Lactococcus garvieae in fish: a review

Lactococcus garvieae is the etiological agent of Lactococcosis, an emergent disease which affects many fish species and causes important economic losses both in marine and freshwater aquaculture when water temperature increases over 16 degrees C in summer months. Normally, it causes a hyperacute and haemorrhagic septicemia. This paper presents a state of the art review of fish Lactococcosis including aspects such as pathogen characterization, pathogenesis, epidemiology, diagnosis and control measures of the disease in farmed fish.     

Lactococcus garvieae Endocarditis on a Prosthetic Biological Aortic Valve

Lactococcus garvieae (LG) endocarditis is a rare disease in humans. There are only about 16 reported cases in the world. We report a 76‐year‐old male patient with LG endocarditis. In depth interview with the patient revealed that 2 weeks prior to admission, he had eaten sushi containing raw fish. Unlike many of the other infections reported, which were on a native mitral valve, our patient's vegetation was on a prosthetic aortic valve.     

Antibiotic resistance in Lactococcus species from bovine milk: presence of a mutated multidrug transporter mdt(A) gene in susceptible Lactococcus garvieae strains

A total of 72 Lactococcus strains (41 Lactococcus lactis and 31 Lactococcus garvieae) isolated from bovine milk were tested for susceptibility to 17 antibiotics and screened for the presence of antibiotic resistance genes using a microarray. Resistance to tetracycline, clindamycin, erythromycin, streptomycin, nitrofurantoin were found. The tetracycline-resistant L. garvieae and L. lactis harbored tet(M) and tet(S). L. lactis that were resistant to clindamycin were also resistant to erythromycin and possessed the erm(B) gene. The multidrug transporter mdt(A), originally described in L. lactis, was detected for the first time in L. garvieae and does not confer decreased susceptibility to erythromycin nor tetracycline in this species. Mdt(A) of L. garvieae contains one mutation in each antiporter motif C, which is known to play an essential role in drug efflux antiporters. This suggests that the mutations found in the C-motifs of Mdt(A) from L. garvieae may be responsible for susceptibility. The study revealed the presence of antibiotic resistance genes in non-pathogenic and pathogenic lactococci from bovine milk, including a mutated multidrug transporter in L. garvieae.     

Safety and efficacy of an inactivated vaccine against Lactococcus garvieae in rainbow trout (Oncorhynchus mykiss)

We studied the safety and efficacy of an inactivated vaccine (Ichtiovac-Lg) against Lactococcus garvieae in rainbow trout (Oncorhynchus mykiss). In an initial dose-response experiment to test safety, we injected 50 rainbow trout weighing 30-40 g with a double dose of vaccine (0.2 ml) intraperitoneally. We observed these fish three times a day until day 50 post-vaccination when they were killed to evaluate visceral reactions, adhesions and intraperitoneal absorption. Survival was 100% in both the treatment and control groups and no significant differences were found in percentage of severe adhesions and pigmentation of peritonea and viscera. In a second trial, we injected 50 rainbow trout weighing 30-40 g with 0.1 ml of vaccine and a control group was injected with 0.1 ml of PBS intraperitoneally. On day 29 post-vaccination, both groups were challenged by intraperitoneal injection with 0.1 ml of a virulent heterologous strain of L. garvieae at 3 x 10(6) cfu ml(-1) and fish were observed for a further 21 days. At the end of the experiment, the survivals of the vaccinated fish and control group were 94 and 4%, respectively.     

Induction, characterisation and pathogenicity in rainbow trout Oncorhynchus mykiss (Walbaum) of Lactococcus garvieae L-forms.

