control, inanimate P. pentosaceus (105 CFU g feed-1) at low concentration (IPL) and inanimate P. pentosaceus (106 CFU g feed-1) at high concentrations (IPH). The diets of IPL and IPH significantly increased final weight, specific growth rate and production compared to the control group. Shrimp fed with IPL and IPH significantly utilized feed more efficiently than those fed the control diet. The IPH treatment significantly lowered the cumulative mortality rate compared to the control and IPL diet following Vibrio parahaemolyticus infection. No significant difference was observed for Vibrio-like and lactic acid bacteria in intestine of shrimp fed with the control diet and the experimental diets. Adding inanimate P. pentosaceus significantly improved immune responses such as lysozyme and phagocytic activity compared to the control group. However, the total hemocyte count, phenoloxidase activity, respiratory burst, and superoxide dismutase activity were not significantly different among treatments. The immune-related genes alf, pen3a, and pen4 expression were significantly higher in shrimp fed IPL diet compared with control and IPH. Taxonomic identification of bacterial genera in all dietary groups belonged to two predominant phyla, Proteobacteria and Bacteroidota. An abundance of Photobacterium, Motilimonas, Litorilituus, and Firmicutes bacterium ZOR0006 were identified in the intestine of shrimp fed postbiotic diets. Unique microbes such as Cohaesibacter was discovered in the shrimp fed IPL while Candidatus Campbellbacteria, uncultured Verrucomicrobium DEV114 and Paenalcaligenes were discovered in the intestines of shrimp fed IPH diet. Collectively, these data suggest that including heat-killed P. pentosaceus, particularly IPH, can enhance growth performance, promote microbial diversity, elevate immune responses, and increase shrimp's resistance to V. parahaemolyticus.

First identification and histopathological analysis of Lactococcus garvieae infection in whiteleg shrimp, Penaeus vannamei cultured in low salinity water.

The isolation and characterization of bacterial species Lactococcus garvieae, previously unreported in whiteleg shrimp, Penaeus vannamei, has now been identified in the species. The pathogen was recovered from an affected shrimp farm in southern Taiwan. Bacterial characterization first identified the isolate as Gram-positive cocci, and biochemical profiles demonstrated that the causative agent of mortality was 97% L. garvieae. The bacterial cell DNA resulted in amplification of 1522 bp with 99.6% confirmation by PCR analysis. The phylogenetic tree revealed 100% evolutionary similarity among previously isolated strains. Experimental infection further confirmed higher susceptibility of whiteleg shrimp to L. garvieae in waters of lower salinity, particularly 5 ppt, than in higher salinity. Histopathological analysis showed severely damaged hepatopancreas with necrotized, elongated, collapsed tubules, dislodged membranes and granuloma formation in infected shrimp. Transmission electron microscopy observation indicated a hyaluronic acid capsular layer surrounding bacterial cell which is a virulence factor of L. garvieae and likely responsible for immunosuppression and higher mortality of shrimp cultured in lower salinity. Collectively, these findings report the first isolation of L. garvieae from whiteleg shrimp and shed new light on the disease that threatens the highly valuable species and accentuates the need for finding a solution.

Dietary SYNSEA probiotic improves the growth of white shrimp, Litopenaeus vannamei and reduces the risk of Vibrio infection via improving immunity and intestinal microbiota of shrimp.

