Limitation of the spread and impact of infectious coryza through the use of a continuous disinfection programme

The effect of a continuous disinfection programme, using the non-toxic disinfectant Virukill, in layers, on the spread and impact of infectious coryza, caused by Haemophilus paragallinarum was evaluated. In this experiment, both unvaccinated layers and layers vaccinated against infectious coryza were used. Duplicate smaller groups of vaccinated and unvaccinated chickens were challenged with different serovars of both NAD-dependent as well as NAD-independent isolates of Haemophilus paragallinarum. One group of chickens challenged with each of the different becterial serovars was treated with the continuous disinfection programme, while the other group remained as the untreated controls. The clinical signs of infectious coryza were evaluated over a period of 20 days in each group. The egg production over this period was also evaluated. It was found in all experimental challenges, that the severity of the symptoms was reduced in the birds receiving the continuous disinfection programme. The drop in egg production was also found to be less severe in the treated groups when compared to the untreated control groups. The duration of infection was found to be either unchanged, or shorter in the birds treated with the continuous disinfection programme. In none of the experimental challenges was the duration or expression of clinical signs of IC increased due to the continuous disinfection programme.     

Serovar identification, antimicrobial sensitivity, and virulence of Avibacterium paragallinarum isolated from chickens in Thailand

Avibacterium paragallinarum causes infectious coryza in chickens, an acute respiratory disease that has worldwide economic significance. The objectives of this study were to determine the serovars, antimicrobial resistance, and pathogenicity of A. paragallinarum isolated from chickens in Thailand. Eighteen field isolates of A. paragallinarum were confirmed by PCR. When examined by serotyping in a hemagglutination inhibition test, 10 isolates were serovar A, five isolates were serovar B, and three isolates were serovar C. The susceptibility of the isolates to 16 antimicrobial agents was tested by a disk diffusion method. All isolates were susceptible to amoxicillin-clavulanic acid. There was a high level of resistance to lincomycin and erythromycin. All isolates were resistant to cloxacillin and neomycin. A study of bacterial entry into, and survival within, chicken macrophages showed variation between isolates but no clear connection to serovar. A virulence test was performed by challenging 4-wk-old layers via the nasal route with 400 dl of bacteria (10(8) colony-forming units/ml). Clinical signs were observed daily for 7 days, and the birds were subjected to a postmortem necropsy at 7 days postchallenge. All 18 field isolates caused the typical clinical signs of infectious coryza and could be re-isolated at 7 days after challenge. There was no significant difference in the clinical scores of the isolates except that two isolates (112179 and 102984, serovars A and B, respectively) gave a significantly higher score than did isolate CMU1009 (a serovar A isolate). No correlation between serovar and severity of clinical signs was found.     

Development of an enzyme-linked immunosorbent assay for the measurement of antibodies against infectious coryza vaccine

Infectious coryza is an acute respiratory disease caused by infection with Avibacterium (Haemophilus) paragallinarum. It is characterized by nasal discharge and facial swelling and is associated with growth retardation and a reduction in egg production. Hemagglutination inhibition (HI) tests are used to estimate vaccine-induced immunity against infectious coryza in vitro; however, these procedures are complicated and their sensitivity is insufficient. To address these problems, an enzyme-linked immunosorbent assay (ELISA) technique using serovar-specific regions of HMTp210 (210 kDa), an outer-membrane protein of A. paragallinarum, was developed to measure the antibodies against infectious coryza. Chickens with an ELISA titer of 0.3 or more did not exhibit clinical signs of infectious coryza against challenge with A. paragallinarum, although their HI antibody titers were negative. On the other hand, chickens with an ELISA titer below 0.3 exhibited clinical signs of the disease with one exception. Antibody prevalence rates on ELISA were 80% and 60% against infection with serovars A and C, respectively, and ELISA also detected antibodies in chickens infected with A. paragallinarum with a sensitivity higher than that of HI tests. Taken together, the ELISA technique developed in this study is a valuable tool for the measurement of antibodies produced against the infectious coryza vaccine or in response to an infection with A. paragallinarum.     