Difficulties with induction and cultivation of L-forms, particularly those derived from Gram positive parent cells, have constrained to some degree the ability to evaluate the pathogenicity of these morphotypes. Induction of L-forms of Lactococcus garvieae was undertaken using either charcoal or inactivated horse serum media supplemented with ampicillin, benzylpenicillin or erythromycin, the drug of choice for treatment of infections in rainbow trout, Oncorhynchus mykiss, (Walbaum), and NaCl as an osmotic stabiliser. Lysozyme treated cells could be cultured in a cell wall deficient state using media consisting of charcoal, NaCl and either ampicillin or benzylpenicillin. The influence of some amino acids for induction of L-forms was assessed by disc diffusion and combined interaction. Analysis of variance of colony counts indicated that the amino acids glycine, DL-methionine, L-threonine and L-serine (P<0.03), and the presence of charcoal were beneficial and that inactivated horse serum was detrimental to L-form development. Electron microscopy revealed that the cell wall of L-forms was missing and this cell had a greatly expanded volume compared to parent cells. Electrophoresis of whole cell proteins showed some variation of electropherotype between parent and L-form cells. L-forms expressed greater quantities of proteins with molecular mass of 36 and 66 kDa and parent cells contained greater quantities of proteins of molecular mass 29, 43 and 60 kDa. Additional proteins of molecular mass 32, 44 and 53 kDa were present in L-form extracts, and in parent cells of 34, 38, 40, 42, 85 and 123 kDa which may represent cell wall associated proteins or alterations in expression due to different growth rates. Intraperitoneal challenge of rainbow trout with L-forms failed to produce overt infection even in immune-suppressed fish, but L-forms were shown by indirect fluorescent antibody test to remain inkidney tissue. Fish were susceptible to infection when challenged with parent cells of L. garvieae.     

Effect of a potential probiotics Lactococcus garvieae B301 on the growth performance,immune parameters and caecum microflora of broiler chickens

In this study, a novel Lactococcus garvieae B301 was isolated from the intestinal tract of a healthy piglet. L. garvieae B301 was tolerant to acid pH, simulated gastric and small intestinal transit juices, indicating that it was capable of surviving in the gastrointestinal tract. L. garvieae B301 was safe and beneficial to broilers, as broiler chickens supplemented with L. garvieae B301 had lower diarrhoea incidence and mortality than the Control. Moreover, supplementation of broiler diets with L. garvieae B301 resulted in an increase in body weight and the number of caecum lactic acid bacteria and Bifidobacterium spp., and decrease in feed‐to‐gain ratio and the number of caecum coliforms. It also had a positive effect on the thymus index and bursa of Fabricius index and enhanced serum levels of immune globulins. All these results showed that L. garvieae B301 could enhance the growth performance of broiler chickens and improve their health. Thus, L. garvieae B301 could be a promising feed additive for broiler chickens.     

Antigen recognition by rainbow trout (Oncorhynchus mykiss) of whole cell proteins expressed by Lactococcus garvieae when obtained directly from fish and under iron limited culture conditions

Infection by Lactococcus garvieae has become a widely recognised problem associated with intensively cultured fish. Long-term control of fish infections may be possible by vaccination providing a suitable and efficacious epitope is expressed during production of cells used for vaccine preparation. The identification of novel vaccine candidates must, therefore, consider how the host species recognises and responds to bacterial cell components. L. garvieae was cultured in iron deficient, limited and haem iron enriched media and the whole cell proteins expressed under these conditions were compared with those expressed in bacteria extracted with Percoll gradients directly from spleen tissue of infected rainbow trout (Oncorhynchus mykiss). SDS-PAGE of the cell proteins showed the existence of several different electropherotypes according to the iron status of the culture media. Only minor differences in cell protein profile were detected in bacteria obtained directly from fish spleens, but when the electropherograms were analysed by Western blots using L. garvieae hyperimmune fish sera, several proteins could be identified that were expressed only when L. garvieae was growing in vivo. Siderophore could be detected in culture supernatant of iron deficient, limited and haem iron enriched media but not in media with higher nutrient concentrations. The siderophore could not be identified as a type of catechol or hydroxymate. Rainbow trout recognise proteins in the range of approximately 50-80 kDa for bacterial cells obtained without subculture from infected fish and culture conditions can influence protein profiles for this pathogen.     

