Minimization of pathologic changes in Ornithobacterium rhinotracheale infection in turkeys by temperature-sensitive mutant strain

The protection elicited by a temperature-sensitive (Ts) mutant of Ornithobacterium rhinotracheale (ORT) vaccine against challenge with pathogenic strain was investigated. In Experiment 1, specific serologic response to ORT was detected in 12%-19% of Ts-vaccinated birds at 3 wk postvaccination by either drinking water or oculo-nasal instillation. At 7 days postchallenge, 100% of Ts-vaccinated turkeys of all groups were able to respond with an ORT-specific antibody response, but the control group was not, suggesting the potential of Ts strain to evoke immune protection. The study also revealed a statistically significant ability of the Ts strain to protect vaccinated turkeys against gross lesions caused by the pathogenic strain of ORT in treated groups vs. control. In Experiment 2, seroconversion was detected by enzyme-linked immunosorbent assay in birds after they were given the Ts strain in drinking water in field conditions. The results of the field study showed mean scores of gross lesions of nonvaccinated/challenged groups to be up to seven times higher than those of the vaccinated/challenged group. In addition, reisolation rates and quantification of ORT colonies per gram of lung tissue were significantly lower for vaccinated/challenged than for nonvaccinated/challenged turkeys. In conclusion, results from laboratory and field experiments suggest that use of the Ts mutant strain of ORT as a live vaccine would be a suitable method to evoke protection against ORT infection in turkeys.     

Outer membrane proteins for serologic detection of Ornithobacterium rhinotracheale infection in turkeys

Ornithobacterium rhinotracheale (ORT) is a bacterium responsible for a respiratory disease in turkeys and chickens and has been identified as one of the emerging respiratory bacterial pathogens. The clinical signs and lesions caused by ORT are very similar to those caused by other respiratory infectious agents; therefore, an accurate diagnostic test is necessary to identify the infection. In this study, we have investigated the use of outer membrane proteins of ORT in an indirect enzyme-linked immunosorbent assay (ELISA) to detect the exposure to ORT infection. Outer membrane proteins of ORT were extracted and used as an antigen in ELISA to detect infection in turkeys exposed to different serotypes of ORT. The ELISA results were compared with the conventional serum plate agglutination test. The agglutination test detected specific antibodies for ORT in 65% of experimentally infected turkeys during the first 2 wk of infection. The ELISA detected up to 100% of infected birds for 8 wk postinfection. The results suggest that ELISA is able to detect the exposure to ORT in later stages of the infection and this assay can be used in serologic surveillance of ORT infection for poultry in the field.     

Isolation and differentiation of a cytochrome oxidase-negative strain of Ornithobacterium rhinotracheale from turkeys

The present study describes the first isolation and identification of a cytochrome oxidase-negative strain of Ornithobacterium rhinotracheale. This strain was isolated from 19-week-old turkeys with severe respiratory signs and was identified using biochemical, cytochemical and serological test methods. These findings indicate that cytochrome oxidase-negative Ornithobacterium rhinotracheale strains become probably also an important rule as a causative agent.     

The use of a bacterin vaccine in broiler breeders for the control of Ornithobacterium rhinotracheale in commercial broilers

Ornithobacterium rhinotracheale (ORT) is a recently identified bacterial pathogen of poultry, linked to the respiratory disease complex of broilers and the economic losses associated with that disease complex. Present control measures applied for the disease include the continuous use of in-feed antibiotics. A recently developed bacterin vaccine that is applied to broiler-breeder hens to pass on protective immunity to their broiler progeny was tested under large-scale commercial conditions in South Africa. An indirect ELISA test for antibodies to ORT, optimised for use in South Africa, was used to determine antibody levels in breeders and broilers. ELISA test results showed that the vaccine stimulated the development of high antibody titre levels in broiler breeders. The efficacy of the vaccine in protecting the progeny of these birds from ORT challenge could not be determined during the trial, although the progeny of vaccinated hens appeared to perform slightly better under commercial conditions than the progeny of unvaccinated hens.     

