Agalactiae Infection Research Articles

Ancestral T Cells in Fish Require mTORC1-Coupled Immune Signals and Metabolic Programming for Proper Activation and Function.

T cells suddenly appeared in jawed fish ∼450 million years ago. Biological studies of fish T cells may provide helpful evidence to understand evolution of adaptive immune systems. To this end, using a Nile tilapia (Oreochromis niloticus) model, we revealed the regulatory mechanism of adaptive immunity mediated by ancestral T cells in jawed fish. Nile tilapia T cells as well as a tightly regulated mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway participate in the cellular adaptive immune response during Streptococcus agalactiae infection. Blockade of mTORC1 signaling by rapamycin impairs T cell activation and Ag-induced proliferation in this early vertebrate. More critically, we show that signals from mTORC1 are indispensable for primordial effector T cells to eliminate infection by promoting the expression of proinflammatory cytokines, cytotoxic-related molecules, and proapoptotic genes. Mechanistically, teleost mTORC1 directs effector T cell function by coordinating multiple metabolic programs, including glycolysis, glutaminolysis, and lipogenesis through activating key transcription factors c-Myc, HIF-1α, and sterol regulatory element-binding proteins, and thus links immune signals to metabolic reprogramming in jawed fish. To our knowledge, these results represent the first description of the regulatory mechanism for T cell-mediated adaptive immunity in a fish species. From an evolutionary viewpoint, our study suggests that primordial T cells are armed with sophisticated regulatory strategies like those in modern T cells prior to the divergence of bony fish from the tetrapod lineage. Therefore, our findings fill in an important gap regarding evolution of the adaptive immune system.

Effects of Assam tea extract on growth, skin mucus, serum immunity and disease resistance of Nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae

The present study aimed to assess the possible effects of Assam tea (Camellia sinensis) extract (ATE) on growth performances, immune responses, and disease resistance of Nile tilapia, Oreochromis niloticus against Streptococcus agalactiae. Five levels of ATE were supplemented into the based diet at 0, 1, 2, 4, and 8 g kg−1 feed of Nile tilapia fingerlings (10.9 ± 0.04 g initial weight) in triplicate. After four and eight weeks of feeding, fish were sampled to determine the effects of the tea supplements upon their growth performance, as well as serum and mucosal immune responses. A disease challenge using S. agalactiae was conducted at the end of the feeding trial. Fish fed ATE revealed significantly improved serum lysozyme, peroxidase, alternative complement (ACH50), phagocytosis, and respiratory burst activities compared to the basal control fed fish (P < 0.05). The mucus lysozyme and peroxidase activities were ameliorated through ATE supplementation in the tilapia diets. Supplementation of ATE significantly (P < 0.05) enhanced final body weight, weight gain, and specific growth rate; while a decreased feed conversion ratio was revealed at 2 g kg−1 inclusion level, after four and eight weeks. Challenge test showed that the relative percent survival (RSP) of fish in each treatment was 33.33%, 60.00%, 83.33%, 76.68%, and 66.68% in groups fed 0, 1, 2, 4, and 8 g kg−1, respectively. In summary, diets supplemented with ATE especially at 2 g kg−1 increased the humoral and mucosal immunity, enhanced growth performance, and offered higher resistance against S. agalactiae infection in Nile tilapia.

Expression and functional analysis of tumor necrosis factor receptor (TNFR)-associated factor 5 from Nile tilapia, Oreochromis niloticus

Nile tilapia (Oreochromis niloticus) is a pivotal economic fish that has been plagued by Streptococcus infections. Tumor necrosis factor receptor-associated factor 5 (TRAF5) is a crucial adaptor molecule, which can trigger downstream signaling cascades involved in immune pathway. In this study, Nile tilapia TRAF5 coding sequence (named OnTRAF5) was obtained, which contained typical functional domains, such as RING, zinc finger, coiled-coil and MATH domain. Different from other TRAF molecules, OnTRAF5 had shown relatively low identify with its homolog, and it was clustered into other teleost TRAF5 proteins. qRT-PCR was used to analysis the expression level of OnTRAF5 in gill, skin, muscle, head kidney, heart, intestine, thymus, liver, spleen and brain, In healthy Nile tilapia, the expression level of OnTRAF5 in intestine, gill and spleen were significantly higher than other tissues. While under Streptococcus agalactiae infection, the expression level of OnTRAF5 was improved significantly in all detected organs. Additionally, over-expression WT OnTRAF5 activated NF-κB, deletion of RING or zinc finger caused the activity impaired. In conclusion, OnTRAF5 participate in anti-bacteria immune response and is crucial for the signaling transduction.