The growth performance, immunological status, and intestinal microbiology of white shrimp, Litopenaeus vannamei, were evaluated after dietary administration of the commercial probiotic SYNSEA. Shrimp were fed a control diet (without probiotic supplement) and two levels of SYNSEA probiotic, a low concentration of SYNSEA (LSL) containing 105 CFU (g diet)-1Bacillus subtilis and 105 CFU (g diet)-1 lactic acid bacteria (LAB), and a high concentration of SYNSEA (LSH) containing 106 CFU (g diet)-1B. subtilis and 106 CFU (g diet)-1 LAB, for 12 weeks. Shrimp fed with the LSL diet significantly increased growth performance as well as final weight and feed efficiency compared to the control, but not the LSH diet. After being orally challenged with Vibrio parahaemolyticus, shrimp fed with LSL diet prior to the challenge or fed with LSL and pathogen simultaneously showed significantly lower mortality compared to the control. SYNSEA probiotic significantly improved shrimp immune response, including lysozyme activity in LSL and LSH groups, and phagocytic activity in the LSL group in comparison to the control. In addition, the gene expressions of anti-lipopolysaccharide factor 2 in LSL and LSH groups, and penaeidin 4 in LSL were also up-regulated. Although there was no significant difference among groups for hepatopancreas and intestinal morphology, the muscular layer thickness and villi height were slightly improved in the intestines of shrimp fed SYNSEA. The 16S rDNA gene amplicon sequence analysis using next-generation sequencing revealed a significant decrease in α-diversity (Margalef's species richness) after oral administration of SYNSEA due to an increase in the relative abundance of beneficial bacteria in the gut flora of shrimp, such as Lactobacillus, Shewanella, and Bradymonadales and a decrease in harmful bacteria, such as Vibrio, Candidatus_Berkiella, and Acinetobacter baumannii. Together the data suggest that the provision of SYNSEA probiotic at 105 CFU (g diet)-1B. subtilis and 105 CFU (g diet)-1 LAB can improve shrimp growth, enhance immunity, and disease resistance status of the host. In addition, these findings conclude that SYNSEA probiotic has great preventive and therapeutic potential for Vibrio infection in shrimp aquaculture.

Combined Treatment with Cryptocaryone and Ultraviolet C Promotes Antiproliferation and Apoptosis of Oral Cancer Cells.

Cryptocaryone (CPC) was previously reported as preferential for killing natural products in oral cancer cells. However, its radiosensitizing potential combined with ultraviolet C (UVC) cell killing of oral cancer cells remains unclear. This study evaluates the combined anti-proliferation effect and clarifies the mechanism of combined UVC/CPC effects on oral cancer cells. UVC/CPC shows higher anti-proliferation than individual and control treatments in a low cytotoxic environment on normal oral cells. Mechanistically, combined UVC/CPC generates high levels of reactive oxygen species and induces mitochondrial dysfunction by generating mitochondrial superoxide, increasing mitochondrial mass and causing the potential destruction of the mitochondrial membrane compared to individual treatments. Moreover, combined UVC/CPC causes higher G2/M arrest and triggers apoptosis, with greater evidence of cell cycle disturbance, annexin V, pancaspase, caspases 3/7 expression or activity in oral cancer cells than individual treatments. Western blotting further indicates that UVC/CPC induces overexpression for cleaved types of poly (ADP-ribose) polymerase and caspase 3 more than individual treatments. Additionally, UVC/CPC highly induces γH2AX and 8-hydroxy-2'-deoxyguanosine adducts as DNA damage in oral cancer cells. Taken together, CPC shows a radiosensitizing anti-proliferation effect on UVC irradiated oral cancer cells with combined effects through oxidative stress, apoptosis and DNA damage.

Immunization with Streptococcus suis bacterin plus recombinant Sao protein in sows conveys passive immunity to their piglets.

BACKGROUND: Streptococcus suis (S. suis) causes arthritis, meningitis, septicemia, and sudden death in pigs and is also an zoonotic agent for humans. The present study demonstrated that immunization with recombinant Sao-L (surface antigen one-L, rSao-L) protein from a strain of S. suis serotype 2 in pigs was able to increase cross-serotype protection against S. suis serotype 1 and 2 challenge. Since weaning piglets are more susceptible to S. suis infections due to the stresses associated with weaning, prepartum immunization in sows may convey passive immunity to piglets and provide protection. RESULTS: Pregnant sows were immunized with a vaccine containing inactivated S. suis serotype 2 plus rSao as the antigens. Blood samples were collected from their piglets after birth for analysis of antigen-specific antibody titers and levels of various cytokines. Results demonstrated that the titers of S. suis and rSao-specific antibodies were significantly (p < 0.05) higher in the vaccinated piglets in comparison with that of piglets in the control group. The serum levels of interferon (IFN)-γ, interleukin (IL)-4, IL-6, and IL-12 were significantly (p < 0.05) increased in piglets born from vaccinated sows when compared to piglets from unvaccinated sows. In addition, piglets were challenged by heterologous and homologous S. suis. All piglets from unvaccinated sows developed severe symptoms of bacteremia, fever, anorexia, depression, and arthritis. On the other hand, piglets from vaccinated sows had significantly (p < 0.05) reduced clinical symptoms and lesion score (by 75 and 81%). CONCLUSIONS: Our results revealed that immunizing pregnant sows with the vaccine containing inactivated S. suis bacterin plus rSao as the antigens is able to enhance passive immunity against heterologous and homologous S. suis challenge in their piglets.

