Abstract
The study of Mycoplasma gallisepticum (MG) infection is needed, not only to understand the disease process but also to understand the mechanisms by which MG vaccines protect the host. Many model systems have been used to study the MG disease process. This work compared two different routes of infection (intratracheal versus eye drop) in commercial pullets, looking for differences in the pathology (air sac and tracheal lesion scores, and tracheal mucosal thickness) and the humoral immune response (measured by serum plate agglutination) of the host. The impact of concurrent infectious bronchitis virus vaccination on disease outcomes was also determined. Results showed that the intratracheal route of MG infection caused increased air sac and tracheal lesion scores and tracheal mucosal thickness at one week post infection, whereas the eye drop route produced no noticeable pathology. However, tracheal mucosal thicknesses of intratracheally challenged pullets were not statistically different from those of the eye drop challenged or control pullets at two and three weeks post infection. Concurrent infectious bronchitis virus vaccination had a negligible outcome on disease pathology. Vaccination of specific-pathogen-free chickens with the F-strain MG vaccine completely protected them against the effects of MG intratracheal infectious challenge, as evidenced by a lack of significant difference in air sac and tracheal lesion scores and tracheal mucosal thickness with those of unchallenged media control chickens.
Introduction
Mycoplasma gallisepticum (MG) is the cause of chronic respiratory disease in chickens and infectious sinusitis in turkeys (Bradbury, 1984; Evans et al., 2005). MG infection results in significant losses for poultry producers, including mortality, decreased egg production, reduced hatchability, and increased carcass condemnation (Ley, 2003; Evans et al., 2005).
Many model systems have been used to study MG infection in avian species. Routes of infection include aerosol, eye drop, intranasal, intratracheal, direct air sac injection, and spray application (Rodriguez & Kleven, 1980; Lin & Kleven, 1982; Levisohn et al., 1983; Levisohn & Dykstra, 1987; Nunoya et al., 1987; Naylor et al., 1992; Whithear et al., 1996; Ganapathy & Bradbury, 1998; Kollias et al., 2004; Feberwee et al., 2005). Co-infectious agents, including Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and Escherichia coli, have also been used to study their effects on or the enhancement of the disease process (Rodriguez & Kleven, 1980; Bradbury, 1984; Nakamura et al., 1994). Outcomes of these infections were measured through serology, blood parameters, re-isolation of MG and pathology including air sac lesions, tracheal lesions, increased tracheal mucosal thickness, and conjunctivitis (Rodriguez & Kleven, 1980; Nunoya et al., 1987; Nakamura et al., 1994; Branton et al., 1997b; Kollias et al., 2004). Infection results have also been measured in terms of commercial production parameters including egg production, egg quality characteristics, weight gain, and feed conversion (Branton & Deaton, 1985).
The experiments described here were designed to test two routes of MG infection, eye drop and intratracheal, and the time needed to obtain optimal infection results as measured by air sac lesion score, tracheal mucosal thickness, and tracheal lesion score. A further experiment was conducted to determine the impact of concurrent IBV vaccination on the resulting MG pathology. Therefore, the goal of this work was to compare two MG infection routes and the impact that concurrent IBV vaccination would have on chickens with the ultimate goal of identifying an infection route that could be used in an infection model for studying the protection afforded by MG vaccines.
Materials and Methods
Chicken housing and management
Chickens for experiments were obtained from two different sources. For Experiment 1, 1 day-old Hy-Line W-36 pullets were obtained from a commercial, National Poultry Improvement Plan monitored, MG-free and Mycoplasma synoviae-free source. Pullets were housed on clean pine shavings as previously described (Burnham et al., 2002). Feed and water were provided ad libitum as described previously (Burnham et al., 2002). Temperature and lighting conditions were set to follow the Hy-Line guidelines for the W-36 pullet variety. At seven weeks of age, 20 pullets were bled and the serum was screened for antibodies to MG by serum plate agglutination (SPA) assay (Branton et al., 1984). At 9 weeks of age, pullets were moved to the isolation facility and housed at seven pullets per isolation unit (Branton & Simmons, 1992).