Dietary supplementation with Bacillus subtilis LT3‐1 enhance the growth, immunity and disease resistance against Streptococcus agalactiae infection in genetically improved farmed tilapia, Oreochromis niloticus

A feeding trial was conducted to investigate the effect of different levels of Bacillus subtilis LT3‐1 in diets on growth, immune parameters, intestinal morphology and disease resistance in genetically improved farmed tilapia, Oreochromis niloticus. Fish (46.91 ± 0.17 g) were fed with a basal diet supplemented with B. subtilis LT3‐1 at 0 (B0), 3.8 × 10¹⁰ (B1), 7.6 × 10¹⁰ (B2), 1.14 × 10¹¹ (B3) and 1.52 × 10¹¹ (B4) CFU kg⁻¹ for 6 weeks. The results showed that the weight gain of fish in B1 group was significantly enhanced compared to that in B0 group (p < 0.05). The addition of B. subtilis significantly affected serum biochemical indices (total protein, albumin, aspartate aminotransferase, alkaline phosphatase). Besides, the haematocrit, total counts of red and white blood cells, as well as the serum catalase and lysozyme activities, were increased, whereas the serum malondialdehyde, the serum immunoglobulin M and complement three contents were reduced. Parameters for intestinal morphology suggested a healthier intestine for the fish fed B. subtilis‐supplemented diets than fish fed the control diet. The survival rate after Streptococcus agalactiae challenge increased in tilapia fed with B. subtilis. The present study demonstrated B. subtilis can effectively improve growth, immunological status and resistance against S. agalactiae infection in tilapia farming.

Molecular characterisation and expression analysis of scavenger receptor class B member 1 (SR-B1) gene in Nile tilapia Oreochromis niloticus (Linnaeus, 1758)

The scavenger receptor class B member 1 (SR-B1) plays an important role in the first level of host defense against invading pathogens. In the present study, we cloned and characterized SR-B1 gene from Oreochromis niloticus (Linnaeus, 1758). The sequence of SR-B1 is 2248-bp long and contains a 1404-bp ORF encoded 467 amino acids. The sequence alignment showed that SR-B1 gene contains ten exons and nine introns. A multiple alignment analysis suggested that SR-B1 protein contains the conserved CXXS redox and GXXXG motifs, and shared high similarities with that of other species. On a phylogenetic tree, SR-B1 clustered with those of other teleosts and formed a separate fish clade. Real-time quantitative PCR analysis showed that SR-B1 was differentially expressed in various tissues and the expression levels in spleen and intestine were significantly upregulated by Streptococcus agalactiae infection. The results suggest that SR-B1 may be involved in the immune response against bacteria in Nile tilapia.

Effectivity of Streptococcus agalactiae bacterial vaccine with different coatings for increasing the immunity system on nile tilapia Oreochromis niloticus (Linnaeus, 1758)

Vaccination is one of the most effective control measure in preventing Streptococcus agalactiae infection. In this study, vaccine coatings were prepared with certain biomaterials such as chitosan, skim milk, and maltodextrin at concentration 1 % or 10 %, and further freeze-dried. Vaccine selected (in vitro) by tests: viability cell, solubility, protein concentration and protein molecular weight. The result of in vitro test showed that chitosan coating at doses 1 % and 10 % were the best in solubility and protein concentration test. Vaccine coated with chitosan was administrated again by injection (in vivo) in nile tilapia (Oreochromis niloticus) before challenged by S. agalactiae. This test consists of six treatments and three replications, i.e. the fish were injected with liquid vaccine (A), Phospat Buffered Saline (PBS) (B), vaccine with chitosan coating 1 % (C), vaccine with chitosan coating 10 % (D), chitosan 1 % (E), and chitosan 10 % (F). Parameters were observed including survival rate, relative percent survival (RPS), antibody titre, and total leucocyte. The results showed that treatment C showed a significant difference (P &lt; 0,05) in survival rate 92.22 ± 3.85 %, RPS 85.21 ± 7.20 %, and antibody titre highest result, but not significant (P &gt; 0,05) to increase total leukocyte in experimental fish against S. agalactiae infection. In conclusion, vaccine freeze dry with chitosan coated 1 % is effective to improve immunity system of nile tilapia.