Ear fibroblasts derived from Taiwan yellow cattle are more heat resistant than those from Holstein cattle.

The objective of this study was to compare the thermotolerances of ear fibroblasts derived from Holstein (H) and Taiwan yellow cattle (Y) and their apoptosis-related protein expressions with (1, 3, 6, 12, and 24h) or without heat shock treatment. The results showed that the vaginal temperatures of Y (38.4-38.5°C) were (P<0.05) lower than that of H (38.8°C) during the hot season. The apoptotic rates of ear fibroblasts derived from Y (6h: 1.1%; 12h: 1.6%; 24h: 2.6%) were lower (P<0.05) than those of cells derived from H (6h: 1.8%; 12h: 4.0%; 24h: 6.9%), respectively, after heat shock (42°C). The expression level of apoptosis inducing factor (AIF) in ear fibroblasts derived from H was higher (P<0.05) than those derived from Y after the heat shock treatment for 6h and 12h, respectively. The level of cytochrome c of ear fibroblasts derived from H was higher (P<0.05) than those derived from Y after the heat shock treatment for 1-12h, respectively. The abundances of Caspase-3, Caspase-8 and Caspase-9 of ear fibroblasts derived from H were higher (P<0.05) than those of cells derived from Y after 12h and 24h of heat shock, respectively; the Bcl-2/Bax ratios of ear fibroblasts derived from H were lower (P<0.05) than those from Y-derived fibroblasts after heated for 1-24h. The expression level of HSP-70 of Y-derived ear fibroblasts was also higher (P<0.05) than that from H after the same duration of heat shock treatments. Taken together, the thermotolerance of ear fibroblasts derived from Taiwan yellow cattle was better than that of cells derived from Holstein cattle.

Identification and expression analysis of cobia (Rachycentron canadum) Toll-like receptor 9 gene.

Cobia culture is hindered by bacterial infection (Photobacterium damselae subsp. piscicida) and in order to study the effect of P. damselae subsp. piscicida challenge and CpG ODN stimulation on cobia Toll like receptor 9 (RCTLR9), we used PCR to clone RCTLR9 gene and qRT-PCR to quantify gene expression. The results indicated that RCTLR9 cDNA contains 3141 bp. It encodes 1047 amino acids containing 16 typical structures of leucine-rich repeats (LRRs) including an LRRTYP, LRRCT and a motif involved in PAMP binding was identified at position 240-253 amino acid. Broad expression of RCTLR9 was found in larval, juvenile and adult stages irrespective of the tissues. In larval stage, RCTLR9 mRNA expression decreased at 5 d and then increased at 10 dph. At juvenile stage cobia, the expression was significantly high (p < 0.05) in spleen and intestine compared to gill, kidney, liver and skin. However, at adult stage, the significant high expression was found in gill and intestine. Cobia challenged with P. damselae subsp. piscicida showed significant increase in RCTLR9 expression at 24 h post challenge in intestine, spleen and liver, while in kidney the expression was peak at 12 h and later it decreased at 24 h. The highest expression was 40 fold increase in spleen and the lowest expression was ∼3.6 fold increase in liver. Cobia stimulated with CpG oligonucleotides showed that the induction of these genes was CpG ODN type and time dependent. In spleen and liver, CpG ODNs 1668 and 2006 injected group showed high expression of RCTLR9, IL-1ß, chemokine CC compared to other groups. Meanwhile, CpG ODN 2006 has induced high expression of IgM. The CpG ODNs 2395 have induced significant high expression of Mx in spleen and liver. These results demonstrates the potential of using CpG ODN to enhance cobia resistance to P. damselae subsp. piscicida infection and use as an adjuvant in vaccine development.

Identification of Molecular Profile of Ear Fibroblasts Derived from Spindle-Transferred Holstein Cattle with Ooplasts from Taiwan Yellow Cattle under Heat Stress.

