Abstract
This study aimed to develop an enzyme-linked immunosorbent assay to detect antibody associated with Staphylococcus aureus that is produced during the chicken immune response to this organism. The protein nuclease was tested for suitability as an antigen to detect antibody in sera from broiler chickens that had been exposed to aerosolized S. aureus on day 1 post hatch during experiments to reproduce bacterial chondronecrosis and osteomyelitis. An enzyme-linked immunosorbent assay was developed to measure the levels of nuclease antibody in 509 chicken sera from various experiments, which also enabled the examination of the influence of factors such as the S. aureus infection status and co-infection with chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) on nuclease antibody levels. Positive levels of nuclease antibody were detected in 71% of serum samples from chickens inoculated with S. aureus, CAV and IBDV, while positive levels were detected in 35% of chickens inoculated with S. aureus only. The influence of CAV and IBDV on the number of chickens with positive levels was most prominent in chickens aged up to 42 days. The study showed that nuclease-specific antibodies form part of the humoral immune response in broiler chickens that have been exposed to S. aureus. Co-infection with CAV and IBDV appeared to promote development of antibody in birds younger than 42 days; however, the presence of antibody did not necessarily prevent systemic infection.
Réponse immunitaire humorale des poulets de chair exposés à Staphylococcus aureus , au virus de la bursite infectieuse aviaire et au virus de l'anémie du poulet dans un modèle expérimental pour la chondronécrose et l'ostéomyélite
Cette étude avait pour but de développer un test d'immunoadsorption à enzyme conjuguée pour détecter les anticorps anti Staphylococcus aureus qui sont produits durant la réponse immunitaire à cet agent. La protéine nucléase a été testée pour savoir si elle pouvait être utilisée comme antigène pour détecter les anticorps dans les sérums de poulets de chair qui ont été exposés à un aérosol de S. aureus, un jour après l'éclosion, au cours de différentes expérimentations destinées à reproduire la chondronécrose bactérienne et l'ostéomyélite. Un test ELISA a été développé pour mesurer les niveaux des anticorps anti nucléase à partir de 509 sérums de poulets de différentes expérimentations qui ont également permis l'examen de l'influence des facteurs tels le statut de l'infection par S. aureus et la co-infection par le virus de l'anémie infectieuse du poulet (CAV) et par le virus de la bursite infectieuse aviaire (IBDV) sur le niveau des anticorps anti nucléase. Des niveaux positifs d'anticorps anti nucléase ont été détectés dans 71% des échantillons de sérum de poulets inoculés avec S. aureus, CAV et IBDV, alors que des niveaux positifs ont été détectés chez 35% des poulets inoculés avec S. aureus seul. L'influence du CAV et de l'IBDV sur le nombre de poulets avec des niveaux positifs a été plus marquée chez les poulets âgés de moins de 42 jours. L'étude a montré que les anticorps spécifiques de la nucléase formaient une part de la réponse immunitaire humorale chez les poulets de chair qui ont été exposés à S. aureus. La co-infection par le CAV et l'IBDV a semblé favoriser le développement d'anticorps chez des sujets âgés de moins de 42 jours, cependant la présence d'anticorps n'a pas nécessairement prévenue l'infection systémique.