Virulence of the nine serovar reference strains of Haemophilus paragallinarum

The virulence of the reference strains of the nine currently recognized Kume serovars of Haemophilus paragallinarum was investigated. The capacity of the H. paragallinarum strains to cause the typical clinical signs of upper respiratory tract disease associated with infectious coryza in unvaccinated, nasal-challenged chickens was assessed. Differences in virulence were assessed by means of a standardized scoring system for clinical signs. All nine strains were pathogenic to chickens, producing typical clinical signs of infectious coryza. The highest clinical signs score was obtained for serovar C-1 (1.72), while the lowest clinical signs score was obtained for serovar C-4 (0.32). Our results indicate that virulence differences exist among the serovars of H. paragallinarum.     

Vaccination of one-day-old broiler chicks against infectious coryza

In order to evaluate the protection conferred by an experimental inactivated vaccine against infectious coryza, three challenge trials were undertaken using 112 1-day-old broilers. The vaccine "Hepa Inmuno NC" included bacterial antigens of Avibacterium paragallinarum (serogroups A, B, variant B, and C) as well as antigens of Newcastle virus and hepatitis virus. Fifty-six broiler chicks were vaccinated at the first day of life at the hatchery while another 56 chicks were left unvaccinated. Three infection trials were conducted simultaneously using each of the three serogroups A, B, or C of Av. paragallinarum. In each trial, 17 vaccinated and 17 unvaccinated broilers were used. Challenge was performed at day 31 of life by injection, into the left infraorbital sinus, of approximately 1 x 10(5) colony forming units of the corresponding Av. paragallinarum strain. Clinical signs were recorded on day 2 postchallenge. All broilers were euthanatized and both infraorbital sinuses were bacteriologically examined for the presence of Av. paragallinarum on day 5 postchallenge. In comparison with the unvaccinated broilers, the vaccine significantly reduced the number of broilers with clinical signs after challenge with serogroup B, and significantly fewer vaccinated broilers were positive for the presence of Av. paragallinarum after challenge with serogroup C. On the other hand, no significant protection was observed when broilers were challenged with Av. paragallinarum from serogroup A. Despite the high infection rates in vaccinated chicks after an experimental infection with Av. paragallinarum, it was possible to reduce colonization of Av. paragallinarum (serogroup B) and clinical signs (serogroup C) in broiler chicks by vaccination at the first day of life. Further cross-protection trials should be done, including other Av. paragallinarum strains in the vaccine, especially those from serogroup A.     

An evaluation of the cross-protection afforded by inactivated infectious coryza vaccines

The cross-protection afforded by three inactivated infectious coryza vaccines was evaluated. Each vaccine contained one of the following strains of Haemophilus paragallinarum, HP31, HP60 and HP14. Strain HP31 belongs to Kume serovar C-2, strain HP60 belongs to Kume serovar C-4 while strain HP14 belongs to Kume serovar A-4. Four groups of twenty six-week-old specific-pathogen-free chickens were either given a single dose of an aluminium-hydroxide based vaccine (three groups) or left as unvaccinated controls. Three weeks after vaccination, all four groups were challenged with virulent H paragallinarum. One half of each group was challenged with the strain HP31 and the other half with strain HP60. The efficacy of the vaccines was assessed in terms of the prevention of the typical clinical signs and macroscopic lesions of infectious coryza as well as the prevention of any colonisation by the challenge organism. The serovar C-2 and serovar C-4 vaccines protected more than 50% of birds against either a serovar C-2 or C-4 challenge while the serovar A-4 vaccine protected less than 50% of birds.     

Effects of differences in virulence of different serovars of Haemophilus paragallinarum on perceived vaccine efficacy

The virulence of four South African field isolates of NAD-dependent Haemophilus paragallinarum and two field isolates of NAD-independent H. paragallinarum has previously been tested in unvaccinated chickens. In this study, the disease profiles caused by the NAD-dependent isolates of H. paragallinarum in vaccinated chickens were studied. It was shown that the clinical signs induced in the vaccinated chickens were substantially less severe than were those in unvaccinated chickens, as was expected. However, due to the high virulence of the serovar C-3 isolates, clinical signs in the vaccinated chickens challenged with this isolate were still detected. These were as severe as those occurring in unvaccinated chickens challenged with serovar B-1 isolates. Although the clinical signs induced in unvaccinated birds challenged with serovar A-1 were more severe than those occurring when vaccinated birds were challenged with serovar C-3, the overall disease profiles were similar. Substantial clinical signs were recorded in vaccinated birds challenged with serovar C-3. This could be interpreted as vaccination failure if the disease profile obtained in unvaccinated birds is not considered. It was found that a high level of protection was provided by this vaccine against challenge by serovar C-3. The high virulence of this serovar resulted in the development of clinical signs in vaccinated birds. These findings could possibly explain the large number of so-called vaccination failures that are reported in South Africa.     