Molecular serotyping and antimicrobial susceptibility of Streptococcus agalactiae isolated from fish in China

Streptococcus agalactiae (group B streptococcus, GBS) is a major pathogenic bacterium frequently associated with septicemia and meningoencephalitis in fish. In this study, we used molecular typing and the disc diffusion method to determine serotypes and antimicrobial susceptibilities of 28 S. agalactiae isolates from diseased farmed fish (tilapia, Schizothorax prenanti and Schizopygopsis pylzovi) in China. We found that the 28 S. agalactiae isolates belonged only to two serotypes (Ia and III), with serotype Ia being significantly more prevalent than serotype III. Antimicrobial susceptibility results showed that all of the isolates were susceptible to vancomycin, cephalexin and florfenicol. The S. agalactiae isolates examined showed resistance to erythromycin (42.9%), penicillin (35.7%), clindamycin (28.6%), enrofloxacin (35.7%), tetracycline (32.1%), gentamicin (28.6%) norfloxacin (28.6%), ofloxacin (14.3%) and doxycycline (7.14%), but the serotype III isolates showed higher proportions of antibiotic resistance than serotype Ia isolates. The macrolide resistance gene (ermB) was detected in all the 28 isolates and the tetracycline resistance gene (tetM) was detected in 78.6% isolates. All quinolone-resistant S. agalactiae isolates carried mostly double point mutations in DNA with inferred amino acid substitutions including the change of Ser-81 to Leu in the product of gyrA and Ser-79 to Tyr in the product of parC. Our study is a valuable reference for effective antibiotic application of S. agalactiae infections of farmed fish in China, and illustrates the importance of better use of antibiotics to prevent the widespread emergence of resistance S. agalactiae.

Antimicrobial activities of chitosan nanoparticles against pathogenic microorganisms in Nile tilapia, Oreochromis niloticus

Nanotechnology is a recent unique technique generally used for nutrition and therapy purposes among others. In this respect, the present study was carried out to evaluate the antimicrobial activity of chitosan nanoparticles (CNP) against various microorganisms (fungi and bacteria) isolated from diseased or health Nile tilapia, Oreochromis niloticus. The CNP was prepared based on the ionic gelation of chitosan with tripolyphosphate anion and its mean size was 35 nm with a narrow size distribution and zeta potential of 61.2 mV. The lethal dose of pathogenic bacterial isolates for Nile tilapia was successfully standardized. Clinical signs including weakness, slower movement, swimming closer to the surface, fin hemorrhages, and red patches at the gut regions were observed. Enlargement of spleen followed by tissue necrosis along with signs of hemorrhagic septicemia was also seen in infected fish. Fungal and bacterial isolates were exposed to different CNP doses and it is noticed that CNP inhibited all examined fungal and bacterial isolates in a dose-dependent manner. However, high CNP doses (80 μg/ml) gave highest inhibition zones where Aspergillus flavus, Mucor sp., and Candida sp. were more susceptible, whereas Aspergillus niger, A. fumigatus, and Fusarium sp. were more resistant. Similarly, largest inhibition zones of tested bacteria were obtained at high CNP dose (20 μg/ml). And Aeromonas sobria, A. hydrophila, and Pseudomonas aeruginosa were the most susceptible bacterial strain; meanwhile, Staphylococcus aureus and Pseudomonas fluorescens were the most resistant ones. The minimal inhibitory concentration of CNP against the examined bacteria ranged from 0.156 to 2.5 μg/ml causing their minimal counts. The transmission electron microscope images revealed that CNP showed antagonistic action against A. hydrophila causing disruption of cell membranes and the leakage of cytoplasm. In a practical experiment, Nile tilapia fed dietary CNP at levels of 0.0 and 1.0 g/kg diet for 3 weeks and post-challenged with different pathogenic bacteria via intraperitoneal injection. It is noticed that fish fed a CNP-enriched diet showed less mortality with all bacterial strains (6.7–20%), while, fish fed a CNP-free diet showed highest mortality (66.7–100%). The dietary CNP protected Nile tilapia efficiently against A. hydrophila, A. sobria, and Streptococcus agalactiae infections with relative level of protection (RLP) value of 93.3%, while the RLP against Staphylococcus aureus was 70.0%.