For Experiment 2, fertilized eggs for commercial specific-pathogen-free (SPF) Leghorn chickens (Babcock variety) were obtained from Charles River Avian Products and Services (North Franklin, Connecticut, USA). Eggs were incubated according to Charles River guidelines and according to incubator instructions (Charles River, 2011). On day 18, eggs were placed in hatching trays. Hatched chicks were allowed to remain overnight in the incubator. One-day-old chicks were housed on 0.25-inch wire mesh in an isolation unit. The temperature and lighting schedule for Hy-Line W-36 pullets was followed and feed and water were provided ad libitum. At six weeks of age, chickens were subdivided to four pullets per isolation unit. Two chickens from each isolation unit were bled and the serum was screened by SPA assay for MG infection. All procedures used in this work were approved by the Mississippi State location US Department of Agriculture—Agricultural Research Service Animal Care and Use Committee.
Experimental protocols
Experiment 1
Ninety-eight commercial Hy-Line W-36 pullets were used. At 9 weeks of age, pullets were divided into seven groups: untreated control (Control), intratracheal media control (IT media), eye drop MG infection (Eye MG), intratracheal MG infection (IT MG), eye drop MG infection with IBV vaccination (Eye MG, IBV), intratracheal MG infection with IBV vaccination (IT MG, IBV), and IBV vaccination (IBV control). An eye drop medium control was not included due to space constraints in the isolation facility and the likely minimal impact on air sac lesions and tracheal pathology compared with what could be obtained by intratracheal infection. IBV vaccine (Mildvac™-M+ARK; Intervet Inc., Millsboro, Delaware, USA) was rehydrated in phosphate-buffered saline at 20 µl per manufacturer's dose and pullets were vaccinated with 20 µl dropped into the left eye. MG infection was carried out using frozen stocks of MG strain R-low, a low-passage virulent MG strain (Rodriguez & Kleven, 1980). Each infectious dose contained 3.3×105 colour change units. For the eye drop route of infection, the infectious dose was contained in 20 µl Frey's medium (Frey et al., 1968) and was dropped into the right eye of each pullet. For the intratracheal route of infection, each infectious dose was diluted into 1 ml sterile Frey's broth base (2.8 g Frey's Mycoplasma broth base and 0.038 g dextrose per 100 ml dH2O). Each infected pullet received a 1 ml infectious dose instilled into the trachea using a disposable plastic 2 ml pipette. At weeks 10, 11, and 12 (7, 14, and 21 days post infection), four pullets from each treatment group were bled for serum and killed by cervical dislocation. Air sacs of these pullets were examined for lesions. The 2 cm of trachea proximal to the syrinx were collected for histology as they were the least likely to be impacted by cervical dislocation, and they were stored in buffered 10% formalin.
Experiment 2
Sixteen mixed-sex SPF chickens were used. SPF chickens were selected for Experiment 2 due to a temporary problem obtaining Hy-Line W-36 pullets from a commercial source. Chickens were divided into the following groups: intratracheal media (unvaccinated sham infected) control (Control), unvaccinated intratracheal MG R-low infection (IT MG), F-strain vaccinated with intratracheal MG R-low infection (F, IT MG), and F-strain vaccinated sham infected (F MG). Four chickens were used in each treatment group. Poulvac® MycoF (Fort Dodge Animal Health, Fort Dodge, Iowa, USA; now Pfizer Animal Health) was rehydrated using phosphate-buffered saline to a final concentration of one dose per 20 µl. The IT MG and F MG groups were vaccinated at 6 weeks of age by instilling one dose (20 µl) in the right eye. The vaccine titre was 7.3×106 colour change units per dose. At 12 weeks of age, two chickens from each treatment group were bled and the serum was used for SPA analysis. The IT MG and F, IT MG groups were then infected at 12 weeks of age by intratracheal route with MG strain R-low as in Experiment 1. At 7 days post infection, chickens were bled for serum and killed by cervical dislocation. Euthanized chickens were examined for air sac lesions and 2 cm of trachea proximal to the syrinx were stored in buffered 10% formalin for histology. The serum was used for SPA analysis.