Global warming has a significant impact on the dairy farming industry, as heat stress causes reproductive endocrine imbalances and leads to substantial economic losses, particularly in tropical-subtropical regions. The Holstein breed, which is widely used for dairy production, is highly susceptible to heat stress, resulting in a dramatic reduction in milk production during hot seasons. However, previous studies have shown that cells of cows produced from reconstructed embryos containing cytoplasm (o) from Taiwan yellow cattle (Y) have improved thermotolerance despite their nuclei (n) being derived from heat-sensitive Holstein cattle (H). Using spindle transfer (ST) technology, we successfully produced ST-Yo-Hn cattle and proved that the thermotolerance of their ear fibroblasts is similar to that of Y and significantly better than that of H (p < 0.05). Despite these findings, the genes and molecules responsible for the different sensitivities of cells derived from ST-Yo-Hn and H cattle have not been extensively investigated. In the present study, ear fibroblasts from ST-Yo-Hn and H cattle were isolated, and differentially expressed protein and gene profiles were compared with or without heat stress (hs) (42 °C for 12 h). The results revealed that the relative protein expression levels of pro-apoptotic factors, including Caspase-3, -8, and -9, in the ear fibroblasts from the ST-Yo-Hn-hs group were significantly lower (p < 0.05) than those from the H-hs group. Conversely, the relative expression levels of anti-apoptotic factors, including GNA14 protein and the CRELD2 and PRKCQ genes, were significantly higher (p < 0.05) in the ear fibroblasts from the ST-Yo-Hn-hs group compared to those from the H-hs group. Analysis of oxidative phosphorylation-related factors revealed that the relative expression levels of the GPX1 gene and Complex-I, Complex-IV, CAT, and PGLS proteins were significantly higher (p < 0.05) in the ear fibroblasts from the ST-Yo-Hn-hs group compared to those from the H-hs group. Taken together, these findings suggest that ear fibroblasts from ST-Yo-Hn cattle have superior thermotolerance compared to those from H cattle due to their lower expression of pro-apoptotic factors and higher expression of oxidative phosphorylation and antioxidant factors. Moreover, this improved thermotolerance is attributed, at least partially, to the cytoplasm derived from more heat-tolerant Y cattle. Hence, using ST technology to produce more heat-tolerant H cattle containing Y cytoplasm could be a feasible approach to alleviate the negative impacts of heat stress on dairy cattle in tropical-subtropical regions.

Ultrasonicated Enterococcus faecium SF68 enhances neutrophil free radical production and udder innate immunity of drying-off dairy cows.

Proper dry cow management is critical not only for subsequent milk production and fertility but also for mastitis control. A phenomenon of immunosuppression was commonly observed in transition cows, an example being the high susceptibility of the mammary gland during early the dry period to new infectious agents. Polymorphonuclear neutrophils (PMN) play important defence roles in the mammary gland of newly dried cows. One of the bactericidal mechanisms of PMN is through producing reactive oxygen species (ROS), which can be efficiently quantified by chemiluminescence (CL) assay. In the current study, the potential of intramammary application of a commercial Enterococcus faecium SF68 (SF68) product to enhance the local innate immunity of newly dried mammary glands was evaluated based on the CL assay. The preliminary experiments in vitro indicated virtual dose-responsiveness of ROS generation from three different cell preparations, bovine blood PMN, bovine blood PMN pre-conditioned with cow milk, and the post-diapedesis model somatic cells from cow milk, on their exposure to phorbol 12-myristate 13-acetate (PMA), viable SF68, and ultrasonicated SF68, but not dry-heated SF68. Because ultrasonication treatment was found to profoundly enhance the immunogenicity of SF68 in vitro, in the following animal trial, single infusion of either 5 or 10×107 original cfu of ultrasonicated SF68 was randomly applied to the front quarters and phosphate-bufferedsaline (PBS) applied to the rear quarters of each of the four experimental cows on the first day of milk stasis. The results showed that within the first post-infusion week, ultrasonicated SF68 induced a faster and greater (P<0·05) recruitment of PMN into mammary lumen with no apparent local or systemic inflammatory sign. Meanwhile, ultrasonicated SF68 also induced a greater (P<0·05) ROS production in response to PMA challenge by in situ somatic cells of mammary secretion. Taken together, ultrasonicated SF68 modulated ROS generation of bovine neutrophils, and would be a potential enhancer of udder innate immunity in drying-off dairy cows. More thorough work is warranted.

The influence of subclinical mastitis on the protein composition and protease activities of raw milk from lactating Thai-crossbred dairy cows.