Use of ovotransferrin as an antimicrobial in turkeys naturally infected with Chlamydia psittaci, avian metapneumovirus and Ornithobacterium rhinotracheale

Respiratory pathogens are difficult to control in large-scale turkey production. This report describes a clinical trial of antimicrobial ovoTF aerosol on a large Belgian turkey farm. ovoTF was administered to reduce Chlamydia psittaci (C. psittaci) infections and to study the impact of this action on the occurrence of Ornithobacterium rhinotracheale (O. rhinotracheale) and avian metapneumovirus (aMPV) infections. Two subsequent broods were included; (i) a control brood receiving no ovoTF and (ii) an ovoTF brood receiving ovoTF aerosol (5mg/animal) at the age of 2 weeks, continuing daily for 12 days. Twenty-four one-day-old toms of the control and ovoTF brood were tagged and monitored for 15 weeks. The control brood experienced two periods of respiratory disease, the first (2-3 weeks of age) due to C. psittaci and the second (8-17 weeks of age) in the presence of C. psittaci, O. rhinotracheale and maybe aMPV. Extensive antibiotic treatment was needed in 2, 8 and 9 week-old toms. In the ovoTF brood, toms stayed healthy until the age of 9 weeks, whereafter respiratory disease occurred in the presence of C. psittaci, O rhinotracheale and aMPV. OvoTF administration: (i) reduced the amount of C. psittaci in the air as demonstrated by bioaerosol monitoring, (ii) prevented respiratory disease during the first half of the brood period, (iii) was associated with 46% reduction of mortality, and (iv) reduced the antibiotic cost. Our results justify additional clinical trials to explore the use of this innovative antimicrobial strategy for poultry.     

Isolation and characterization of Ornithobacterium rhinotracheale from chickens in Brazil

Ornithobacterium rhinotracheale (ORT) is a recently described species of bacterium associated with respiratory disease, growth retardation, mortality, and decreased egg production in chickens and turkeys. Pneumonia, pleuritis, and airsacculitis characterise the infection. ORT has been isolated in many countries but it is still considered exotic in Brazil. Up to date it is prohibited to import and produce reagents for diagnostic and vaccination control. The aim of this study was to isolate and identify the bacteria in chickens. Four isolates were obtained from tracheal swabs of broilers. They were isolated in blood agar with gentamicin and showed biochemical, morphological, antigenic and genetic characteristics of ORT. The results confirm that ORT is present in Brazil.     

Studies of the transmission routes of Ornithobacterium rhinotracheale and immunoprophylaxis to prevent infection in young meat turkeys

The importance and prevention of the horizontal as well as the vertical transmission of Ornithobacterium rhinotracheale were investigated. In our first experiment we observed that specific-pathogen-free broiler chickens that were placed in hatching incubators at a commercial turkey hatchery during hatch showed respiratory tract lesions at postmortem examination that were positive for O. rhinotracheale by bacteriology and immunohistology. It appeared that vertical transmission occurred and that horizontal transmission of O. rhinotracheale is possible. In a second experiment, the turkeys derived from vaccinated parents showed significantly fewer respiratory tract lesions at postmortem examination at 16 days of age than the birds derived from nonvaccinated parents. In a third experiment, all vaccinated young birds, regardless of the vaccination state of their parents, showed significantly fewer respiratory tract lesions at 6 wk of age. We concluded that vaccination of the breeders reduces vertical transmission and that vaccination of the progeny is needed to resist challenge at 6 wk of age.     

Phenotypic and molecular characterization of isolates of Ornithobacterium rhinotracheale from chickens and pigeons in Taiwan

Forty Ornithobacterium rhinotracheale (ORT) strains were isolated from 28 chickens and 12 pigeons for the first time in Taiwan. All isolates reacted positively in the p-nitrophenyl-beta-D-galactopyranoside (PNPG) and oxidase tests, showing an API 20NE identification system biocode 0-0-2-0-0-0-4. All the pigeon isolates and 85.7% (24 of 28) of the chicken isolates belonged to serotype A. Compared to the ORT ATCC 51464 strain, 14.3% (4 of 28) of chicken isolates and 58.3% (7 of 12) of pigeon isolates showed smaller colonies after 72 hr incubation. Most of the chicken isolates (22 of 28), but none of the pigeon isolates, could agglutinate chicken and pigeon red blood cells. There appears to be a correlation that ORT isolates with a larger colony size tend to be more able to agglutinate red blood cells than the ORT isolates with a smaller colony size. A majority of isolates was sensitive to amoxicillin, ampicillin, ceftiofur, penicillin, and oxytetracycline. The 16S ribosomal RNA (rRNA) sequences of 23 Taiwanese ORT isolates showed high identity (98%-100%) to sequences in GenBank. Phylogenetic analysis of these sequences showed that pigeon isolates formed a distinctive cluster, while chicken isolates and all other 16S rRNA sequences obtained from GenBank belonged to another two clusters. The results indicate that pigeon ORT isolates are different from most chicken isolates in regard to a number of phenotypic and molecular traits.     