A novel bioengineered derivative of nisin displays enhanced antimicrobial activity against clinical Streptococcus agalactiae isolates.

Streptococcus agalactiae is the leading cause of neonatal disease worldwide, and infections caused by this opportunistic pathogen are becoming increasingly more prevalent in adults. With the global incidence of antimicrobial resistance continuing to rise, there is a recognised need for new therapeutic agents. Nisin is a potent antimicrobial peptide with demonstrated broad-spectrum activity against a range of clinically significant pathogens. This study aimed to examine the efficacy of nisin against a clinical population of S. agalactiae isolates and further to investigate the bioactivity of a novel bioengineered derivative of the peptide, designated nisin PV. A deferred antagonism assay was used to assess the bioactivity of wild-type nisin and nisin PV against 122 S. agalactiae isolates. Minimum inhibitory concentrations (MICs) were evaluated to determine the specific activity of both peptides. The genetic basis of nisin resistance among the isolate collection was investigated by PCR detection of the nsr gene. In total, 91.0% (111/122) of the collection showed some level of susceptibility to nisin, whilst 9.0% (11/122) displayed complete resistance. Interestingly, the nisin derivative exhibited enhanced antimicrobial activity for 64.8% of the isolates. The frequency of the nsr gene conferring nisin resistance was 98.4% (120/122), suggesting that resistance may be linked to levels of expression of the protein or other regulatory elements. This study indicates that there is potential for the use of nisin and its derivatives as therapeutic agents against S. agalactiae infections.

Efficacy of live attenuated vaccine derived from the Streptococcus agalactiae on the immune responses of Oreochromis niloticus

Streptococcus agalactiae species have been recognized as the main pathogen causing high mortality in fish leading to significant worldwide economical losses to the aquaculture industries. Vaccine development has become a priority in combating multidrug resistance in bacteria; however, there is a lack of commercial live attenuated vaccine (LAV) against S. agalactiae in Malaysia. The aim of this study is to compare two methods using attenuated bacteria as live vaccine and to evaluate the efficacy of selected LAV on the immune responses and resistance of Oreochromis niloticus (tilapia) against S. agalactiae. The LAV derived from S. agalactiae had been weakened using the chemical agent Acriflavine dye (LAV1), whereas the second vaccine was weakened using serial passages of bacteria on broth media (LAV2). Initial immunization was carried out only on day one, given twice-in the morning and evening, for the 42 day period. Serum samples were collected to determine the systemic antibody (IgM) responses and lysozymal (LSZ) activity using ELISA. On day 43 after immunization, the fish were injected intraperitoneally (i.p) with 0.1 mL of S. agalactiae at LD50 = 1.5 × 105 (CFU)/fish. Fish were monitored daily for 10 days. Clinical signs, mortality and the relative percent of survival (RPS) were recorded. Trial 1 results showed a significant increased (P < 0.05) in serum IgM titers and LSZ activity as compared to LAV2 and the control group (unvaccinated fish). The efficacy of LAV1 was proven effective as determined by the RPS values, LAV1 at 81.58% as compared to LAV2 at 65.79%. Trial 2 of LAV1 and control group were further determined by administering primary and booster doses revealed a RPS value for LAV1 of 82.05%, with the significant enhancement on the immune responses of tilapia as compared to control group. In conclusion, LAV revealed to elevate antibody IgM levels, LSZ activity and provide long-term protection when added to feed. LAV is a low-cost vaccine shown to rapidly increase the immune response of fish and increase survival rates of fish against S. agalactiae infection.