Background and Aim: Mastitis in dairy cattle is associated with a high rate of morbidity and death, which has major implications for milk production and quality. This study aimed to investigate the protein component and the activity of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9) in raw milk samples with different testing scores determined using the California mastitis test (CMT). Materials and Methods: Thirty cows were employed in the study, and milk from each quarter was tested for subclinical mastitis (SCM). According to the results of CMT, raw milk samples were classified into five categories: Healthy (score 0), trace (score T), weakly positive (score 1), distinctly positive (score 2), and strongly positive (score 3) for somatic cell count (SCC). The total milk protein was analyzed using the Bio-Rad protein assay, and the milk protein composition was determined using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique. In addition, gelatin zymography was used to evaluate changes in proteolytic abilities. Results: Milk samples with CMT scores of 1 and 3 had the highest total milk protein levels (32.25 ± 12.60 g/L and 32.50 ± 7.67 g/L, respectively), while the samples from healthy cows (CMT score 0) were only 6.75 ± 1.64 g/L. Globulin and lactoferrin were significantly increased in samples with a CMT score of 3 compared with those with other CMT scores. The bovine serum albumin level in samples with a CMT score of 2 was significantly (p < 0.05) higher than those with other CMT scores. No significant differences in casein abundance were found among samples with different CMT scores. Results from analysis of proteolytic activities demonstrated that the level of MMP-9 in samples with a CMT score of 3 was significantly (p < 0.05) higher than those with other CMT scores. Conclusion: The protein content and gelatinolytic activity of milk were drastically altered by the number of SCC, mainly due to SCM.

Effects of a novel recombinant Gonadotropin-Releasing Hormone-1 vaccine on the reproductive function of mixed-breed dogs (Canis familiaris) in Taiwan.

Immunocastration is an effective alternative to surgical castration for controlling the population of animals. As gonadotropin-releasing hormone (GnRH) regulates the reproductive endocrine system in mammals, it is a target antigen for vaccine formulation. Through this study, we evaluated the effectiveness of a recombinant subunit GnRH-1 vaccine for the immunocastration of the reproductive function of 16 mixed-breed dogs (Canis familiaris) provided voluntarily by different households. All the dogs were deemed clinically healthy prior to and during the experiment. A specific anti-GnRH immune response was detected at Week 4, which was maintained for at least 24 weeks after vaccination. Moreover, decreased levels of sexual hormones (testosterone as well as progesterone and estrogen, respectively) were observed in both male and female dogs. Estrous suppression was apparent in female dogs, and testicular atrophy and poor semen quality (concentration, abnormality, and viability) were observed in male dogs. In conclusion, the recombinant subunit GnRH-1 vaccine could successfully suppress fertility and delay the estrous cycle in canines. These results support the efficacy of the recombinant subunit GnRH-1 vaccine; thus, it is a suitable candidate for fertility control in dogs.

Effects of intracytoplasmic sperm injection timing and fertilization methods on the development of bovine spindle transferred embryos.

Cytoplasmic replacement by spindle transfer (ST) technique can be applied to improve the developmental competence of poor qualitied or aged oocytes. In cattle, ST technology has not been well established for producing embryos and the calves successfully using intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). The objective of this study was to develop a novel procedure for producing bovine ST embryos, which could be fundamental to applying ST technology in other mammals. In the present study, the efficacies of performing ICSI before (ICSI-ST) or after (ST-ICSI) and IVF on the development of ST bovine embryos were investigated. Results indicated that the blastocyst rate of ST embryos produced by ICSI-ST (24.7%) was higher (P < 0.05) than that produced by ST-ICSI (5.9%). On the other hand, ST-IVF had the highest fertilization rate (97.3%), polyspermy rate (24.7%), and lowest blastocyst rate (22.7%) when compared to denuded oocytes (DO), zona cut oocytes (ZC), and cumulus-oocyte complexes (COCs)-IVF groups. Finally, the in vitro development rates of ICSI-ST (24.5%) and ST-IVF (25.2%) were not significantly different (P > 0.05). However, the pregnancy rate (46.7%) and birth rate (33.3%) of ST-IVF were higher (P < 0.05) than those of ICSI-ST (6.3% and 0%, respectively). The percentage of mitochondrial DNA (mtDNA) heteroplasmy derived from donor karyoplasts of the 5 claves was between 2% and 18%. Taken together, performing ICSI prior to ST can improve the embryonic development of ST bovine embryos. Moreover, using IVF, instead of ICSI, for ST oocyte fertilization dramatically increased the pregnancy rate and birth rate of ST calves, in which mtDNA heteroplasmy derived from donor karyoplasts exists.