鸭疫里默氏杆菌类似crp基因的鉴定及其突变株对雏鸭免疫效果的评价

旨在鉴定鸭疫里默氏杆菌(Riemerella anatipestifer)中的crp基因,并探究该基因与菌株毒力的关系,了解crp基因突变株的免疫保护效力,为开发新型R.anatipestifer减毒活疫苗奠定基础。采用P-BLAST在R.anatipestifer蛋白质组中搜寻与沙门菌crp基因相似的蛋白基因,发现R.anatipestifer CH-1株的B739-0373基因与沙门菌crp基因相似度为45%;将B739-0373基因克隆到表达载体上,并回补到沙门菌Δcrp突变株中,利用麦芽糖-麦康凯培养基颜色反应对其糖代谢功能进行鉴定,结果发现该基因可完全回补沙门菌自身crp的缺失,推测R.anatipestifer CH-1株的B739-0373基因具有类似沙门菌crp基因的功能;利用同源重组和自杀质粒,构建R.anatipestifer CH-1Δcrp突变株,比较野生及突变株的生长情况和毒力(包括黏附率、侵染率、LD_(50)、在雏鸭肝脑中的定殖能力),结果表明,crp基因的缺失使菌株生长变得缓慢,黏附力(P0.01)和侵染力(P0.001)显著下降,对雏鸭的半数致死量(LD_(50))显著提高,在雏鸭肝脏、脑中定殖能力也显著下降(P0.01);在CH-1Δcrp突变株免疫雏鸭后7,14,21 d,检测雏鸭血清中IgG水平,并以R.anatipestifer野生株对免疫后的雏鸭进行肌注攻毒,观察其临床症状和免疫保护率,发现Δcrp突变株能刺激雏鸭产生高水平的IgG抗体(P0.01),对雏鸭的免疫保护率为100%。综上表明, B739-0373基因鉴定为R.anatipestifer CH-1株的类似crp基因,可全局调控该菌株的生长和毒力;安全剂量的CH-1Δcrp突变株可诱导雏鸭产生较强的免疫保护效力,可作为新型减毒活疫苗的候选菌株。     

Efficacy of enrofloxacin, florfenicol and amoxicillin against Ornithobacterium rhinotracheale and Escherichia coli O2:K1 dual infection in turkeys following APV priming

Experimental groups of 15 susceptible 3-week-old turkeys were inoculated oculonasally with avian metapneumovirus (APV) subtype A and susceptible Escherichia coli O2:K1 and Ornithobacterium rhinotracheale (ORT) bacteria, with a 3 days interval between viral and bacterial inoculation and approximately 8h between the two bacterial inoculations. The aims of the present study were to assess the efficacy of drinking-water administration of enrofloxacin for 3 and 5 days, amoxicillin for 5 days and florfenicol for 5 days for the treatment of the resulting respiratory disease, based on clinical and bacteriological examinations. Antimicrobial treatment started 1 day after dual bacterial inoculation. After infection, the birds were examined and scored for clinical signs daily, weighed at different times, and their tracheae swabbed daily. Five birds were euthanised and examined for macroscopic lesions at necropsy at 5 days post-bacterial inoculation (dpbi) and the remainder at 15dpbi. Samples of the turbinates, trachea, lungs, sinuses, air sacs, heart, pericardium and liver were collected for bacteriological examination. Recovery from respiratory disease caused by an APV/E. coli/ORT triple infection in 3-week-old turkey poults was overall most successful after enrofloxacin treatment, irrespective of treatment duration, followed by florfenicol treatment. Compared with the untreated group, clinical signs as well as ORT and E. coli multiplication in the respiratory tract were significantly reduced by both enrofloxacin treatments and the florfenicol treatment, with the enrofloxacin treatments showing significantly better reductions than the florfenicol treatment. Five-day treatment with amoxicillin, compared with the untreated group, did not cause a significant reduction in any of the aforementioned parameters.     

马耳它布氏杆菌bp26基因缺失株的构建及鉴定

为了建立马耳他布氏杆菌M5-90株弱毒疫苗株与野生株的鉴别诊断,本研究以bp26基因作为重组靶位点,以M5-90为亲本,利用bp26基因ORF外侧序列作为同源序列,将卡那霉素抗性基因(Kan')整合到细菌基因组中,双交叉重组阳性菌株,经SDS-PAGE和western blot试验表明迁移率约为29 ku的BP26蛋白在亲本菌中表达并可被鼠抗BP26高免血清识别,而突变株M5-90-26反应结果为阴性,表明BP26蛋白在突变疫苗株M5-90-26中未表达.M5-90-26具备从血清学角度区分M5-90-26免疫与野生型布氏杆菌感染,对布氏杆菌病防制、监测及净化具有重要的意义.     