Humorale Anti-Nuklease-Immunantwort bei mit Staphylococcus aureus und den Viren der infektiösen Bursitis und der Hühneranämie infizierten Broilern in einem Versuchsmodell für die bakteriell bedingte Chondronekrose und Osteomyelitis
Diese Studie hatte zum Ziel, einen Enzymimmunoassay (ELISA) zum Nachweis von Antikörpern gegen Staphylococcus aureus, die als Immunantwort des Huhns gegen diesen Organismus gebildet werden, zu entwickeln. Die Proteinnuklease wurde auf ihre Eignung als Antigen für den Nachweis von Antikörpern in Seren von Broilern, die in Versuchen zur Reproduktion bakteriell bedingter Chondronekrose und Osteomyelitis mittels Aerosol am 1. Lebenstag mit S. aureus infiziert worden waren, getestet. Es wurde ein ELISA entwickelt zur Bestimmung der Antikörpertiter gegen Nuklease in 509 Hühnerseren aus verschiedenen Experimenten, wodurch auch die Untersuchung von Einflussfaktoren wie eine Infektion mit S. aureus und eine Koinfektion mit dem Virus der Hühneranämie (CAV) und dem Virus der infektiösen Bursitis (IBDV) auf die Antikörpertiter gegen Nuklease möglich wurde. Positive Nuklease-Antikörpertiter wurden in 71% der Serumproben der mit S. aureus, CAV und IBDV inokulierten Hühner nachgewiesen, während bei den nur mit S. aureus inokulierten Hühnern 35% der Seren positiv waren. Der Einfluss von CAV und IBDV auf die Zahl der positiven Reagenten war am deutlichsten bei den Hühnern, die bis zu 42 Tage alt waren. Diese Studie zeigt, dass Nuklease-spezifische Antikörper Teil der humoralen Immunantwort von Broilern gegen S. aureus sind. Die Koinfektion mit CAV und IBDV schien die Antikörperbildung in bis zu 42 Tage alten Broilern zu verstärken. Die Anwesenheit von Antikörpern verhinderte jedoch durchaus nicht die systemische Infektion.
Respuesta inmune humoral anti-nucleasa de pollos de engorde expuestos a Staphylococcus aureus, virus de la bursitis infecciosa aviar, y virus de anemia infecciosa en un modelo experimental para osteomielitis y condronecrosis bacterianas
Este estudio tenía como objetivo desarrollar un ELISA para la detección de anticuerpos asociados a Staphylococcus aureus que se producen durante la respuesta inmune del pollo frente a este organismo. Se testó la idoneidad de la proteína nucleasa como antígeno para detectar anticuerpos en el suero de pollos de engorde que habían sido expuestos a S. aureus vía aerosol al día de nacimiento en pruebas para reproducir osteomielitis y condronecrosis bacterianas. Se desarrolló un ELISA para cuantificar los niveles de anticuerpos frente a la proteína nucleasa en sueros de 509 pollos procedentes de diversos ensayos que también permitieron la valoración de la influencia de otros factores como son el estatus de la infección por S. aureus y la co-infección con virus de anemia infecciosa (CAV) y virus de bursitis infecciosa aviar (IBDV) en los niveles de anticuerpos frente a la proteína nucleasa. Se detectaron niveles positivos de anticuerpos frente a la proteína nucleasa en el 71% de las muestras de pollos inoculados con S. aureus, CAV e IBDV, mientras que tan sólo se detectaron niveles positivos en el 35% de los pollos inoculados solamente con S. aureus. La influencia de CAV e IBDV en el número de pollos con niveles positivos fue más importante en pollos mayores de 42 días. Este estudio mostró que los anticuerpos específicos frente a nucleasa están involucrados en la respuesta inmune humoral en pollos que han sido expuestos a S. aureus. La coinfección con CAV e IBDV pareció promover la producción de anticuerpos en aves menores de 42 días, aunque la presencia de anticuerpos no previno necesariamente la infección sistémica.
Introduction
Staphylococcus aureus can cause several disease conditions in poultry including septicaemia and skeletal infections in broiler chickens (Jordan, 1996). In a survey of Northern Ireland broiler chickens, the predominant cause of lameness was identified as bacterial chondronecrosis and osteomyelitis (BCO) of the femur and tibiotarsus that was associated with S. aureus (McNamee et al., 1998). S. aureus has often been recovered from poultry regardless of their health status (Devriese et al., 1975; Thompson et al., 1980), however, suggesting that factors additional to the presence of the bacterium in a flock are important for the initiation of disease.
In a model of S. aureus-induced BCO in broiler chickens developed by McNamee et al. (1999), an increase in BCO incidence was observed in broilers that were also exposed to chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV). Because these viral agents have immunosuppressive qualities (Qureshi et al., 1998; Adair, 2000), the function of the avian immune system was likely to be a key factor in the initiation of this S. aureus-associated disease. While the innate immune response is believed to play a crucial role in preventing S. aureus infections in poultry (Zhu & Hester, 2000), the importance of the adaptive immune response has not been as widely investigated. A number of recent studies have examined cell-mediated responses following exposure to S. aureus but studies detailing the humoral response are rare (Zhu & Hester, 2000).
