Association of Enterococcus cecorum with vertebral osteomyelitis and spondylolisthesis in broiler parent chicks

Enterococcus cecorum is the most frequently occurring enterococcal species in the intestine of chickens of over 12 weeks of age, and there are few reports on its isolation from the skeleton of broiler parent chicks. In the present study, observations on vertebral osteomyelitis and spondylolisthesis ('kinky back syndrome') showing high incidence in 8 broiler parent flocks in different parts of Hungary are summarised. Clinical signs were seen only in roosters between 5 and 13 weeks of age. Diseased birds were alert and remained sitting on their hocks with their feet slightly raised off the ground. Incidence of the disease among male birds ranged from 8% to 30% depending on flocks. Enlargement and distortion of the body of the 6th vertebra were seen as the main pathological lesions. The cavity of the spinal canal was constricted by the distorted vertebral bodies. Resorption of bone tissue and sequestrum formation, signs of increased osteoclast activity, proliferation of fibrotic tissues, infiltration with heterophils and formation of sclerotic layers were detected in the vertebral bodies. From all 24 samples collected from the vertebral lesions, Enterococcus cecorum was isolated and identified using metabolic fingerprinting as well as 16S rRNA gene sequencing. Demonstration of E. cecorum from the vertebral lesions in all examined broiler breeder roosters showing the same clinical and pathological findings in different flocks suggested the pathogenic role of this microorganism for the first time in Hungary.     

Effect of subtherapeutic antimicrobials on genetic diversity of Enterococcus faecium from chickens

The effect of growth promotants (bacitracin, virginiamycin, and flavomycin) on the genetic population of Enterococcusfaecium isolated from a commercially integrated poultry farm was examined. A total of 551 E. faecium were isolated from chick boxliners (n=16), litter (n=334), feed (n=67), and carcass rinse (n=134) samples from four chicken houses. Two houses on the farm were control houses and did not use any antimicrobials while two other houses on the farm used flavomycin, virginiamycin, and bacitracin during six different chicken grow outs. BOX-PCR and pulsed-field gel electrophoresis (PFGE) results indicated that E. faecium strains had a high degree of genetic diversity as overall clustering was independent of source, house, or grow out. Similarity of > or =60% for the majority of BOX-PCR genogroups and > or =80% for the majority of PFGE genogroups was observed for a subset of carcass rinse samples (n=45) examined. Seventy-nine percent (19/24) of isolates in BOX-PCR genogroup 2 also clustered in PFGE genogroup 2, although no association between the isolates and house or grow out was observed. These results suggest that E. faecium from chicken are genetically diverse and that growth-promoting antimicrobials do not affect the genetic population of E. faecium.     

Transmission and genetic diversity of Enterococcus faecalis among layer chickens during hatch

Background

Studies on transmission of Enterococcus faecalis among chickens during hatch have not been carried out so far. Information about vertical transmission and subsequent spreading and colonization of the cloacal mucosa through cloacal ''drinking'' during hatch are important to understand the epidemiology of E. faecalis infections. In the present investigation vertical transmission and subsequent spreading and colonization of the cloacal mucosa of chickens by E. faecalis through cloacal ''drinking'' were examined.

Methods

Two different batches of layer chickens originating from 45 weeks old Brown and White Lohmann parents, respectively from the same farm were sampled in the hatcher. Isolates were confirmed to be E. faecalis by polymerase chain reaction (PCR) and further by multilocus sequence typing (MLST) to state their population structure and comparison made to sequence types previously obtained from chicken.

Results

A total of 480 chickens were swabbed from the cloacae just after hatch and after 24 hours. A total of 101 isolates were confirmed as E. faecalis by a species specific PCR. The prevalence of E. faecalis increased from 14% at 0 h to 97% after 24 h for the Brown Lohmann chickens and from 0.5% to 23% for the White Lohmann flock. The 84 isolates analysed by MLST were distributed on 14 sequence types (ST). Three ST (401, 82 and 249) accounted for 64% of all isolates analysed by MLST after 24 h. ST 82 has previously been reported from amyloid arthropathy and other lesions in poultry.

Conclusions

The present findings demonstrated a high potential of a few contaminated eggs or embryos to rapidly facilitate the spread of E. faecalis to almost all chickens during hatch.     