The role of rpoS,hmp, and ssrAB in Salmonella enterica Gallinarum and evaluation of a triple-deletion mutant as a live vaccine candidate in Lohmann layer chickens

Salmonella enterica Gallinarum (SG) causes fowl typhoid (FT), a septicemic disease in avian species. We constructed deletion mutants lacking the stress sigma factor RpoS, the nitric oxide (NO)-detoxifying flavohemoglobin Hmp, and the SsrA/SsrB regulator to confirm the functions of these factors in SG. All gene products were fully functional in wild-type (WT) SG whereas mutants harboring single mutations or a combination of rpoS, hmp, and ssrAB mutations showed hypersusceptibility to H₂O₂, loss of NO metabolism, and absence of Salmonella pathogenicity island (SPI)-2 expression, respectively. A triple-deletion mutant, SGΔ3 (SGΔrpoSΔhmpΔssrAB), was evaluated for attenuated virulence and protection efficacy in two-week-old Lohmann layer chickens. The SGΔ3 mutant did not cause any mortality after inoculation with either 1 × 10⁶ or 1 × 10⁸ colony-forming units (CFUs) of bacteria. Significantly lower numbers of salmonellae were recovered from the liver and spleen of chickens inoculated with the SGΔ3 mutant compared to chickens inoculated with WT SG. Vaccination with the SGΔ3 mutant conferred complete protection against challenge with virulent SG on the chickens comparable to the group vaccinated with a conventional vaccine strain, SG9R. Overall, these results indicate that SGΔ3 could be a promising candidate for a live Salmonella vaccine against FT.     

Evaluation of a protease activation mutant of Sendai virus as a potent live vaccine.

A protease activation mutant of Sendai virus, TR-5, was investigated as a candidate for a live vaccine. Vaccination with TR-5 which had been activated by chymotrypsin beforehand (active TR-5) elicited protective immunity against otherwise lethal challenge infection with wild-type Sendai virus in DBA/2, C3H and ICR strains of mice. Less of the active TR-5 was required to confer protection on mice compared with an ordinary ether-inactivated Sendai virus vaccine (split vaccine). The protective immunity elicited by TR-5 lasted longer and the booster effect was more prominent compared to the split vaccine. No seroconversion was observed with contact mice when housed in a cage with mice vaccinated with the active TR-5. The overall results show that the active TR-5 is an effective and safe live vaccine of Sendai virus in mice.     

Potential use of a transposon Tn916-generated mutant of Aeromonas hydrophila J-1 defective in some exoproducts as a live attenuated vaccine

Aeromonas hydrophila is a Gram-negative opportunistic pathogen that causes disease in a wide range of hosts due to its multifactorial virulence. Here we describe the application of transposon insertion mutagenesis approach to obtain an exoenzyme mutant of A. hydrophila strain J-1. Immunization of swordtail fish (Xiphophorus helleri Heckel) with the highly attenuated mutant provided protection (survival of 27 out of 35 fish, compared to survival of only 13 out of 35 control fish) in the fish given the highest immunization dose (10(7) CFU) against intraperitoneal challenge with the wild J-1 strain. Immunization with doses of 10(5) or 10(3) did not provide significant protection.     

Isolation and characterization of temperature-sensitive mutants of Streptococcus suis: efficacy trial of the mutant vaccine in mice

A model of experimental Streptococcus suis infection was developed in young mice. Minimum lethal dose (MLD) values were calculated for four virulent serotypes (1/2, 1, 2, 3) of S. suis using this model. Temperature-sensitive (ts) mutants of S. suis serotypes 1/2 and 1-8 were isolated and characterized on the basis of their growth kinetics and reversion rates. Ts mutants of S. suis 1/2, 1, 2, and 3 were tested as vaccines against the virulent homologous and heterologous challenges in mice. The protection provided was evaluated by analyzing the clinical signs, death or survival. Homologous but not heterologous protection was noted in all mice vaccinated with the mutant strains. Ts mutants of S. suis 1/2 provided 100% protection against challenge by virulent strains of S. suis 1/2, 1, and 2.     