Serology and pathology
The serum from blood was tested by SPA assay using MG SPA antigen produced by Intervet Inc. The 0 to 4 scale recommended by the manufacturer was followed. All clinical disease results were scored in a blinded manner as follows. After the pullets were sacrificed, air sacs were examined for gross pathology. Air sac pathology was scored on a scale of 0 to 3 as described by Nunoya et al. (1987): 0 = no significant changes; 1 = cloudy appearance or several yellowish foci; 2 = cloudy thickening and/or more than several yellowish foci; and 3 = diffuse yellowish thickening with caseous exudates. Two centimetres of trachea proximal to the syrinx was stored in buffered 10% formalin for histology. Tracheal sections were stained with haematoxylin and eosin. Both tracheal mucosal thickness measurements and tracheal lesion scores were determined blind. The tracheal mucosal thickness was measured at four points offset 90° for each section. For each pullet, three non-contiguous sections were measured for a total of 12 measurements, which where averaged together to obtain the tracheal mucosal thickness for each pullet. Tracheal lesion scores were determined from the same three non-contiguous sections of trachea. Tracheal lesions scores were also calculated on a 0 to 3 scoring system published by Nunoya et al. (1987): 0 = no significant change; 0.5 = very small aggregates (one to two foci) or very slight, diffuse infiltration of lymphocytes; 1 = small aggregates (more than several foci) of lymphocytes or slight thickening of the mucosa due to diffuse lymphocytic infiltration; 2 = moderate thickening of the mucosa due to heterophil and lymphocyte infiltration and oedema accompanied by epithelial degeneration with or without luminal exudation; and 3 = extensive thickening due to heterophil and lymphocyte infiltration and oedema with squamous metaplasia or degeneration of epithelia with luminal exudation.
Statistical analysis
Tracheal mucosal thickness data were subjected to log10 transformation prior to analysis. There was no difference in results between non-transformed data and log10-transformed data for SPA, air sac and tracheal lesion results, so the non-transformed data were used for analysis. All data were analysed using the one-way analysis of variance procedure of SAS Analyst (SAS Institute, 2003). Significance of differences in the treatment means were separated using Tukey's studentized range test. Differences were considered significant at P≤0.05.
Results
Experiment 1
At 1 week post infection, exposure by the eye drop route resulted in very similar SPA scores to that by the intratracheal route with strain R-low (Figure 1a and Table 1). Concurrent vaccination with IBV vaccine had no impact on SPA results at 1 week post infection by the intratracheal route. However, concurrent IBV vaccination significantly decreased the SPA scores of the eye drop infected pullets (P=0.0029). At 2 and 3 weeks post infection, all MG infected groups had equivalent SPA results (Table 1). One caveat of these results was that the media control group was also SPA-positive at 1 week post infection, which made all SPA results post intratracheal infection questionable at that time point. However, the average SPA reaction of the IT media control group was significantly decreased at 2 and 3 weeks when compared with 1 week post infection (P=0.0101 and P=0.0232, respectively).
Published online:
01 October 2012Figure 1. Results of Experiment 1: infection with MG strain R-low at 1, 2, and 3 weeks post infection. 1a: Average serum plate agglutination (SPA) score. 1b: Average air sac lesion score. 1c: Average tracheal mucosal thickness. 1d: Average tracheal lesion score. Bars with different superscript letters are significantly different (P≤ 0.05). Error bars represent the standard error of the mean.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2012/cavp20.v041.i05/03079457.2012.721925/production/images/medium/cavp_a_721925_o_f0001g.gif"})
Figure 1. Results of Experiment 1: infection with MG strain R-low at 1, 2, and 3 weeks post infection. 1a: Average serum plate agglutination (SPA) score. 1b: Average air sac lesion score. 1c: Average tracheal mucosal thickness. 1d: Average tracheal lesion score. Bars with different superscript letters are significantly different (P≤ 0.05). Error bars represent the standard error of the mean.