Antibiotic resistance in Escherichia coli and Enterococcus spp. isolates from commercial broiler chickens receiving growth-promoting doses of bacitracin or virginiamycin

Antibacterial agents such as zinc bacitracin (ZB) and virginiamycin (VG) are used as growth promoting agents (GP) in broiler chicken production. The objective of this study was to evaluate the effect of the use of ZB and VG on the emergence of antibacterial resistance in a commercial broiler chicken farm. Three trials were conducted using 3 different diets: one without antibacterial agents, one containing VG, and one with ZB. Escherichia coli and Enterococcus spp. strains were isolated and tested for their susceptibility to various antibacterial agents. The occurrence of the resistance genes vatD, ermB, and bcrR in Enterococcus spp. isolates was determined by polymerase chain reaction (PCR). Comparative quantification of vatD and bcrR genes in total deoxyribonucleic acid (DNA) extracts from litter was done by SYBR Green Real-Time PCR (QPCR). Escherichia coli and Enterococcus spp. isolates from diet groups had different levels of resistance to various antibacterial agents over time. These GPs did not select for specific antibacterial agent resistance (AAR) in Enterococcus spp. The use of GPs seemed to lower the percentage of E. coli isolates resistant to some antibacterial agents. The presence of the bcrR gene could not explain all resistant phenotypes to ZB. Genes other than vatD and ermB might be involved in the resistance to VG in Enterococcus spp. Use of GPs was not associated with presence of the bcrR gene in DNA extracts from litter, but use of VG was associated with vatD presence.     

Isolation of Mycoplasma meleagridis from chickens

Mycoplasma meleagridis (MM) is a major cause of disease and economic loss in turkeys. Formerly it was thought that this species was very host specific and only restricted to turkey. In this study, we report on the recovery of MM from breeding flocks of chickens located near a turkey breeding unit. Ten MM field strains were isolated (by culture on Frey broth medium) from tracheal swabs of chickens displaying clinical signs of mycoplasmosis-essentially respiratory symptoms and poor performance. Assignment of the isolated field strains to MM was confirmed by a growth inhibition assay using MM-specific polyclonal antiserum and by PCR amplification targeting the 16S rRNA sequence as well as the Mm14 sequence, a MM-species-specific DNA fragment previously identified and characterized in our laboratory. The nucleotide sequence of Mm14 proved to be highly conserved among the 10 MM field strains, indicating a common source of infection. However, on the basis of slight differences in sodium dodecyl sulfate-polyacrylamide gel electrophoresis whole-cell proteins and western blot profiles, two groups of the isolated MM field strains could be distinguished. Evidence of MM infection of chickens was further provided by serology, since 13.77% (35/254) of sera proved positive to MM by either rapid serum agglutination or recombinant antigen-based enzyme-linked immunosorbent assay. In addition, sera of all chickens from which MM was isolated were positive for antibodies to MM. Collectively, the data unambiguously show that MM could infect chickens; thus, MM warrants further exploration to determine its pathogenicity in this unusual host.     

Study on the vertical transmission of arthropathic and amyloidogenic Enterococcus faecalis in a flock of brown layer chickens

Enterococcus faecalis-related amyloid arthropathy was diagnosed in a sample of birds from a flock of brown layer parent chickens aged 57 weeks. E. faecalis was isolated from amyloidotic knee joints and from blood samples from lame birds. From this flock a smaller one was kept for production purposes to study the vertical transmission of arthropathic and amyloidogenic E. faecalis. Three batches of non-soiled and thoroughly disinfected eggs, to prevent egg-shell contamination, were collected for 6 weeks and submitted for incubation every 2 weeks. No joint pathology was found in the offspring chicks (n = 458) monitored for joint disease until 16 or 21 weeks of age. Fresh and candled eggs (infertiles and dead embryos) obtained at day 18 of the incubation period had negative results at bacteriological examination of the yolk sac, except one non-hatched egg (late embryonal death in shell) from which E. faecalis was isolated. Genotyping by pulsed-field gel electrophoresis (PFGE) of all E. faecalis isolates obtained from blood and joints (except one) of the parent birds and of the non-hatched egg after Sma I digestion showed that all isolates were genetically closely related or identical to a known arthropathic and amyloidogenic E. faecalis strain. In this study, vertical transmission of E. faecalis, although it may occur on a small scale as shown by PFGE, did not seem to play a significant role. Conversely, the chronicity of the condition and the development of an immune response may have affected the efficiency of its transmission.     

Clonality of Enterococcus faecalis associated with amyloid arthropathy in chickens evaluated by multilocus sequence typing (MLST)

The aim of the study was to evaluate the clonality of 21 strains of Enterococcus faecalis associated with arthritis and amyloid arthropathy by multilocus sequence typing (MLST). The strains originated from five countries. Fifteen of the 21 strains demonstrated the same sequence type, ST82, including a reference amyloid arthropathy strain from the Netherlands. Some of the demonstrated sequence types (ST36, ST59 and ST82) have also been described from human clinical samples while other types are reported for the first time. The results confirm previous observations that outbreaks of amyloid arthropathy seem to be clonally related and indicate a wide distribution of the predominant sequence type which was demonstrated in four countries in Europe and in the USA.     