Characterisation of isolates of Haemophilus paragallinarum from Indonesia

OBJECTIVE: To characterise 18 isolates of Haemophilus paragallinarum isolated from chickens in Indonesia. PROCEDURE: The isolates were identified to species level by traditional phenotypic methods. Six of the isolates were also identified by a species-specific polymerase chain reaction. Fourteen of the isolates were examined for resistance to a panel of seven antimicrobial agents using a disc diffusion method. All 18 isolates were serotyped according to the Page scheme using reference antisera in a haemagglutination inhibition test. RESULTS: Four of the 18 isolates were obtained from indigenous (kampung) chickens, with the remainder being from typical intensive poultry production systems. The 18 isolates were obtained from 11 outbreaks that showed the typical clinical signs of infectious coryza and 11 of the isolates were obtained from chickens that had been vaccinated with infectious coryza vaccines. All 18 isolates were confirmed as H paragallinarum by biochemical testing and six isolates were also identified as H paragallinarum by the polymerase chain reaction test. Eleven isolates were resistant to erythromycin and streptomycin, 10 to neomycin, eight to oxytetracycline, five isolates to doxycycline, three to sulphamethoxazoltrimethoprim but only one to ampicillin. Seven isolates were Page serovar A, four were Page serovar B and seven were Page serovar C. CONCLUSION: The presence of all three Page serovars (A, B and C) has been confirmed for the first time in Indonesian chickens. As the majority of the infectious coryza vaccines in use in Indonesia contain only serovar A and C, the presence of serovar B in chickens indicates that the protection by these bivalent vaccines would be reduced. The use of trivalent infectious coryza vaccines that contain serovars A, B and C is recommended for use in Indonesia.     

Efficacy of a commercially available coryza vaccine against challenge with recent South African NAD-independent isolates of Haemophilus paragallinarum in chickens

In South Africa the incidence of NAD-independent Haemnophilus paragallinarum isolation from clinical cases is increasing. This study was carried out to test whether a commercially available coryza vaccine (Nobilis Coryza, Intervet International BV) could protect chickens against challenge with recent NAD-independent isolates. SPF chickens were vaccinated twice at 3 and 7 weeks of age and were challenged at 9 weeks of age with 5 different NAD-independent isolates of serotype A or C-3. The results after challenge show that the coryza vaccine induces good protection against challenge with the different South African NAD-independent isolates of H. paragallinarum, including serotype C-3.     

Monoclonal antibody characterization of two field strains of Haemophilus paragallinarum isolated from vaccinated layer hens

An oil-based bacterin, containing strains 083 and 0222 of Haemophilus paragallinarum, is commonly used in South Africa to vaccinate laying flocks against infectious coryza. Two strains of H. paragallinarum, designated M85 and SB86, were isolated from infected but vaccinated commercial laying flocks in two incidental outbreaks of coryza in 1985 and 1986. A panel of five monoclonal antibodies was established which clearly distinguished the vaccine strains from the field isolates. One of these reacted with only vaccine strains A and B, another reacted with only field strains M85 and SB86, and the remaining three cross-reacted to various degrees with all four strains or isolates. Immunoassays were performed by enzyme-linked immunosorbent assay using whole bacteria as solid-phase antigen. These monoclonal antibodies may aid in serotyping new field isolates of H. paragallinarum and in improved standardization of vaccine strains.     

单抗阻断ELISA检测副鸡嗜血杆菌血清型特异性抗体的研究

本研究在副鸡嗜血杆菌Ａ、Ｃ型特异性单克隆抗体的基础上成功地建立了能区分Ａ、Ｃ血清型抗体的阻断ＥＬＩＳ诊断方法,并应用此法对９种常见病原体阳性血清及多份免疫鸡。     

Virulence of South African isolates of Haemophilus paragallinarum. Part 2: naturally occurring NAD-independent field isolates

Naturally occurring NAD-independent variants of Haemophilus paragallinarum, which have been isolates from poultry showing clinical signs of infectious coryza, were used to determine their virulence using a newly developed challenge model for infectious coryza. It was established that the NAD-independent isolates belonging to a particular serogroup, were less virulent when compared to the virulence of the NAD-dependent isolates from the same serogroup. It was shown that the virulence of the NAD-independent isolates belonging to serogroup C and serogroup A were very similar to each other. This differs to the results obtained with NAD-dependent isolates reported on previously, in which the serogroup C isolates were found to be more virulent then the serogroup A isolates.     