Table 1. Serology and pathology results from differing routes of experimental MG infection with or without the presence of concurrent IBV vaccination.
The eye drop route of infection did not result in any disease pathology as measured by air sac lesion score, tracheal mucosal thickness, or tracheal lesion score at any of the time points (Figure 1b to d and Table 1). Unlike the eye drop route, the intratracheal route of infection yielded consistent disease pathology, including increased air sac lesion scores, increased tracheal mucosal thickness, and increased tracheal lesion scores. Air sac lesion scores remained significantly increased for the intratracheal route of infection at both 2 and 3 weeks post infection (P≤0.0001 and P=0.0202, respectively) although appeared lower compared with day 7, indicating resolution of lesions. Air sac pathology at 2 and 3 weeks post infection was limited to caseous exudates, indicating that lesions are being cleared from the air sacs (data not shown). The results for tracheal mucosal thickness and tracheal lesion score at 2 and 3 weeks followed this trend. The scores decreased such that they were no longer significantly different from eye drop infected pullets or even the control pullets by 3 weeks post infection (tracheal thickness P=0.0579 and P=0.7703, tracheal lesion score P=0.0289 and P=0.3098, compared with the IT media control group for weeks 2 and 3 post vaccination, respectively).
Concurrent vaccination with IBV vaccine had minimal impact on the disease pathology caused by MG infection alone. At 1 week post infection, IBV vaccination was found to have decreased the severity of air sac lesions following infection by the intratracheal route (Figure 1b and Table 1) (P=0.0002 when compared with the intratracheal route of infection without concurrent IBV vaccination). However, at 2 and 3 weeks post infection there was no difference between pullets without IBV and those vaccinated with IBV. Results were somewhat different for the tracheal mucosal thickness and tracheal lesion score. At 2 and 3 weeks post infection, there was no statistical difference in tracheal lesion scores or tracheal mucosal thicknesses between the IT MG and IT MG, IBV treatment groups at any of the time points (P=0.668, P=0.8324, and P=0.6398 for tracheal thickness and P=0.9285, P=0.2350, and P=0.9381 for tracheal lesion scores at 1, 2, and 3 weeks post infection, respectively) (Figure 1c,d and Table 1). There was no significant difference in air sac lesion scores between IBV vaccinated intratracheally infected pullets (IT MG, IBV group) and unvaccinated intratracheally infected pullets (IT MG group) at 2 and 3 weeks post infection (P=0.1622 and P=0.8767, respectively).
Experiment 2
Prior to infection, all unvaccinated chickens had SPA scores of zero, and vaccinated chickens had SPA scores of two or greater (data not shown). At 1 week post infection, all chickens had SPA scores of two or greater including the intratracheal media control chickens, consistent with Experiment 1 (data not shown). Interestingly, sera from media control chickens in this experiment were negative (SPA score of 0) when tested with a SPA antigen manufactured by Charles River Laboratories. When serum from the other treatment groups were tested with the Charles River Laboratories SPA antigen, the results obtained were consistent with the results obtained with the Intervet Inc. SPA antigen, except that one of the IT MG group chickens scored zero instead of two (data not shown).