Study on Streptococcus agalactiae infection in Javanese medaka (Oryzias javanicus Bleeker, 1854) model

This study determines the median lethal dose, and describes the clinico-pathological changes and disease development following Streptococcus agalactiae infection in Javanese medaka model. Javanese medakas were infected with S. agalactiae via intraperitoneal (IP) from 104 to 108 CFU/mL, and immersion (IM) route from 103 to 107 CFU/mL. The LD50–240h and clinico-pathological changes of the fish was determined until 240 h post infection (hpi). Next, the disease development was determined for 96 hpi in the fish following IP and IM infection at 103 CFU/mL and 104 CFU/mL, respectively. The LD50–240h of S. agalactiae in Javanese medaka was lower following IP injection (4.5 × 102 CFU/mL), compared to IM route (3.5 × 103 CFU/mL). The clinical signs included separating from the schooling group, swimming at the surface of water column, lethargy, erratic swimming pattern, corneal opacity and exophthalmia. Histopathological examinations revealed generalized congestion in almost all internal organs, particularly in liver and brain, while the kidney displayed tubular necrosis. Both IP and IM routes showed significant positive correlation (p < 0.05) between the CFU/g of S. agalactiae in the fish tissue and fish deaths. Moreover, the lesions for histopathological scoring in selected organs following IP and IM challenges were also reflecting the CFU/g and fish deaths. This study indicates the capability of Javanese medaka as a model organism in study of streptococcosis development.

Expert evaluation of different infection types in dairy cow quarters naturally infected with Staphylococcus aureus or Streptococcus agalactiae

The purpose of this study was to improve the diagnostic recommendations for Staphylococcus aureus and Streptococcus agalactiae control using bacterial culture (BC), polymerase chain reaction (PCR) and somatic cell count (SCC) as diagnostic methods. The study was carried out in three steps: firstly, diagnostic test patterns for naturally infected quarters with Staph. aureus (24 quarters) and Strep. agalactiae (16 quarters) were created by sampling the quarters each day for 21 days and analysing the daily quarter milk samples using BC, PCR and SCC. Secondly, 30 mastitis experts were asked to group and describe the diagnostic test patterns and to establish a diagnosis for each group. The experts’ statements regarding the groups they established were subsequently examined using qualitative content analysis to assign “infection types” to the statements. Lastly, the test performance was estimated for BC, PCR and SCC using generalised logistic regression models with the interpreted statements as a reference for infection.The experts mainly identified the Staph. aureus quarter-patterns as persistent infections, while some had more dynamic patterns. Strep. agalactiae quarter-patterns mainly involved persistent infection, yet some appeared hard to diagnose and were assigned to almost all different infection types, while experts did not agree on the interpretation.Estimates of Se for detection of Staph. aureus infection were 95.9% [93.7; 97.3] for BC, 99.5% [98.3; 99.8] for PCR, and 96.1% [94.0; 97.5] for SCC. The corresponding Sp estimates were 74.5% [65.7; 81.7], 66% [57.2; 73.8] and 43.7% [36.2; 51.5] for BC, PCR and SCC, respectively.The Se estimates of BC and PCR for Strep. agalactiae infection were 100% [83.5; 100] and 99.9% [99.6; 100], respectively, whereas the Se of SCC detecting Strep. agalactiae infection was only 34.3% [26.4; 43.3]. This indicated that Strep. agalactiae-positive BC and PCR test results were more important than SCC results to the experts when diagnosing a quarter as infected. The Sp estimates of BC, PCR and SCC for Strep. agalactiae infection were 99% [72.8; 100], 97.7% [62.1; 99.9], and 65.7% [56.7; 73.7], respectively.We conclude that PCR and BC are highly sensitive in the detection of persistent and new infections as defined by the experts, although the Se was not always 100%. An accepted lower Sp suggests that experts place less emphasis on false-positive results. We recommend that efforts are made to develop consistent terminology to characterise intramammary infections over time so that the course of infection can be taken into account at diagnosis.

Dietary supplementation of dry oregano leaves increases the innate immunity and resistance of Nile tilapia against Streptococcus agalactiae infection

This study aimed to evaluate the influence of increasing concentrations of dietary dry oregano leaves (DOL), ranging from 0 to 0.15%, on different performance parameters of Nile tilapia. Hence, two separate experimental trials testing growth factors (Trial 1) and innate immunity and disease resistance against Streptococcus agalactiae infection (Trial 2) were conducted with a total of 464 tilapia juveniles and two to four replicates. As a result, dietary inclusion of DOL did not induce significant effects on growth performance, hematological parameters, and fillet composition. However, there were significant differences in lysozyme activity between groups of DOL at 0.075% and the control (0%). Moreover, the bactericidal index of the positive control and groups supplemented with DOL were significantly higher than baseline treatment groups. For disease resistance, there was a reduction of 15% in the relative risk of streptococcosis in fish juveniles after dietary inclusion of oregano at 0.15%. In conclusion, the inclusion of DOL in tilapia diets resulted in no significant gains in terms of growth; nevertheless, the immune system and resistance of tilapia against S. agalactiae infection were significantly improved.