Williams-Smith (1954) reported a humoral response after subcutaneously inoculating chickens with a live broth culture of S. aureus. However, no studies have been published that describe the humoral response in chickens exposed to S. aureus by a natural route. In studies aimed at detecting S. aureus antibody in other host species, enzyme-linked immunosorbent assay (ELISA) methods have used many different antigens, and no single antigen has been shown to be clearly indicative of infection (Scott-Adams et al., 1988; Ryding et al., 2002). An extra-cellular protein, nuclease, secreted by S. aureus was identified by Scott-Adams et al. (1988) as an immunodominant protein associated with bovine mastitis. Proteins with nuclease activity are secreted by S. aureus, Staphlococcus hyicus and certain other staphylococci (Kloos & Bannermann, 1999), and may constitute suitable ELISA antigens to detect specific immune responses in poultry to these organisms.
In this study, a commercially purified preparation of the S. aureus protein nuclease was tested for suitability as an antigen to detect antibody associated with S. aureus in sera from chickens that had been exposed to aerosolized S. aureus during experiments concerning the model for BCO in broiler chickens (McNamee et al., 1999). The influence of the age of the bird, S. aureus infection status and the CAV and IBDV infection status on the levels of S. aureus antibody present was also determined.
Materials and Methods
S. aureus and virus strains
A S. aureus strain recovered from a naturally occurring case of BCO, designated Isolate 24, was used (McNamee et al., 1999). Pulsed field gel electrophoresis has characterized this strain as the predominant type recovered from broilers with clinical disease in Northern Ireland (McCullagh et al., 1998). The CAV and IBDV strains used were CUX1 and G9, respectively (McNamee et al., 1999).
Sera
All sera tested were collected from broiler chickens that were examined and bled at necropsy during the development of the model for BCO (McNamee et al., 1999) and subsequent experiments (Table 1). The broilers (Ross, Midlothian, UK) were hatched and reared in negative pressure isolators from day 1 until necropsy. They were observed twice daily for lameness and any lame birds were removed immediately and bled at necropsy. In general, two to five non-lame birds were removed from each group and bled during necropsy at 20 days post exposure to S. aureus and then every 5 days thereafter until the end of the experiment, to avoid overcrowding in the isolators. All work was undertaken in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 under the auspices of the Veterinary Sciences Division Ethics Review Committee.
Table 1. Details of the sera tested by the ELISA for nuclease antibody
All of the chickens inoculated with S. aureus were exposed to live broth culture of the bacteria by aerosol from a trigger sprayer at 1 day old (day 1 of the experiment). All of the chickens that were inoculated with either CAV alone, IBDV alone or both viruses were inoculated on day 21 of the experiment. Inoculation for CAV was by intramuscular injection and inoculation for IBDV was by eye drop.
Three hundred and thirty of the 417 chickens that donated sera and were inoculated with S. aureus alone or S. aureus and CAV and/or IBDV were examined by culture for the presence of S. aureus. At postmortem examination, samples of the proximal end of the femur and tibiotarsus from each leg and the liver were cultured individually for S. aureus as described previously (McNamee et al., 1999). Chickens that were identified as having S. aureus present in either the liver or leg bone samples were classed as positive by bacteriology.
ELISA methods
All reagents were used at 100 µl per microtitre well, except the final sulphuric acid stage to stop the enzyme reaction, which was used at 50 µl per well. The optimum dilution of each reagent was determined by titration. The ELISA used phosphate-buffered saline (PBS) (0.1 M, pH 7.2) to prepare the wash fluid (PBS + 0.05% Tween-80) and reagent/sample dilution buffer PTN (PBS with an additional 2.85% NaCl and 0.04% Tween-80).
