Immunogenicity of Riemerella anatipestifer broth

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12 Nov 2007 - SUMMARY. Strains belonging to Riemerella anatipestifer serotype 1 are responsible for most of the major disease outbreaks caused by this ...
Avian Pathology

ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: http://www.tandfonline.com/loi/cavp20

Immunogenicity of Riemerella anatipestifer broth culture bacterin and cell‐free culture filtrate in ducks Pornpen Pathanasophon , Takuo Sawada , Tarika Pramoolsinsap & Tipa Tanticharoenyos To cite this article: Pornpen Pathanasophon , Takuo Sawada , Tarika Pramoolsinsap & Tipa Tanticharoenyos (1996) Immunogenicity of Riemerella anatipestifer broth culture bacterin and cell‐free culture filtrate in ducks, Avian Pathology, 25:4, 705-719, DOI: 10.1080/03079459608419176 To link to this article: http://dx.doi.org/10.1080/03079459608419176

Published online: 12 Nov 2007.

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Date: 29 January 2016, At: 14:52

Avian Pathology (1996) 25, 705-719

Immunogenicity of Riemerella anatipestifer broth culture bacterin and cell-free culture filtrate in ducks PORNPEN PATHANASOPHON 1 , TAKUO SAWADA2, TARIKA PRAMOOLSINSAP 1 & TIPA TANTICHAROENYOS 1

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1

National Institute of Animal Health, Department of Livestock Development, Kasetklang, Bangkhen, Bangkok 10900, Thailand, and 2Department of Veterinary Microbiology, Nippon Veterinary and Animal Science University, Musashino, Tokyo, 180 Japan

SUMMARY Strains belonging to Riemerella anatipestifer serotype 1 are responsible for most of the major disease outbreaks caused by this organism in ducks in Thailand. Therefore, the immunogenicity induced by a broth culture bacterin prepared from a local strain (1081) of this serotype was studied in ducks and a protective index (PI) was derived. The inactivated vaccine contained the equivalent of 5 X 109 colony-forming units of bacteria per 0.5-ml dose. A single subcutaneous inoculation of the broth culture bacterin in 2-week-old Khaki Campbell ducklings gave adequate protection (PI = 88) against challenge with the homologous strain 7 days after vaccination, while the highest protection (PI = 95.6) was obtained 2 weeks after vaccination. The duration of immunity was at least 6 months. Inoculation of the vaccine at both 1 and 5 weeks of age gave the greatest protection. The vaccine was found to be safe in one-week-old ducklings and it also gave significant protection (PI values from 66.7 to 100) against challenge with virulent strains of the homologous serotype. The immunogenicity of broth culture bacterin prepared from local strains of various serotypes was also studied. Strains of serotypes 1, 6, 11, 14 and 19 gave no significant protection against challenge with strains of heterologous serotypes. Concentrated cell-free culture filtrates prepared from strain 1081 of serotype 1 and strain 328 of serotype 19 induced highly significant protection against homologous challenge (P < 0.01), but cross-protection between the two strains was not significant.

INTRODUCTION Among the diseases of domestic ducks, Riemerella anatipestifer infection has been well documented as a cause of considerable economic loss to the duck industry. So far twenty one serotypes of R. anatipestifer have been reported (Sandhu & Leister, 1991; Loh et al, 1992; Pathanasophon et al., 1995) but serotype 1 organisms are responsible for most of the major outbreaks in Thailand. Received 30 October 1995; Accepted 9 April 1996. 0307-9457/96/040705-15 © 1996 Houghton Trust Ltd

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P. PATHANASOPHON ETAL.

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Several attempts have been made to immunize ducks against anatipestifer infection, using either inactivated bacterin or live vaccines. Most vaccines required more than one injection (Sandhu, 1979, 1991; Layton & Sandhu, 1984). There are no reports on the protective role of cell-free culture filtrate prepared from the organism, although such preparations of Erysipeloihrix rhusiopaihiae (White & Verwey, 1970; Rothe, 1982; Sawada & Takahashi, 1987) and PasteureUa multocida (Ficken et al., 1991) have been shown to be immunogenic. In the present study we have investigated the efficacy of broth culture bacterin produced from strain 1081 of serotype 1 and have obtained results which differed from previous reports. We have also shown concentrated culture filtrate (CF) to be highly effective in protection against artificial challenge. MATERIALS AND METHODS Bacterial strains RiemereUa anatipestifer strain 1081 of serotype 1, was used as the representative strain for this study. This is a local strain which is highly virulent for ducks and which, in pilot studies, gave the highest protection against challenge by the homologous strain. Strains 1080, 1192 and 1154 of serotype 1 were also used as challenge strains in one experiment. Other strains used in cross-protection studies were strain 283 (serotype 6), strain 1204 (serotype 11), strain 1233 (serotype 14) and strain 328 of serotype 19. Bacterin preparation The appropriate culture was grown on tryptic soy agar (Difco Laboratories, Detroit, USA) containing 5% defibrinated sheep blood (TSAB) and one or two colonies were inoculated into 10 ml of tryptic soy broth (Difco) containing 0.3% yeast extract (Difco) (TSBY) and incubated in a shaker bath at 37°C for 24 h. This was then transferred to one 11 of TSBY medium in a 2-1 flask and incubated as above. Purity was checked by Gram-staining, and part of the culture was diluted and plated on TSAB to check its purity and also to determine the number of colony-forming units (CFU/ml). The optical density (OD) was read at 540 nm (Spectronic 20 A, Shimadzu, Millón Roy Company) and the culture was then inactivated with 0.3% formalin at room temperature for 24 h after which it was standardized to an OD of 2 at 540 nm, which was approximately equivalent to 1.4 X 1010 CFU/ml). Aluminium hydroxide gel was added to give a final concentration of 25% v/v. The adjuvant was provided by the Foot and Mouth Disease Centre, Veterinary Biologies Division, Department of Livestock Development, Thailand.