Lyn is involved in host defense against S. agalactiae infection and BCR signaling in Nile tilapia (Oreochromis niloticus)

Lyn, a member of Src protein kinase family, plays a crucial role in immune reactions against pathogenic infection. In this study, Lyn from Nile tilapia (Oreochromis niloticus) (OnLyn) was identified and characterized at expression pattern against bacterial infection, and regulation function in BCR signaling. The open reading frame of OnLyn contained 1536 bp of nucleotide sequence encoded a protein of 511 amino acids. The OnLyn protein was highly conversed to other species Lyn, including SH3, SH2 and a catalytic Tyr kinase (TyrKc) domain. Transcriptional expression analysis revealed that OnLyn was detected in all examined tissues and was highly expressed in the head kidney. The up-regulation OnLyn expression was observed in the head kidney and spleen following challenge with Streptococcus agalactiae (S. agalactiae) in vivo, and was also displayed in head kidney leukocytes challenge with S. agalactiae and LPS in vitro. In addition, after induction with mouse anti-OnIgM mAb in vitro, the OnLyn expression and phosphorylation of OnLyn (Y507) were significantly up-regulated in the head kidney leukocytes. Moreover, after treatment with AZD0530 and mouse anti-OnIgM monoclonal antibody, the down-regulation of cytoplasmic free-Ca2+ concentration was detected in the head kidney leukocytes in vitro. Taken together, the findings of this study revealed that OnLyn might play potential roles in BCR signaling and get involved in host defense against bacterial infection in Nile tilapia.

Prevalence and capsular type distribution of Streptococcus agalactiae isolated from pregnant women in Namibia and South Africa

BackgroundStreptococcus agalactiae or Group B Streptococcus (GBS) is the leading cause of neonatal morbidity and mortality resulting in septicaemia, bacteraemia and meningitis. Long term problems in children range from loss of hearing to mental retardation. While Intrapartum Antibiotic Prophylaxis (IAP) has reduced the incidence of S. agalactiae infection, it still remains the leading cause of disease in neonates. GBS has ten capsular types whose distribution varies across the world. Therefore, this study sought to determine the prevalence of GBS in Namibia and South Africa amongst pregnant women between 35 and 37 weeks gestation and elucidate the capsular types.MethodsLower vaginal and rectal swabs were collected from pregnant women between 35 and 37 weeks gestation. Five hundred and thirty pregnant women were recruited into the study in Windhoek, Namibia while one hundred pregnant women were recruited in the Eastern Cape, South Africa. The swabs were cultured on 5% sheep blood agar (Biomerieux, New Jersey, USA) for isolation of GBS. Presumptive isolates were confirmed using both the Vitek (2) and molecular techniques targeting the scpB gene. Capsular typing was performed in a multiplex PCR with capsular specific primer pairs.ResultsThe prevalence of GBS in Namibia was 13.6 and 37% in South Africa respectively. In both countries most women were dually colonised with GBS. Capsular types II, III and V were the most prevalent.ConclusionsThe prevalence of GBS in Namibia was lower than in South Africa in this study. The prevalence in both countries was not different from those reported in other African countries and around the world. The predominant capsular types in this study are the ones commonly associated with adverse maternal outcomes.