The antigen used for the ELISA was purified micrococcal nuclease (Sigma-Aldrich, Poole, UK) isolated from the S. aureus strain Foggi. The nuclease was stored in aliquots at −20°C at a concentration of 8.4 mg/ml distilled water.
Each chicken serum ELISA test included three controls: an antigen-coated well + conjugate-only control, an uncoated well with chicken serum + conjugate-only control, and an uncoated well with conjugate-only control. Duplicate wells were used for both the chicken serum ELISA tests and for the uncoated well with chicken serum + conjugate-only controls.
Nuclease diluted 1:800 in coating buffer (0.05 M carbonate buffer, pH 9.5) was used to coat wells overnight at 4°C. The wells were emptied and 3% bovine serum albumin in coating buffer was added to each well for 1 h at 37°C. Following washing with five changes of wash fluid, the chicken serum diluted 1:1000 in PTN was added for 1 h at 37°C. The wells were washed and the peroxidase-conjugated rabbit anti-chicken IgY (Jackson Immunoresearch Laboratories Inc., Pennsylvania, USA) diluted 1:8000 in PTN was added for 1 h at 37°C. After a final wash, the tetramethylbenzidine substrate (E solution; Chemicon, California, USA) was added to each well and held at 37°C. After 10 min the reaction was stopped with 2.5 M sulphuric acid and the absorbance read at 450 µm using an Original Multiskan RC ELISA plate reader (Thermolabsystems, Finland). Each microtitre plate included a positive control chicken serum (high nuclease antibody level) and a negative control chicken serum (low nuclease antibody level) that had been selected from stored samples during development of the ELISA. Non-specific antibody binding in the chicken serum + conjugate-only control was highly variable between sera, and therefore the test result for each serum was calculated as the average chicken serum value minus the average chicken serum + conjugate-only control value. The relative antibody level (RAL) for each serum was then calculated by the following equation: RAL=(chicken serum result – negative control chicken serum result)/(positive control serum chicken result – negative control chicken serum result)
The positive/negative cut-off point for the ELISA was calculated as the average RAL for the 56 sera from control birds aged between 15 and 49 days that had not been inoculated with S. aureus, plus four times the standard deviation. The 56 sera from control birds produced a mean RAL of −0.0012 with a standard deviation of 0.0113. Therefore sera with a RAL of 0.044 or higher were classed as positive in the ELISA.
Results
Nuclease ELISA survey results for 1-day-old chickens
The ELISA detected positive levels of nuclease antibody in 13 of 36 (36%) 1-day-old chicks. The average RAL for 1-day-old chicks was 0.07 and the average positive RAL for the 1-day-old chicks was 0.2.
Nuclease ELISA survey results for birds inoculated with S. aureus alone or with viruses
The ELISA detected positive levels of nuclease antibody in 64% of the 417 sera samples taken from chickens that were inoculated with either S. aureus alone or with CAV and/or IBDV. The RALs were highest in the older chickens of all the groups tested (Figures 1–4). A summary of results obtained for sera samples taken from chicken inoculated with S. aureus alone or S. aureus and CAV and/or IBDV is presented in Table 2. The overall percentage of positive results in sera from chickens inoculated with S. aureus alone was 35% and the average positive RAL was 0.49. The percentage of positive results from chickens inoculated with S. aureus and CAV + IBDV was 71% and the average positive RAL was 0.47. In chickens aged up to 42 days, positive reactors were detected in 8% of birds exposed to S. aureus alone and in 68% of those exposed to S. aureus, CAV + IBDV. In chickens aged above 42 days, positive reactors were detected in 83% of birds exposed to S. aureus alone and in 74% of those exposed to S. aureus, CAV + IBDV. In chickens from the S. aureus+CAV group, positive reactions were detected in 85% of birds with an average positive RAL of 0.42. Positive reactions were detected in 66% of chickens from the S. aureus+IBDV group, with an average positive RAL of 0.46.
Published online:
18 January 2007Figure 1. Nuclease antibody in sera from chickens of various ages after inoculation with S. aureus on day 1.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2006/cavp20.v035.i04/03079450600821174/20160222/images/medium/cavp_a_182057_o_f0001g.gif"})
Figure 1. Nuclease antibody in sera from chickens of various ages after inoculation with S. aureus on day 1.