猪源粪肠球菌的分离、鉴定及应用

通过培养特征、形态观察、生理生化鉴定、16SrDNA序列分析和种特异性片段扩增等方法,从健康猪直肠内容物中分离到3株粪肠球菌,其中菌株NA14对常见病原微生物具有较强的拮抗能力;进一步研究表明该菌株具有较强的耐受人工胃液和耐胆汁的能力,能够耐受60℃、30min;动物试验表明饲料中添加该粪肠球菌能够降低仔猪腹泻,改善仔猪生产性能,增强猪瘟疫苗的免疫效果;因此该菌株作为动物益生菌具有好的应用前景。     

鸭源肠球菌的致病性试验

以临床分离的2株鸭源肠球菌分别接种小白鼠,观察感染后小白鼠的主要临床症状和病变特点,并测定感染小白鼠的发病率、死亡率及半数致死量,旨在探讨这2个菌株对小白鼠的致病性。结果表明,2株肠球菌均能造成小白鼠发病。该病原主要侵害小白鼠肝脏、脾脏和肾脏,最明显的病变特征是引起脾肿大、纤维素性渗出性炎症;肝脏淤血、出血甚至坏死;肾间质血管淤血等。从而揭示了肠球菌可引起小鼠感染发病并导致组织病变而死亡。     

鸡霉形体的分离鉴定

从沈阳地区 2个鸡场的 6只病鸡取样 ,进行了霉形体的分离鉴定。通过培养特性、生化特性及血清学特性鉴定 ,分离出鸡败血霉形体 4株 ,分别命名为Sy1 株 ,Sy2 株 ,Sy4 株 ,Sy5株     

Susceptibility of Escherichia coli and Enterococcus faecium isolated from pigs and broiler chickens to tetracycline degradation products and distribution of tetracycline resistance determinants in E. coli from food animals

One hundred Escherichia coli isolates from diseased and healthy pigs, cattle and broiler chickens were screened for the presence of tetracycline resistance genes tet(A), (B), (C), (D) or (E). The tet(A) gene was the most abundant (71% of the 100 isolates) followed by tet(B) (25%). The predominance of tet(A) and tet(B) applied to all three animal species, and there was no difference between the distribution of tet(A) and tet(B) genes among non-pathogenic and pathogenic E. coli in any of the animal species. The susceptibility of 20 of these isolates together with 10 tetracycline sensitive E. coli and 18 tetracycline resistant and 10 sensitive Enterococcus faecium to tetracyclines and tetracycline degradation products was determined. The resistant isolates showed reduced resistance to anhydrotetracycline, 4-epi-anhydrotetracycline, anhydrochlortetracycline and 4-epi-anhydrochlortetracycline. In general both the tetracycline resistant and susceptible E. faecium were more susceptible to the compounds tested than E. coli.     

Isolation of Mycoplasma capricolum-like strains from chickens

Members of the genus Mycoplasma infect a wide range of hosts, but individual Mycoplasma species tend to exhibit a considerable degree of host specificity. We characterized Mycoplasma strain 700, isolated from a kidney of a layer hen in Spain and Mycoplasma strains ULB-A and ULB-B, isolated from the air sac and from the bile of stunted broiler chickens in Slovenia. The serologic examination showed that these three strains are antigenically unrelated to all of the recognized Mycoplasma species of avian origin, but closely related to the ruminant mycoplasma Mycoplasma capricolum subspecies capricolum (M. capricolum). The comparison of their 16S rRNA gene sequences with the sequence of M. capricolum (California kid) revealed 99.66% sequence identity for the strain 700 and 99.59% identity for strains ULB-A and ULB-B. Moreover, the predicted DnaK sequences of the M. capricolum-like strains, isolated from chickens, were identical to DnaK sequences of M. capricolum. Comparison of their dnaK gene sequences with M. capricolum showed 99.64% sequence identity for strain 700 and 99.27% identity for strains ULB-A and ULB-B. In the flock from which M. capricolum-like strains ULB-A and ULB-B were isolated, the majority of chickens (83% of the chickens examined) raised antibodies reacting with M. capricolum antigens. Notably, the infection of chickens with M. capricolum-like strains represents an unusual exception to the range of Mycoplasma species host specificity.     