Immunoproteomic analysis of the antibody response obtained in Nile tilapia following vaccination with a Streptococcus iniae vaccine

Streptococcus iniae is one of the most economically important Gram-positive pathogens in cultured fish species worldwide. The USDA-ARS Aquatic Animal Health Research Unit developed a modified (contains concentrated culture supernatant) S. iniae bacterin that has been demonstrated to be efficacious, and protection is mediated by specific anti-S. iniae antibodies. Although effective, the specific vaccine components important for efficacy are not known. In the present study, an immunoproteomic approach was utilized to identify whole-cell lysate proteins of S. iniae that stimulated specific antibody production in Nile tilapia (Oreochromis niloticus) following vaccination. Groups of tilapia were vaccinated by intraperitoneal injection with the modified S. iniae bacterin or were mock-vaccinated, and at 30 d post-vaccination sera samples were obtained from individual fish. Vaccination of tilapia with the S. iniae vaccine stimulated significantly elevated specific antibody responses against proteins of the bacterium and passive immunization of tilapia with this serum demonstrated the antibodies were highly protective. Whole-cell lysate proteins of S. iniae were separated by 2D-PAGE and were probed with a pooled serum sample from vaccinated tilapia. A total of eleven unique immunogenic proteins were positively identified by mass spectrometry. Based on research conducted on homologous proteins in other Streptococcus spp., antibodies specific for three of the identified proteins, enolase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-bisphosphate aldolase, are likely involved in protection from streptococcosis caused by S. iniae.     

Comparative single‐dose pharmacokinetics of orally administered florfenicol in rainbow trout (Oncorhynchus mykiss,Walbaum, 1792) at health and experimental infection with Streptococcus iniae or Lactococcus garvieae

This study evaluates changes in the pharmacokinetic behavior of a single oral dose of florfenicol in rainbow trouts experimentally infected with Lactococcus garvieae or Streptococcus iniae. One hundred and fifty fish were randomly divided into three equal groups: 1—healthy fish, 2—fish inoculated with S. iniae (2.87 × 10⁷ CFU/ml, i.p.), and 3—fish inoculated with L. garvieae (6.8 × 10⁵ CFU/ml, i.p.). Florfenicol was administered to all groups at 15 mg/kg by oral gavage. Blood sampling was performed at 0, 2, 3, 6, 8, 12, 24, 48, 72, and 120 hr after drug administration to each group, and plasma concentration of florfenicol was assayed by HPLC method. The MICs of florfenicol were 1.2 μg/ml and 5 μg/ml against L. garviae and S. iniae, respectively. Healthy fish showed higher values for most of the PK/PD parameters as compared to fish infected with L. garvieae which was reversed in fish infected with S. iniae. Fish infected with L. garvieae showed decreased relative bioavailability accompanied by increased volume of distribution at steady‐state (Vd_ss) and total body clearance (Cl_B)_. Infection with S. iniae increased the peak concentration of drug after administration (Cmax) and decreased elimination half‐life (T_{1/2 β})_, central compartment volume (V_c), and Vd_ss. In conclusion, infection with these bacteria can affect the pharmacokinetic behavior of florfenicol in rainbow trouts as shown by decreased bioavailability and increased total body clearance and volume of distribution in L. garvieae infection and decreased volume of distribution accompanied by increased Cmax in S. iniae‐infected fish.     

Immunoproteomic analyses of outer membrane antigens of Actinobacillus pleuropneumoniae grown under iron-restricted conditions

Actinobacillus pleuropneumoniae, a bacterial pathogen of swine and agent of porcine pneumonia, causes a highly infectious disease of economic importance in the pig industry. Commercial vaccines for A. pleuropneumoniae include whole-cell bacterins and second generation subunit vaccines but they only confer partial protective immunity. Our search for new vaccine candidates identified antigens that are expressed during conditions that mimic infection; the outer membrane (OM) proteome of A. pleuropneumoniae serotype 5b was examined under iron restriction. Quantitative profiling by 2D-DiGE technology revealed that iron restriction induced expression of previously described transferrin binding proteins (TbpA, TbpB) plus four lipoproteins including spermidine/putrescine binding periplasmic protein 1 precursor (PotD2). Immunoproteomic analyses with antisera from na?ve animals and from infected pigs were able to differentiate antigens within the OM proteome that were specifically recognized during A. pleuropneumoniae infection. Immunoblots of iron-restricted profiles detected PotD2, heme-binding protein A (HbpA), and capsule polysaccharide export protein (CpxD) as well as surface antigens TbpA, TbpB, and OmlA. These data identify OM proteins that demonstrate immunogenicity and upregulation under conditions mimicking infection, providing emphasis on lipoproteins as an important class of antigens to exploit for vaccine development for A. pleuropneumoniae.     