Ocurrence and risk factors associated with Mycoplasma agalactiae infection in dairy goat herds of Paraíba State, Brazil

ABSTRACT: Mycoplasmosis is a disease that may cause severe economical losses in goat and sheep herds, and it is associated with mastitis, polyarthritis, agalactia, conjunctivitis, pneumonia and reproductive failure. The objective of this study was to determine the occurrence of Mycoplasma agalactiae in milk samples and investigate the main risk factors associated with infection in goats from farms of the state of Paraíba, Brazil. For Mycoplasma agalactiae diagnosis, 251 milk samples were submitted to DNA extraction using a commercially available kit, following the manufacturer’s instructions and Polymerase Chain Reaction (PCR) was performed. In addition, questionnaires were applied to identify the main risk factors associated with contagious agalactia. Out of the two hundred fifty-one samples analyzed, 50 (19.9%, I.C. 15.1-25.4%) were PCR positive for M. agalactiae. In the risk factors analysis, some associations were observed for the following variables: size of the herd (P&lt;0.001, OR=7.1, I.C. 2.4-20.6), replacement of farm animals (P&lt;0.001, OR=4.7, I.C. 1.8-12.2) and participation of animals in fairs and exhibitions (P=0.029, OR=2.0, I.C.1.0-3.9). The results allowed confirming the occurrence of Mycoplasma agalactiae in milk samples of goats from Paraíba. Therefore, it is strictly necessary to monitor dairy goat flocks and to raise the awareness of farmers about the economic importance of the disease, since it causes severe economic losses for producers of the state. Identification of risk factors is essential for adoption of control measures and for the correction of the management factors in farms where there are animals with positive diagnosis, avoiding, so, pathogen dissemination.

Identification and expression profiling analysis of microRNAs in Nile tilapia (Oreochromis niloticus) in response to Streptococcus agalactiae infection

MicroRNAs (miRNAs) play vital regulatory roles in various biological processes, including in immune responses. Nile tilapia (Oreochromis niloticus) is an important commercial fish species in China. To identify immune-related miRNAs of O. niloticus, 4 libraries from liver during S. agalactiae infection (0 h, 5 h, 50 h, and 7 d) were sequenced by high-throughput sequencing technology in tilapia. We obtained 10,703,531, 11,507,163, 11,180,179 and 13,408,414 clean reads per library, respectively. In our results, a total of 482 miRNAs were identified through bioinformatic analysis, including 220 conserved miRNAs and 262 putative novel miRNAs. Moreover, 21 (4.36%), 50 (10.37%), and 46 (9.54%) miRNAs were significantly differentially expressed at 5 h, 50 h and 7 d, respectively. In addition, 6939 target genes regulated by these differentially expressed miRNAs were predicted, and their functional annotations were predicted by Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, which revealed that a majority of differentially expressed miRNAs were involved in apoptotic process, metabolic process, and immune responses. Finally, Real-time quantitative PCR experiments were performed for 7 miRNAs by stem-loop RT-PCR, and a general agreement was confirmed between the sequencing and RT-qPCR data. To our understanding, this is the first report of comprehensive identification of O. niloticus miRNAs being differentially regulated in liver related to S. agalactiae infection. This work provides an opportunity for further understanding of the molecular mechanisms of miRNA regulation in O. niloticus host–pathogen interactions, and genetic resources for molecular assistant selection for disease resistant breeding program.

B cell receptor accessory molecule CD79 gets involved in response against Streptococcus agalactiae infection and BCR signaling in Nile tilapia (Oreochromis niloticus)

CD79, composed of two distinct chains called CD79a and CD79b, is a transmembrane protein that forms a B cell antigen receptor with membrane immunoglobulin, and generates a signal following antigen recognition by the B cell receptor. In this study, the CD79a (OnCD79a) and CD79b (OnCD79b) were cloned and identified from Nile tilapia (Oreochromis niloticus). The cDNA of ORF for OnCD79a and OnCD79b are 669 and 627 bp, coding 222 and 208 amino acids, respectively. The deduced protein analysis showed that both CD79a andCD79b contain an immunoreceptor tyrosine-based activation motif in their intracellular tails that used to propagate a signal in a B cell. Expression analysis revealed that both CD79a and CD79b expressed at high levels in immune tissues, such as anterior kidney and spleen, and in IgM+ B cells. Upon Streptococcus agalactiae (S. agalactiae) infection, the expressions of OnCD79a and OnCD79b were significantly up-regulated in anterior kidney and spleen. The significant up-regulations of OnCD79a and OnCD79b were also detected in leukocytes after in vitro challenge with S. agalactiae. Further, stimulations of LPS and anti-OnIgM monoclonal antibody induced significant up-regulations of OnCD79a and OnCD79b in leukocytes. Taken together, the results of this study indicated that CD79 molecule, playing roles in BCR signaling, was likely to get involved in host defense against bacterial infection in Nile tilapia.