Preparation of culture fraction vaccines Strains 1081 and 328 were each grown up in 3 1 of broth, and prepared and

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RIEMERELLA ANATIPESTIFER VACCINES IN DUCKS

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inactivated with 0.3% formalin (v/v) as described above. Killed bacterial cells, designated KC and culture supernatant fluid (CS) were separated by centrifugation at 15,000 g for 20 min at 4°C. A portion of cells was resuspended in CS, adjusted to contain the equivalent of 1.4 X 1010 CFU/ml (OD 2.0 at 540 nm) and used as whole culture (WC) vaccine. The remaining CS was filtered through laboratory filter paper and a 0.45 ¿an membrane filter (Amicon Corp., Lexington, MA, USA.). After addition of benzamidine-n-hydrate at a concentration of 1 mM as a preservative the culture nitrate was designated CF. The CF was then concentrated to 30 times the initial volume ( X 30 CF) by ultrafiltration (Amicon standard cell 402) using a diaflomembrane type PM 10 (Amicon). Dilutions were made from the X 30 CF with filtered fluid to obtain 20 times ( X 20 CF) and 10 times (X 10 CF) concentration. The KC fraction was resuspended in the X 10 CF to yield a concentration of organisms equivalent to 1.4 X 10 CFU/ml and designated X 10 CF + KC. The X 30 CF, X 20 CF, X 10 CF and X 10 CF + KC and WC were adsorbed onto aluminium hydroxide gel to obtain a 25% gel suspension (v/v) and used in the immunological investigations described below.

Vaccination and challenge exposure One-day-old male Khakhi Campbell ducklings were obtained. They were bred at Bangpakong Livestock Breeding Station and were free from R. anatipestifer infection by history and by ELISA test. On arrival they were housed in the experimental animal house of the National Institute of Animal Health. Vaccination was carried out subcutaneously (s.c.) into the back of the neck with 0.5 ml of each immunogen. Challenge was given intramuscularly (i.m.) into the thigh using 0.5 ml inoculum containing 5 X 10' viable organisms of the appropriate challenge strain, with the exception of group 3 in experiment 3 which received 1010 CFU. Control birds were raised under the same conditions and all the ducks were observed daily for 14 days after challenge and mortality was recorded. The vaccination and challenge schedules are given below.

Statistics Since there were variable mortalities in control ducks after challenge exposure, the protection was evaluated with a protective index (PI) calculated as described by Sandhu (1979). A chi-square test was performed to compare immunized and control groups. _ % Mortality in controls -% Mortality in vaccinates % Mortality in controls

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Experimental design

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Experiment 1 This was conducted to determine the efficacies of different concentrations of broth culture vaccine. Bacterin prepared from strain 1081 was standardized to a concentration of OD 4.0 at 540 nm containing the equivalent of 2.8 X 1010 CFU/ml ( X 2 concentrated) and then diluted with an equal volume of CF ( X I concentration). Serial 10-fold dilutions were prepared from 10" J to 10~ 3 (designated X0.1, X0.01 and X 0.001) with CF. Five groups of ducklings, each consisting of between 28 and 34 birds, were immunized with the appropriate vaccine at 2 weeks old as described above. Three weeks later the immunized and 20 unimmunized ducklings of the same age were challenged with live culture of strain 1081.

Experiment 2 This experiment was designed to determine the onset and duration of protection after a single inoculation of bacterin prepared from strain 1081. The bacterin contained the equivalent of 1 X 1010 CFU/ml. One hundred and 99 ducklings were immunized at 2 weeks of age and raised in the same facilities as 80 unimmunized control ducklings of the same age. At 7, 14 and 21 days, and also at 2, 3, 4, 5 and 6 months after vaccination, groups of 20 to 25 vaccinated birds along with 10 controls were challenged with strain 1081.

Experiment 3 Since ducklings from 1 to 7 weeks of age are highly susceptible to R. anatipestifer, this experiment was conducted to determine the efficacy of strain 1081 vaccine given at less than 2 weeks of age. For comparison 20 ducklings in group 1 were immunized once at 2 weeks of age, while 24 and 11 ducklings in groups 2 and 3, respectively, were immunized twice at 1 and 5 weeks of age. Three weeks later all vaccinated birds plus unimmunized controls of the same age were challenged with strain 1081. The birds in groups 1 and 2, and their controls were given the normal challenge dose while those in group 3 were given double this dose.

Experiment 4 This experiment was carried out to evaluate the efficacy of vaccine prepared from strain 1081 of serotype 1 against challenge with other strains belonging to this serotype. Thirty-five ducklings were immunized at 2 weeks of age and divided into four groups, each consisting of eight or nine birds. Three weeks later vaccinated and control birds were challenged using strain 1080, 1192, 1154 or 1081.

RIEMERELLA ANATIPESTIFER VACCINES IN DUCKS

709

Table 1. Protection of ducks by different concentrations of bacterin prepared from R. anatipestifer strain 1081 (serotype 1) against challenge with the homologous strain (experiment 1) Group

Vaccine concentration*

1 2 3 4

X2 X1 X0.1 X0.01 X 0.001 Non-immune

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5 6

No. dead/ no. challenged

% Mortality

4/30 4/28 10/31 15/32 15/34 20/20

13.3 14.3 32.3 46.9 44.1 100.0

p l

b

86.7 85.7 67.7C 53.1 d 55.9d

' X I : vaccine at normal concentration; X 2: vaccine at twice normal concentration; X 0.1, X 0.01, X 0.001: vaccine diluted 10, 100 and 1000 times, respectively. "Protective index. °Not significantly different. ""Significantly different (P

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