Genotyping of Toxoplasma gondii isolates from chickens from India

The present study was undertaken to isolate and genotype Toxoplasma gondii from free-range chickens (Gallus domesticus) from villages in Maharashtra and Tamil Nadu states of central and south India, respectively. Blood, heart, and brain from a total of 741 chickens were examined for T. gondii infection. Antibodies to T. gondii, as assayed with the modified agglutination test (MAT >or = 1:5) were found in 133 (17.9%) chickens. Hearts and brains of 186 chickens were bioassayed in mice. Additionally, hearts and/or brains of most of the seronegative (MAT < 1:5) chickens were fed to 20 T. gondii-free cats, while 32 seropositive chickens (MAT 1:5) were fed to 3 cats. T. gondii was not isolated from any of the chickens by mouse bioassay. Five of the cats that were fed seronegative chickens shed oocysts, while isolates were not obtained from any of the other cats fed seropositive chickens. These five isolates, along with the two that were previously isolated in India through cat bioassay, were genetically analyzed. Genotyping using the SAG 2 locus indicated that two isolates were type II and five were type III. Microsatellite analysis revealed allelic differences between and within the lineages. This is the first report of genetic characterization of any T. gondii isolate from India.     

Cryptosporidiosis in chickens from southern Spain

Cryptosporidiosis in chickens from southern Spain is reported. Cryptosporidia were found in the trachea, esophagus, and epithelium of the bursa of Fabricius in chickens from different regions. In one flock, cryptosporidiosis was associated with respiratory problems and high mortality. In another flock, it was associated with low mortality and weight loss.     

Effects of Enterococcus faecium supplementation and floor type on performance, morphology of erythrocytes and intestinal microbiota in broiler chickens

1. The experiment was to study the effects of floor type and probiotic supplementation (Enterococcus faecium) on performance, morphology of erythrocytes and intestinal microbiota of male Ross 308 broiler chickens. 2. The experimental design was a factorial 2 × 2 with 6 replicates. The factors were floor type (wire floor versus wood shaving litter) and the presence or absence of probiotic. 3. Birds housed on wood shavings exhibited significantly improved weight gain and food intake. 4. Addition of E. faecium led to significantly decreased food intake and gizzard weight. Supplementation with E. faecium positively influenced the ileal and caecal microbiota, with a significant decrease in the population of Escherichia coli. 5. Erythrocyte length decreased and erythrocyte width increased in the birds housed on wood shavings.     

鲤鱼源粪肠球菌增菌培养基某的优化

利用SAS软件研究鲤鱼源粪肠球菌(Enterococcus fae clis)YL-1增茵培养基,首先,通过Plackett-Burman试验设计在培养基的各个因子中筛选出对YL-1生长有利的重要因子,然后在Box-Behnken试验设计下确定最大响应值,并通过优化的结果验证.结果表明预测值和实际值具有良好的拟合性,优化模型可靠,确定YL-1最佳培养基为:蛋白胨1.365%、牛肉膏1-25%、酵母浸粉0.625%、麦芽糖2.5%、吐温-80 0.1%、KH2PO4 0.2%、柠檬酸三铵0.16%、MgSO4 0.25%、初始pH值6.4.培养优化后YL-1的茵体浓度达1.71×10.CFU/ml.     

毛皮动物源粪肠球菌的分离鉴定

为获得毛皮动物源粪肠球菌,并评价其益生特性,本研究从健康成年毛皮动物（水貂、狐狸、貉）粪便中分离粪肠球菌,通过形态学观察、生化试验和16S rRNA序列分析等方法进行种属鉴定。测定分离菌株生长曲线、产酸能力、抗菌药敏感性,并挑取部分菌株测定其对于温度、人工胃液、人工胆盐的耐受能力。结果表明,分离菌株中共5株为革兰氏阳性菌,生化特性与粪肠球菌标准株基本相符,且经16S rRNA序列分析鉴定为粪肠球菌。5株菌均于培养后2 h进入对数期,8~10 h进入稳定期,且具有弱产酸能力。分离菌株对四环素、左氟沙星耐药性强,耐药率为100%,其次为青霉素（80%）、红霉素（80%）、庆大霉素（80%）、氯霉素（40%）,对氨苄西林和万古霉素敏感。水貂、狐狸和貉源的粪肠球菌对于60 ℃以下温度处理、pH>3.0的人工胃液、0.3%~0.5%浓度的胆盐耐受能力较强,对70 ℃以上高温和pH<3.0的人工胃液耐受性差。综上所述,本研究共获得5株毛皮动物源（水貂、狐狸、貉）粪肠球菌,分离菌株繁殖较快,适合在毛皮动物肠道中定植并发挥益生作用,且具有较好的益生特性和抗逆性,可作为动物用微生态制剂的候选菌种进一步研究。