The presence of nicotinamide adenine dinucleotide-independent Haemophilus paragallinarum in México

Two isolates of Haemophilus paragallinarum were obtained from a layer chicken in Mexico. The isolates were confirmed as H. paragallinarum by polymerase chain reaction and conventional biochemical identification. The isolates were nicotinamide adenine dinucleotide (NAD) independent-growing on blood agar without the need of a nurse colony as well as on a complex medium that lacked both NAD and chicken serum. Both isolates were pathogenic, causing the typical clinical signs of infectious coryza in susceptible chickens. One isolate was Page serovar B/Kume serovar B-1 and the other isolate was Page serovar C/Kume serovar C-2. The isolates were associated with a field outbreak that involved an egg drop of 20% over a 3-wk period and a doubling of weekly mortality (from 0.1% to 0.2%). This is the first report of NAD-independent H. paragallinarum outside South Africa and is the first time that NAD-independent H. paragallinarum of serovar B has been reported.     

Study on the efficacy and safety of different antigens and oil formulations of infectious coryza vaccines containing an NAD-independent strain of Avibacterium paragallinarum

The present study was designed to assess and compare three different formulations of the new Onderstepoort infectious coryza (IC) quadrivalent vaccine, which contain an NAD-independent strain of Avibacterium paragallinarum (previously known as Haemophilus paragallinarum), and a commercial IC vaccine, not containing an NAD-independent strain, for their safety and ability to protect chickens of varying ages against virulent challenges with four different serovars of A. paragallinarum, including the NAD-independent strain of the C-3 serovar. Four groups of 140 chickens each were vaccinated at the age of 17 weeks and revaccinated at the age of 19 weeks with each of the four vaccine formulations. A similar sized group of non-vaccinated chickens was used as control. Two rounds of challenge were conducted: a group of chicken in each vaccination group was challenged between 31 and 35 weeks of age, while another group was challenged between 51 and 55 weeks of age. The "in-contact" challenge model was used in this experiment. For each vaccination group, the four challenge strains representing four local serovars were used in each challenge round. The efficacy of the vaccines was compared based on overall protection levels obtained and the duration of protection. The safety of the different vaccines was determined by the severity of post-vaccination reactions. The need for the incorporation of the NAD-independent strain in the vaccine was evidenced by the low protection level against NAD-independent challenge recorded in the group of birds vaccinated with the commercial vaccine. The results obtained confirmed not only the variation in virulence of different South African serovars, with serovar C-3 being the most virulent and serovar B having almost no virulence but also the age related increase in susceptibility. The importance of a suitable formulation of the vaccine is discussed.     

Virulence of South African isolates of Haemophilus paragallinarum. Part 1: NAD-dependent field isolates

The virulence of four South African field isolates of NAD-dependent Haemophilus paragallinarum, representing the four serovars known to occur in that country, was investigated. During this study an alternative challenge model for infectious coryza was used, in which the infectivity as well the virulence of different isolates could be evaluated. The challenge model consisted of the direct challenge, via intrasinus injection of one chicken in a row of interconnected layer cages, containing 10 chickens, which are subsequently infected by natural routes. A scoring system of the clinical signs was established in which a score is given to the ability of the isolate to produce clinical signs in the challenge birds. The mean daily disease score for the flock can be calculated and plotted on a graph to give a graphic representation of the disease profile. A mean disease score, calculated over a 20-day examination period can be calculated. Isolates can then be compared to each other, either graphically or by a comparison of the mean disease scores. It has been demonstrated using this scoring system that the South African serogroup C isolates appear to be more virulent than the South African serogroup A or B isolates. It was further established that the serovar C-3 isolate appeared to be the most virulent.     