In vitro and in vivo screening of herbal extracts against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)

This study investigated 354 herbal extracts prepared from 115 herbs via 4 extraction methods. The extracts' antibacterial activities against Streptococcus agalactiae were sequentially evaluated in vitro by two methods: (1) microplate assay and (2) agar well diffusion. Then the herbal extracts with high antibacterial activity in vitro were mixed into feed at 100 mg/g (dry herb/dry feed, w: w) and fed to fishes with S. agalactiae infection for in vivo evaluation of their therapeutic efficacy. Subsequently, the EC50 values (the concentrations result in the relative percent survival (RPS) of 50%) of the three herbal extracts with the highest therapeutic efficacies were calculated according to fit equations. Finally, phytochemical analyses of the herbs with the highest therapeutic efficacies were conducted using ultra performance liquid chromatography-Q Exactive Orbitrap-Mass spectrometry (UPLC-QE-MS). The results showed the following: in detections from the microplate assay method, 62 of the 354 candidate herbal extracts had antibacterial activity with inhibition ratios above 5%; in detections from the agar well diffusion method, 29 of the 62 the candidate herbal extracts showed clear inhibition zones and 33 showed semi-clear inhibition zones. Aristolochia debilis (fruit), Lonicera japonica (flower), Spatholobus suberectus (stem), and Arecae Semen (fruit) extracts prepared by 3 h of maceration at 50 °C had RPSs of 100.0%, 55.6%, 75.0%, and 33.3%, respectively; Panax ginseng (leaf), Rheum palmatum L. (root), and Semen Alpiniae (seed) extracts prepared by 72 h of maceration at room temperature had RPSs of 77.8%, 25.0%, and 12.5%, respectively; and Radix Isatidis (root) extract prepared by 30 min of ultrasonic-extraction at room temperature had an RPS of 62.5%. A. debilis, P. ginseng, and S. suberectus extracts had EC50s of 44.03, 41.19, 39.54 mg/g, respectively. Phytochemical analyses of A. debilis, P. ginseng, and S. suberectus extracts using UPLC-QE-MS produced the following results: the negative model detected 270, 623, and 326 peaks and identified 54, 70, and 52 compounds, respectively; the positive model detected 446, 1401, and 400 peaks and identified 61, 107, and 44 compounds, respectively. Taken together, these results indicate that the extracts of A. debilis, P. ginseng, and S. suberectus could prove useful for the treatment of S. agalactiae infection in Nile tilapia and suggest that appropriate bioactive compounds from these three herbal extracts may be developed as alternates to antibiotics.

Oral vaccination of tilapia against Streptococcus agalactiae using Bacillus subtilis spores expressing Sip

Streptococcus agalactiae infections are becoming an increasing problem in aquaculture because of significant morbidity and mortality, which restricts the healthy development of tilapia aquaculture. To seek safe and effective prevention measures, a Bacillus subtilis GC5 surface displayed vaccine was prepared and applied orally in tilapia. The study first showed that recombinant spores can engraft in the tilapia intestine. Then, the effect of protection and the immune responses were evaluated. The results of ELISA showed that Sip-specific antibody in the sera of GC5-Sip-immunized fish can be detected after the first oral administration when compared to the phosphate buffer saline (PBS) control group, and the levels of specific IgM gradually strengthened with boosting, so does the specific antibody against bacteria, proving that humoral immunity was induced. Quantitative real-time PCR (qRT-PCR) results showed that the immune-related gene expression of the gut and spleen exhibited a different rising trend in the GC5-Sip group, revealing that innate immune response and local as well as systemic cellular immunity were induced. The outcome of fish immunized with GC5-Sip spores provided a relative percent survival (RPS) of 41.7% against S. agalactiae and GC5 group had an RPS of 24.2%, indicating that GC5-Sip was safe and effective in protecting tilapia against bacterial infection. Our study demonstrated that the oral administration of B. subtilis spores expressing Sip could cause an effective immune response and offer good resistance to bacterial infection. Our work may lead to the development of new ideas for immunoprophylaxis against S. agalactiae infection.