幽门螺旋杆菌lpp20-cagA融合基因在乳酸球菌中的表达及免疫原性研究

为研究载体疫苗对幽门螺旋杆菌(HP)感染的免疫保护作用,本实验应用无缝克隆技术将HP的两个候选抗原基因lpp20和cagA直接连接后克隆于表达载体pNZ8149中,构建重组表达质粒pNZ8149-lpp20-cagA。将重组质粒电转化乳酸球菌(L.lactis)NZ3900,构建了不含任何抗生素筛选标记的食品级原核表达系统pNZ8149-lpp20-cagA/NZ3900,经nisin诱导表达,得到的重组蛋白分子量约为50.3ku。将以上得到的重组L.lactis灌胃免疫BALB/c小鼠,利用ELISA方法分别检测各组小鼠血清中IgG、IgG1、IgG2a及细胞因子IL-4、IFN-γ水平。结果显示,与对照组相比,实验组小鼠血清中IgG、IgG1、IL-4水平显著升高,表明重组蛋白Lpp20-CagA可以诱导良好的体液免疫应答;利用HP攻毒经重组菌灌胃免疫的小鼠,检测小鼠胃组织中HP的定植密度及脲酶活性,结果显示重组蛋白Lpp20-CagA在一定程度上可以减少HP在小鼠胃组织中的定植。本实验构建的载体疫苗在预防HP感染方面具有重要的应用价值。     

Restriction endonuclease analysis of Mycoplasma synoviae strains

A diverse group of Mycoplasma synoviae strains from various hosts, pathological processes, and geographic areas collected over about 25 years were analyzed by restriction endonuclease analysis. The results suggest that restriction endonuclease analysis of M. synoviae strains may be a useful strain identification tool to study epidemiological problems.     

产粘乳酸球菌的筛选与鉴定

采用发酵乳粘丝法初筛与发酵乳综合品质对比法复筛相结合的方法,从我国西北部三种不同来源的发酵制品中筛选了三株产粘性能良好的乳酸菌。经鉴定其中两株为嗜热链球菌,一株为乳酸乳球菌乳脂亚种。选取两株嗜热链球菌与保加利亚乳杆菌进行复配发酵试验,以市售酸奶做对照,筛选出使发酵乳粘性好、乳清析出少、感官评价好的菌株,命名为Streptococcus thermophilus ST-1。     

两株猪博卡病毒的检测及遗传演化分析

根据Gen Bank公布的猪博卡病毒序列,设计特异性引物,分别对猪博卡病毒(PBo V)的NS1基因、NP1基因、VP1/VP2全基因进行PCR方法测定,所得到的2个阳性样本分别命名为GD4和GD18。各基因遗传分析表明:GD4和GD18株各基因同源性较低,在34.9%⁵0.5%之间;猪博卡病毒主要分为3个大的分支,GD4和GD18分别处于不同的分支;分别与HQ223038和KF206167亲缘关系较近。     

乳酸乳球菌和魏斯氏菌的分离及鉴定

试验以生牛奶、自制泡菜水、青贮料、市售奶酪为样品,进行乳酸乳球菌和魏斯氏菌的筛选与鉴定。通过培养基中菌落形态观察和镜检细胞形态观察,共筛得6株疑似乳酸球菌（分别命名为ST1、ST2、ST6、ST7、ST8、ST9）。经生理生化、耐盐性、耐热性试验以及16S rD-NA序列分析鉴定,这6株菌分属两个属：ST1、ST2、ST7、ST9为乳酸乳球菌（Lactococcus lac-tis）,其中ST2为乳酸乳球菌乳脂亚种（Lactococcus lactis subsp. Cremoris）,ST7为乳酸乳球菌乳酸亚种（Lactococcus lactis subsp. Lactis ）;ST6、ST8属于魏斯氏菌属（Weissella）,其中ST6为食窦魏斯氏菌（Weissella cibaria）。研究表明,生牛奶和泡菜水分别是乳酸乳球菌和魏斯氏菌的优良生活环境,传统方法与分子生物技术相结合可更准确快速地分离及鉴定菌株。     