Epidemiologic studies on infectious coryza outbreaks in northern New South Wales, Australia, using serotyping, biotyping, and chromosomal DNA restriction endonuclease analysis

The epidemiology of 16 cases of infectious coryza, an upper respiratory tract disease of chickens caused by Haemophilus paragallinarum, was investigated in a retrospective study. The cases occurred over a 14-month period on 10 farms in northern New South Wales. The available field data indicated that the cases formed six unrelated outbreaks. The 16 isolates of H. paragallinarum were subjected to serotyping by the Page and Kume schemes and biotyping based on carbohydrate fermentation and antimicrobial drug-resistance patterns. As well, newer fingerprinting techniques--plasmid profiles, whole-cell protein profiles, immunoblots of whole-cell protein profiles and total DNA restriction endonuclease analysis (REA)--were evaluated. Antimicrobial biotyping and REA profile typing proved most useful, allowing the recognition of three groups among the isolates. The other techniques gave either limited or no subdivision among the isolates. The combined results of the laboratory study indicated that, rather than six unrelated outbreaks, the 16 isolates represented three pairs of related outbreaks. This study represents the first application of sensitive biotyping and fingerprinting techniques to outbreaks of infectious coryza. The results have established that farms can be repeatedly infected with a single strain of H. paragallinarum that re-emerges at intervals. This study also obtained the first detailed evidence that replacement stock are a major source of infectious coryza.     

Amplified 16S ribosomal DNA restriction analysis for identification of Avibacterium paragallinarum

A molecular technique based on the restriction fragment length polymorphism of the 16S ribosomal genes amplified by a polymerase chain reaction (PCR), referred to as amplified 16S ribosomal DNA restriction analysis (ARDRA), was designed to identify 19 Avibacterium paragallinarum strains isolated from infraorbital sinus and nasal turbinate bone samples of broiler chickens, breeders, and laying hens from different regions of Peru. The 16S rDNA was amplified by PCR using a pair of bacterial universal primers and restriction analysis of 16S rDNA sequences was done to select endonucleases with the highest number of cutting points inside the 16S rDNA. The DNA patterns with DdeI and RsaI endonucleases were identical for the 19 A. paragallinarum strains, but differed from those obtained for Ornithobacterium rhinotracheale, a bacterium with a high genetic and phenotypic resemblance to A. paragallinarum, as well as from Escherichia coli, a bacterium associated with infectious coryza. The ARDRA method could prove to be valuable for molecular identification of A. paragallinarum, a microorganism implicated in respiratory diseases in commercial birds.     

Protection conferred by bivalent and trivalent infectious coryza bacterins against prevalent serovars of Avibacterium (Haemophilus) paragallinarum in Mexico

The protection and level of hemagglutination-inhibition (HI) antibodies conferred in infectious coryza bivalent- and trivalent-immunized chickens against Avibacterium (Haemophilus) paragallinarum field isolates of the prevalent serovars in Mexico (A-1, A-2, B-1, and C-2) were investigated. The bivalent bacterin (A-1 and C-1) conferred significant protection and increased HI antibodies against isolates of serovars A-1, A-2, and C-2, but not against a serovar B-1 isolate. The trivalent bacterin (A-1, B-1, and C-2) conferred protection and increased HI antibodies against all four of the isolates. The results confirmed that in poultry areas where serovar B-1 is prevalent, the inclusion of this serovar in bacterins is needed to confer protection against infectious coryza caused by A. (H.) paragallinarum isolates of serovar B-1.     

副鸡嗜血杆菌的分离鉴定及我国鸡传染性鼻炎的流行状况分析

从不同地区免疫失败鸡场的鸡传染性鼻炎疑似鸡体内共分离到19株细菌，经回归试验、PCR等方法鉴定为副鸡嗜血杆菌。又将分离株用血清平板凝集试验、血凝抑制试验和型特异性单抗进行了血清型的鉴定，结果表明分离菌有15株为A型，4株为B型，说明我国鸡传染性鼻炎的流行已经出现了新的特点，在疾病预防和控制中应加以注意。另外大多数鸡场的免疫失败是由于免疫效果不良所致，但也有相当一部分鸡场的免疫失败是由于B血清型的出现引起的。     

Occurrence of V-factor (NAD) independent strains of Haemophilus paragallinarum.

Strains of Bisgaard taxon 31, isolated from chickens in South Africa suffering from a respiratory disease with clinical symptoms and gross lesions similar to infectious coryza, showed great phenotypical similarities with Haemophilus paragallinarum infection except for NAD requirement, beta-galactosidase activity and maltose fermentation. Deoxyribonucleic acid-deoxyribonucleic acid hybridization confirmed a high level of genetic relatedness (DNA binding value, 89%) with Haemophilus paragallinarum. Guanine + cytosine content and genome size data also support the classification of taxon 31 strains within the species Haemophilus paragallinarum.