Efficacy and safety of a cell-culture live virus vaccine for hemorrhagic enteritis of turkeys: laboratory studies

Poults free from hemorrhagic enteritis (HE) antibody were vaccinated by gavage at 1 day or 2 weeks of age with a live HE vaccine virus that had been propagated in a Marek's disease (MD)-induced B-lymphoblastoid cell line of turkey origin. Vaccinated and unvaccinated poults were challenged with a virulent HE virus at various times postvaccination. One hundred tissue-culture-infectious doses of the vaccine virus per poult were sufficient to induce a serological response as well as to protect poults against HE lesions and mortality. Vaccinated poults were protected against the disease as early as 1 week and as late as 8 weeks PV. The vaccine was efficacious by several routes of application. The vaccine virus spread horizontally from vaccinated to contact-exposed poults, as indicated by seroconversion and resistance of contact-exposed poults to challenge. The vaccine had no detectable harmful effects on the humoral immune response to particulate antigens or on weight gain of vaccinated poults. The vaccine proved to be free from MD virus, as indicated by the absence of MD lesions and antibody in 8-week-old chickens inoculated intra-abdominally with the vaccine at hatching. These findings indicate that the cell-culture-propagated HE vaccine is efficacious and safe.     

Seroprevalence of Ornithobacterium rhinotracheale infection in broiler and broiler breeder chickens in West Azerbaijan Province, Iran

Ornithobacterium rhinotracheale is a pleomorphic Gram-negative rod shaped bacterium of the rRNA superfamily V that is associated with respiratory disease in poultry. This study was conducted to determine the seroprevalence of O. rhinotracheale infection in broiler and broiler breeder chickens in West Azerbaijan (Urmia lake region) by using a commercial enzyme-linked immunosorbent assay. In this study, 463 serum samples were obtained from 50 broiler flocks and 472 blood sera from 42 broiler breeder flocks. Results showed that 41 broiler flocks (82%) and 39 broiler breeder flocks (92.8%) were positive. Ornithobacterium rhinotracheale antibodies were detected in 205 (44.2%) of the 463 broiler serum samples. Of the 472 blood sera examined from broiler breeder, 340 (72%) were positive. The results of this study indicated that the prevalence of O. rhinotracheale antibodies is high in the broiler and broiler breeder flocks in West Azerbaijan.     

Efficacy in turkeys of spray vaccination with a temperature-sensitive mutant of Bordetella avium (Art Vax)

Broad-breasted white turkeys were vaccinated with a temperature-sensitive mutant of Bordetella avium (Art Vax) at 2 and 15 days of age and challenged at 22 days of age by contact with infected birds. Necropsy was performed at 35 days of age. Two vaccination protocols (eyedrop/oral and spray cabinet/spray bottle) and two challenge isolates (Arkansas 105 and North Carolina [NC] isolates) were used. Neither the spray nor the eyedrop/oral methods of vaccination prevented infection of the anterior trachea with either of the virulent challenge strains. The spray and eyedrop/oral methods of vaccination were equally effective in reducing the severity of gross lesions in the trachea. The vaccine reduced the severity of gross lesions in the tracheas of turkeys challenged with the NC isolate to a level approximately equal to that observed in unchallenged vaccinated controls, but the vaccine only moderately reduced the severity of lesions in birds challenged with the 105 isolate.     

Auxotrophic, plasmid-cured Salmonella enterica serovar typhimurium for use as a live vaccine in calves

Calves were vaccinated orally, subcutaneously or intraperitoneally with a smooth, plasmid-cured strain of Salmonella enterica serovar typhimurium, strain 81. Oral vaccination was not effective, as only 1/5 calves survived challenge with virulent S. typhimurium. Strain 81 was attenuated for calves, as only a slight rise in rectal temperatures was detected after vaccination. The organism was excreted by some calves in the faeces, but no signs of diarrhoea were observed after vaccination. After parenteral vaccination, strain 81 was able to reach the intestines, gastric associated lymphoid tissues and other internal lymphoid tissues and remained viable for up to 14 days in the bovine host. After oral challenge with a virulent strain, 9/10 vaccinated calves survived challenge as opposed to 4/10 control calves (p<0.5). Diarrhoea was present in all calves of the control groups, but in only 4/10 of the vaccinated calves. The clinical reactions of the vaccinated calves were milder than in the control calves, as the rises in rectal temperatures were lower, diarrhoea was less severe, and the challenge strain was present in fewer organs from vaccinated calves than control calves. This study showed that parenterally administered Salmonella vaccines can induce both mucosal and systemic immunity, and it is postulated that this capability of strain 81 is related to its colonisation of lymphoid tissues and other systemic and intestinal tissues. This study confirmed that plasmid-cured strains were attenuated in the bovine host and conferred significant protection after parenteral vaccination, but not oral vaccination.