Published online:
18 January 2007Figure 2. Nuclease antibody in sera from chickens of various ages after inoculation with S. aureus on day 1, and CAV and IBDV on day 21.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2006/cavp20.v035.i04/03079450600821174/20160222/images/medium/cavp_a_182057_o_f0002g.gif"})
Figure 2. Nuclease antibody in sera from chickens of various ages after inoculation with S. aureus on day 1, and CAV and IBDV on day 21.
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18 January 2007Figure 3. Nuclease antibody in sera from chickens of various ages after inoculation with S. aureus on day 1, and CAV on day 21.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2006/cavp20.v035.i04/03079450600821174/20160222/images/medium/cavp_a_182057_o_f0003g.gif"})
Figure 3. Nuclease antibody in sera from chickens of various ages after inoculation with S. aureus on day 1, and CAV on day 21.
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18 January 2007Figure 4. Nuclease antibody in sera from chicken of various ages after inoculation with S. aureus on day 1, and IBDV on day 21.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2006/cavp20.v035.i04/03079450600821174/20160222/images/medium/cavp_a_182057_o_f0004g.gif"})
Figure 4. Nuclease antibody in sera from chicken of various ages after inoculation with S. aureus on day 1, and IBDV on day 21.
Table 2. Number of chickens from the complete survey with a positive nuclease RAL and the average positive RAL in groups of chickens after inoculation with S. aureus alone or S. aureus with CAV and/or IBDV
Of the 330 chickens that were examined by culture for S. aureus infection, the ELISA detected positive levels of nuclease antibody in 116 of 171 (68%) and 110 of 159 (69%) of sera from chickens that were culture-positive and culture-negative, respectively (Figure 5). The average positive test RAL was 0.47 for both sera taken from culture-positive chickens and that from culture-negative chickens. Within each of the experimental sub groups, similar numbers of chickens with positive levels of nuclease were detected in the both culture-positive and culture-negative chickens (Table 3).
Published online:
18 January 2007Figure 5. Nuclease antibody in sera of chickens that tested either negative or positive by culture for S. aureus following inoculation with S. aureus alone, or S. aureus and CAV and/or IBDV.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2006/cavp20.v035.i04/03079450600821174/20160222/images/medium/cavp_a_182057_o_f0005g.gif"})
Figure 5. Nuclease antibody in sera of chickens that tested either negative or positive by culture for S. aureus following inoculation with S. aureus alone, or S. aureus and CAV and/or IBDV.
Table 3. ercentage of positive nuclease RAL results in chickens that were tested for S. aureus infection by culture
Discussion
The main finding of this study is that nuclease-specific antibodies form part of the humoral immune response in broiler chickens that have been exposed to aerosolized S. aureus in a model of BCO. Furthermore, these nuclease-specific antibodies appeared to develop earlier in birds that were co-infected with CAV and IBDV.
The initial findings of this study showed raised nuclease RAL values in some 1-day-old control chickens, while the RAL values for 56 control chickens aged 15 days and older all had very low RAL values. This suggests transfer of maternal antibody from parents that fades from the circulation by 2 weeks. Similar findings have been observed for maternal transfer of antibody specific for Salmonella spp. and Camplyobacter spp. in chicks (Hassan & Curtiss, 1996; Sahin et al., 2001). Consequently, the positive/negative cut-off point in this study was calculated from RAL levels in control chickens aged 15 days or older to avoid the influence of maternal antibodies.
When results from the chicks younger than 15 days were excluded, the detection of RAL-positive birds increased with time and may be related to the period required to generate an immune response following bacterial challenge and interaction with the immune system. Other studies that have examined chickens infected with bacterial pathogens have reported time periods of 1 to 3 weeks after infection before specific antibody was detected in serum (Lee et al., 1983; Chart et al., 1992).