Recombinant suilysin of Streptococcus suis enhances the protective efficacy of an engineered Pasteurella multocida toxin protein.

Suilysin (Sly) from Streptococcus suis has been shown to elicit strong immune responses and may act as a vaccine adjuvant. In the present study, we tested the adjuvant effect of Sly using an engineered Pasteurella multocida toxin, rPMT-NC, as the antigen. The antigen was also formulated with other conventional adjuvants (aluminum hydroxide, water-in-oil-in-water) for comparison. The efficacy of these vaccine formulations were evaluated in mice. The optimal dosage of purified rSly for enhancing immune responses in mice was first determined to be 40 µg/ml based on significantly (p < 0.05) increased serum antibody titers, expression of cytokines, including interleukin (IL)-4, IL-12, and interferon (IFN)-γ and the survival rate after challenge with P. multocida. Mice immunized with rPMT-NC + rSly had augmented antibody production and cellular immunity compare to those immunized with rPMT-NC plus other adjuvants. In addition, the survival rate of mice immunized with rPMT-NC + rSly was the highest (70% v.s. 30% of mice immunized with rPMT-NC alone) among all groups. In conclusion, rSly has the potential to be used as a biological adjuvant to enhance immune responses and protective efficacy of protein-based vaccines.

Effects of a Recombinant Gonadotropin-Releasing Hormone Vaccine on Reproductive Function in Adult Male ICR Mice.

Gonadotropin-releasing hormone (GnRH) regulates the reproductive endocrine system in mammals. The GnRH immunocontraception vaccine can aid animal population control and management. We evaluated a recombinant GnRH fusion protein with the adjuvant MONTANIDE ISA 206 VG as a GnRH vaccine in adult male ICR mice by evaluating anti-GnRH antibodies; concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone; testis size and histomorphology; and semen quality. Response was assessed after intramuscular administration of the vaccine to mice in weeks 0, 4, and 8. The vaccine induced specific antibody response by week 5, with peak of antibody levels observed by week 13 and a declining level thereafter until the end of the study at week 24. Furthermore, it reduced serum FSH, LH, and testosterone concentrations. The vaccinated mice exhibited testicular atrophy and reduced sperm quality, concentration, morphology, and viability compared to control males. The outcomes of pairings of treated males with untreated females revealed reduced mating, pregnancy rates and number of litters compared to control pairings. Assessment of this GnRH vaccine in different species could assist its development for future applications.

Prokaryotic recombinant hemagglutinin-neuraminidase protein enhances the humoral response and efficacy of commercial Newcastle disease vaccines in chickens.

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays an important role in the pathogenesis of Newcastle disease (ND). Recombinant HN (rHN) protein, produced either by direct injection of recombinant viruses containing HN gene or baculovirus expression systems, has been used to elicit immunity against NDV in chickens. In the present study, a 60.4-kDa rHN was expressed by a prokaryotic expression system and formulated into ND vaccines. Inclusion of rHN (10 microg/ml) into conventional, inactivated ND vaccines significantly (P < 0.05) increased the titer of serum hemagglutination-inhibition Ab in specific-pathogen-free or commercial chickens. Furthermore, when the rHN protein was formulated into ND+IC (infectious coryza) bivalent or ND+IC+FC (fowl cholera) multivalent vaccines, the protection rate of immunized chickens increased from approximately 80%-90% to 100% after being challenged by a velogenic strain of NDV. Our data indicated that inclusion of rHN protein produced by an economical prokaryotic expression system could enhance the immunogenicity of traditional and multivalent inactivated ND vaccines. This approach may be adapted to improve the efficacy of ND vaccines currently used in the poultry industry.

Evaluation of Long-term Antibody Response and Cross-serotype Reaction in Ducks Immunised with Recombinant Riemerella Anatipestifer Outer Membrane Protein A and CpG ODN.