Immunoproteomic assay of membrane-associated proteins of Streptococcus suis type 2 China vaccine strain HA9801

Immunoproteomic approaches were undertaken to study the immunogenicity of the membrane-associated proteins of the Streptococcus suis type 2 (SS2) China vaccine strain HA9801. The membrane-associated proteins were enriched using the Triton X-114 extraction protocol and were analysed by two-dimensional gel electrophoresis (2-DE) and subsequent immunoblotting using the hyperimmune serum of SS2-HA9801-immunized specific pathogen free (SPF) minipigs. A total of 11 proteins were recognized, and the corresponding spots on a duplicate gel were excised and identified by MALDI-TOF MS.     

副猪嗜血杆菌流行优势菌株调查和耐药性分析

为调查研究河南规模化猪场副猪嗜血杆菌(HPS)流行优势菌株及其耐药性情况,2017-2018年从河南规模化猪场分离到30株HPS,根据菌株分离部位统计,肺脏、气管是HPS分离的首要组织部位,肺脏分离14株,占46.67%;气管分离11株,占36.67%。通过PCR分子血清型鉴定,河南流行的优势菌株为血清5,7,4型,其中血清型5型有9株,占30%;血清型7型有5株,占16.67%;血清型4型有4株,占13.33%。通过K-B纸片琼脂法药敏试验,30株分离菌株除对头孢噻呋100%敏感外,对其他17种常用抗生素都有不同程度的耐药现象,多重耐药现象突出,预防控制HPS病选药、用药方面更需慎重和规范。     

3株嗜水气单胞菌弱毒株的毒力相关特性分析

对健康鲫鱼和水体环境中分离的3株嗜水气单胞菌NJ-4、NJ-13和CS-13的毒力相关特性进行分析。采用PCR技术检测该菌5种主要毒力基因：气溶素（aer）、细胞毒性肠毒素（act）、细胞兴奋性肠毒素（alt）、温敏胞外蛋白酶（eprCAI）和丝氨酸蛋白酶（ahp）,并进行溶血性、溶蛋白性、细胞毒性、细胞黏附特性和主要外膜蛋白（MOMP）图谱分析,以及斑马鱼致病性试验。结果显示：NJ-4不合上述5种毒力基因,NJ-13只含有ahp基因,CS-13仅具备aer基因。3个菌株培养上清均不溶血、不溶蛋白;NJ-4和CS-13培养上清不能致细胞病变,NJ-13上清对细胞的毒力效价达1：128;而强毒株BSK-10培养上清的溶血价、溶蛋白效价和细胞毒力效价分别为1：128、1：256和1：256。NJ-4、NJ13和CS-13对HEp-2细胞有一定的黏附能力,但与强毒株BSK-10相比,黏附能力较弱。NJ-4、NJ-13和CS-13的培养液上清均不致斑马鱼死亡,菌体有一定致病作用,对斑马鱼的LD50超过10^8CFU／mL;而BSK-10培养上清和菌体致病性均很强,菌体对斑马鱼的LD50小于10^5CFU／mL。NJ-4、CS-13的MOMP条带与BSK-10相似度很高,而与NJ-13有一定差别,可能与菌株的来源有一定关系。结果表明,3株细菌均属弱毒株,特别是NJ-4的毒力最弱。     

吉林鹿源狂犬病病毒野毒株磷蛋白基因的测序及分析比较

通过对狂犬病病毒鹿源野毒株(DRV)总RNA的提取,用RT-PCR方法扩增磷(N S)蛋白基因并与T载体连接、克隆,将其转化到大肠杆菌JM109细胞中,测定N S蛋白基因的序列,与其他已经发表的国际标准株、疫苗株等进行比较,分析其氨基酸的同源性和N S蛋白的分子生物学功能,为狂犬病病毒野毒株全基因序列的测定奠定基础。