The skin and feathers of birds that are exposed to S. aureus spray as described in this paper were swabbed for culture and have demonstrated S. aureus colonization (unpublished data); however, the degree of colonization will be dependent on the other competitive bacterial flora present. In our study, not all birds exposed to S. aureus in a group became nuclease antibody-positive at the same time, and some birds did not become positive at all. This would suggest that any interaction between the adaptive immune system and the bacterium might not occur on day 1 in all chickens sprayed with S. aureus. This may be a result of the infection method, as the intensity, route and timing of bacterial interactions with the adaptive immune system of each chick were not defined. After spraying, the bacterium has the potential to interact with the adaptive immune system via injury to the skin following colonization of the skin and feathers (Zhu & Hester, 2000). Interactions following spraying may also occur via direct deposition of aerosolized bacterium within the respiratory tract immediately after spraying, and in the longer term the respiratory tract may be exposed to S. aureus present on dust generated from colonized skin and feathers (McNamee et al., 1999).
Therefore, regardless of the route of challenge, an interaction is likely to increase with time as S. aureus probably colonizes the skin and feathers of the chickens for the duration of the experiment. However, early interactions with the chicken immune system may not result in an immediate detectable response as the immune system of newly hatched chicks may be functionally immature in the first week of life (Holt et al., 1999). The significant host/bacteria interactions resulting in a detectable humoral immune response are therefore most likely to occur once the immune system has become functionally mature. Another variable that will influence the immune response following aerosol exposure is the degree of bacterial exposure to the immune system, as humoral responses in chicken to many antigens have been shown to be dose dependent (Gross, 1986). The birds that had negative RALs may have initiated a low level interaction, no interaction or an interaction before the immune system matured.
Thus the aerosol spray inoculation method had potential for variation in the route, timing and intensity of any interaction between the challenging S. aureus and the host. This coupled with variations of host immune status are likely to be the key factors behind the staggered increase in the nuclease RALs over time for birds exposed to S. aureus by spray at day 1.
The percentage of positive nuclease RALs in birds aged under 42 days that had been inoculated with S. aureus and the viruses was much greater than the percentage in similar birds inoculated with S. aureus alone (Table 2). The viral pathogens used in the model can cause periods of immunosuppression in the infected birds. CAV can deplete circulating lymphocytes and heterophils and depress cell-mediated immune responses (Adair, 2000). IBDV can reduce antibody levels in chicks (Qureshi et al., 1998) and suppress the function of heterophils and macrophages (Lam, 1998). Therefore the higher RAL values in birds from groups inoculated with viruses may be due to immunosuppression of the chicken, allowing S. aureus to invade and infect more readily, presumably stimulating the host immune system and allowing the development of detectable response during recovery from the immunosuppressive period. The influence of viral challenge appears less significant after 42 days since similar percentages of positive birds and similar average RAL values were observed in either virus-exposed groups or non-exposed groups (Table 2). However, this period is well after the transient stage of immunosuppression associated with the viral infections (Adair, 2000; Sharma et al., 2000), and is an indication of the invasive capacity of the S. aureus strain used; that is, it was capable of initiating an immune response in birds that were not virus infected that is similar to virus-infected birds, but over a longer time span. After 42 days, birds that were not exposed to viruses had had an increased time for an S. aureus–host interaction and the subsequent immune response to develop. The main influence of the viruses on nuclease RAL may be just to accelerate the antibody development in this model rather than actually promote higher levels in viral-exposed birds.
In groups inoculated with a single virus, the percentage of birds aged up to 42 days with positive nuclease RALs was much higher in the CAV group (91%) compared with the IBDV group (45%), although the numbers in these groups were small. The differences observed between the single virus-exposed groups may suggest that immunosuppressive qualities of CAV were predominantly responsible for the raised percentage of positive birds observed from groups infected with both viruses. Alternatively, the lower level of positive birds in IBDV-infected groups may have been the result of the virus lowering antibody levels to non-detectable levels at the immunosuppressive stage, as has been found in previous studies (Qureshi et al., 1998).