INTRODUCTION: Riemerella anatipestifer (RA) infections can lead to high mortality in ducklings. Inactivated vaccines against RA are commercially available, but they fail to provide cross-protection against various serotypes. We have previously demonstrated that a subunit vaccine containing recombinant outer membrane protein A (rOmpA) antigen of serotype 2 formulated with CpG oligodeoxynucleotides (ODN) as the adjuvant was able to stimulate both humoral and cellular immunities. MATERIAL AND METHODS: In the present study, thirty healthy 7-day-old Pekin ducks were randomly assigned to three equal treatment groups: rOmpA-vaccinated, rOmpA + CpG-vaccinated, and control. Vaccine was injected intramuscularly and a booster dose of the same vaccine was given two weeks after primary immunisation. The long-term antibody response and cross-serotype reaction of this vaccine were evaluated in ducks. RESULTS: Compared to ducks immunised with rOmpA alone, ducks immunised with rOmpA + CpG ODN had significantly (p < 0.05) increased serum antibody titre from two weeks until nine months after primary immunisation. In addition, expression of cytokines including interferon (IFN)-α, IFN-γ, interleukin (IL)-6, and IL-12 was significantly (p < 0.05) enhanced in PBMC of ducks immunised with rOmpA + CpG ODN two weeks after primary immunisation. Antibodies from ducks immunised with the rOmpA + CpG ODN vaccine could also detect RA serotypes 1 and 6 in Western blot analysis. CONCLUSION: Combination of rOmpA and CpG ODN could be a feasible strategy for developing a subunit RA vaccine with long term and broader-ranging protection.

Bovine CD14 gene characterization and relationship between polymorphisms and surface expression on monocytes and polymorphonuclear neutrophils.

BACKGROUND: CD14 is an important player in host innate immunity in that it confers lipopolysaccharide sensitivity to cell types like neutrophils, monocytes and macrophages. The study was aimed at characterizing the CD14 gene of cattle for sequence variations and to determine the effect of variations on the expression of the protein on the surfaces of monocytes and neutrophils in healthy dairy cows. RESULTS: Five SNPs were identified: two within the coding regions (g.A1908G and g.A2318G, numbering is according to GenBank No. EU148609), one in the 5' (g.C1291T) and two in the 3' (g.A2601G and g.G2621T) untranslated regions. SNP 1908 changes amino acid 175 of the protein (p.Asn175Asp, numbering is according to GenBank No. ABV68569), while SNP 2318 involves a synonymous codon change. Coding region SNPs characterized three gene alleles A (GenBank No. EU148609), A1 (GenBank No. EU148610) and B (GenBank No. EU148611) and two deduced protein variants A (ABV68569 and ABV68570) and B (ABV68571). Protein variant A is more common in the breeds analyzed. All SNPs gave rise to 3 haplotypes for the breeds. SNP genotype 1908AG was significantly (P<0.01) associated with a higher percentage of neutrophils expressing more CD14 molecules on their surfaces. The promoter region contains several transcription factor binding sites, including multiple AP-1 and SP1 sites and there is a high conservation of amino acid residues between the proteins of closely related species. CONCLUSION: The study has provided information on sequence variations within the CD14 gene and proteins of cattle. The SNP responsible for an amino acid exchange may play an important role in the expression of CD14 on the surfaces of neutrophils. Further observations involving a larger sample size are required to validate our findings. Our SNP and association analyses have provided baseline information that may be used at defining the role of CD14 in mediating bacterial infections. The computational analysis on the promoter and comparative analysis with other species has revealed regions of regulatory element motifs that may indicate important regulatory effects on the gene.

Development of a novel biochip for rapid multiplex detection of seven mastitis-causing pathogens in bovine milk samples.

To efficiently prevent and treat bovine mastitis and minimize its effect on the dairy industry, a sensitive, rapid, and specific test is required for identifying the mastitis-causing pathogens. In this study, a biochip capable of detecting 7 common species of mastitis-causing pathogens, including Corynebacterium bovis, Mycoplasma bovis, Staphylococcus aureus, and the Streptococcus spp. S. agalactiae, S. bovis, S. dysgalactiae, and S. uberis, within 6 hr was developed. The technique is based on DNA amplification of genes specific to the target pathogens and consists of 4 basic steps: DNA extraction of bacteria, polymerase chain reaction, DNA hybridization, and colorimetric reaction. To examine the accuracy and specificity of this biochip, a preliminary test with 82 random quarter milk samples were analyzed and compared with results from conventional microbiological methods conducted simultaneously. Results from all but 1 sample analyzed by the biochip were in agreement with those analyzed by bacteriology. The biochip could be a feasible tool for rapidly diagnosing mastitis-causing pathogens in milk and providing information for a more effective treatment to cure mastitis.