No relationship was observed between nuclease RAL in an individual bird and the corresponding bacteriology culture result for S. aureus in the liver and the leg bones. As discussed earlier, the nature of bacterial challenge to individual birds may be variable, making assumptions on infection status based on the nuclease RAL impossible, as several different scenarios may have existed. A positive RAL result for a bird may indicate that its adaptive immune system has encountered S. aureus. However, based on the findings from this study, a positive RAL value cannot differentiate between an infected bird and a bird that has resisted a S. aureus challenge. Additionally, negative RAL results may be obtained for birds that have been challenged or infected if the serum samples are taken during the acute phase of an immune response. A similar scenario was observed by Berchieri et al. (1995) when looking for Salmonella-specific antibody in sera from Salmonella-infected poultry flocks, where the organism was isolated from birds before detectable levels of antibody appeared in circulation at 2 weeks post infection. Alternatively a negative nuclease RAL may result from an insufficient interaction between S. aureus and the adaptive immune system or the influence of IBDV on antibody level as discussed earlier.
In summary, this report describes an ELISA that is capable of detecting the presence of nuclease antibodies associated with S. aureus, and provides some information on the humoral response on the BCO model. The ELISA has shown the influence of CAV and IBDV, which appear to raise the percentage of chickens aged up to 42 days that demonstrate a positive nuclease RAL. The ELISA results suggest that maternal nuclease antibody is transferred to newborn chicks but that the maternal antibody does not remain past 2 weeks. No obvious relationship was observed between the RAL values obtained in sera from BCO model experiments and bacteriological culture test results for the presence of S. aureus in the leg bones and liver.
| Sera group | Number of sera | Age of donor birds (days) | Inoculation details |
|---|---|---|---|
| Control | 56 | 15 to 49 | No inoculation |
| 1 day old | 36 | 1 | No inoculation |
| A | 96 | 13 to 50 | S. aureus |
| B | 221 | 23 to 50 | S. aureus+CAV + IBDV |
| C | 41 | 22 to 46 | S. aureus + CAV |
| D | 59 | 27 to 36 | S. aureus + IBDV |
| Sera group | Number tested | Number positive | % positive | Average positive RAL | Number tested up to 42 days old | % positive | Number tested above 42 days old | % positive |
|---|---|---|---|---|---|---|---|---|
| A | 96 | 34 | 35 | 0.49 | 61 | 8 | 35 | 83 |
| B | 221 | 159 | 71 | 0.47 | 112 | 68 | 109 | 74 |
| C | 41 | 35 | 85 | 0.42 | 11 | 91 | 30 | 83 |
| D | 59 | 39 | 66 | 0.46 | 29 | 45 | 30 | 87 |
| All test results based on average values obtained from duplicate test wells. Group A, sera from birds after inoculation with S. aureus on day 1; group B, sera from birds after inoculation with S. aureus on day 1, and CAV + IBDV on day 21; group C, sera from birds after inoculation with S. aureus on day 1, and CAV on day 21; group D, sera from birds after inoculation with S. aureus on day 1, and IBDV on day 21. | ||||||||
| Experimental group | S. aureus bacteriology result | Number tested | Percentage tested with positive RAL for nuclease |
|---|---|---|---|
| A | Positive | 34 | 56 |
| Negative | 26 | 54 | |
| B | Positive | 96 | 67 |
| Negative | 90 | 71 | |
| C | Positive | 25 | 84 |
| Negative | 10 | 80 | |
| D | Positive | 16 | 75 |
| Negative | 33 | 73 | |
| Total | Positive | 171 | 68 |
| Negative | 159 | 69 | |
| Group A, sera from birds after inoculation with S. aureus on day 1; group B, sera from birds after inoculation with S. aureus on day 1, and CAV + IBDV on day 21; group C, sera from birds after inoculation with S. aureus on day 1, and CAV on day 21; group D, sera from birds after inoculation with S. aureus on day 1, and IBDV on day 21. | |||
Acknowledgments
The authors are grateful to Cobb Breeding Company UK Ltd, Moy Park Ltd, Nutec Ltd, Elanco Animal Health Ltd, O'Kane Poultry Ltd and Ross Breeders UK Ltd for financial support, and to the bacteriology staff of the Veterinary Sciences Division, Belfast for technical assistance.
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