Intratracheal infection in SPF chickens yielded similar results as seen with commercial pullets (Figure 2). Caseous exudate was found in the air sacs of all of the unvaccinated infected chickens. Vaccination with F-strain vaccine Poulvac MycoF protected the chickens from MG strain R-low induced air sac lesions (Figure 2a and Table 2). Interestingly, one vaccinated uninfected chicken also showed some air sac lesions (score = 2), but no caseous exudate was found in the air sacs. Results for tracheal mucosal thickness and tracheal lesion scores were very similar to the results obtained in Experiment 1 (Figure 2b,c and Table 2). Tracheal mucosal thickness and tracheal lesion scores were both elevated for MG infected (by intratracheal route) chickens. Vaccination prior to infection (F, IT MG group) decreased the average tracheal mucosal thickness and the tracheal lesion score to levels equivalent to those obtained in the sham vaccinated control and F MG groups (Figure 2b,c and Table 2). However, there were no significant differences between any of the treatments for either tracheal mucosal thickness or tracheal lesion score at 1 week post infection (P=0.1038 and P=0.1828, respectively). While all four chickens in the IT MG group had consistent severe air sac lesions (all were scored three), results for tracheal mucosal thickness and tracheal lesion score were not consistent as two out of the four chickens in this group exhibited increased tracheal mucosal thickness (average 127 vs. 65 µm) and tracheal lesion scores (average 2.44 vs. 0.375) as compared with the other two chickens in the treatment group.
Published online:
01 October 2012Figure 2. Results of Experiment 2: F MG vaccination followed by infection with MG strain R-low at 1 week post infection. 2a: Average air sac lesion score. 2b: Average tracheal mucosal thickness. 2c: Average tracheal lesion score. Bars with different superscript letters are significantly different (P≤ 0.05). Error bars represent the standard error of the mean.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2012/cavp20.v041.i05/03079457.2012.721925/production/images/medium/cavp_a_721925_o_f0002g.gif"})
Figure 2. Results of Experiment 2: F MG vaccination followed by infection with MG strain R-low at 1 week post infection. 2a: Average air sac lesion score. 2b: Average tracheal mucosal thickness. 2c: Average tracheal lesion score. Bars with different superscript letters are significantly different (P≤ 0.05). Error bars represent the standard error of the mean.
Table 2. Serology and pathology results from MG vaccinated and MG experimentally infected chickens.
Discussion
The purpose of this study was to compare differing MG infection models for effectiveness at producing disease and for ease of use. Many infection model systems have been used, including those looking at disease status such as air sac and tracheal pathology (Rodriguez & Kleven, 1980; Levisohn et al., 1986; Nunoya et al., 1987), and those investigating production parameters such as egg production, egg quality, and weight gain (Branton & Deaton, 1985). Production parameter experiments generally require a significantly longer experiment, but the results are directly applicable to real-world conditions. Measuring results based on pathology results in significantly shorter experiments, although the results only infer what would happen in a commercial setting such as table egg production.
Because of previously published research, it was assumed that MG strain R-low would produce disease pathology by either route of infection, and the presence of the IBV vaccine would enhance the disease process (Adler et al., 1962; Bradbury, 1984). However, results of this experiment were not consistent with this expectation. In the short time frame used in this work, the eye drop route of infection with strain R-low had no more impact than eye drop vaccination with F-strain MG. As vaccination with F-strain MG has been documented to result in a decrease in egg production when compared with unvaccinated control chickens (Carpenter et al., 1981), longer or larger studies may identify pathology associated with eye drop vaccination or infection that was not observed in the acute disease process studied in this work.
One difference noted between the two experiments reported here is the decreased effect on tracheal pathology in the IT MG group of Experiment 2 compared with Experiment 1. One possible cause was that two of the chickens were improperly infected. However, all four chickens in the IT MG group in Experiment 2 were SPA-positive and had the same level of air sac lesions, indicating that no error was made during the infection process. Another possibility is that the lack of pathology in the trachea was due to the method of infection. Applying the infectious dose by aerosol instead of in a liquid bolus may result in increased tracheal pathology. It is also possible that the differences in pathology were due to differences in response to MG infection between Hy-Line W-36 pullets and the Babcock strain SPF chickens used in Experiment 2.
The strong MG SPA response in IT media control pullets and chickens that received Frey's broth base were unexpected. As the SPA reaction characterizes the IgM response of the host, it is probable that the Frey's broth base protein components elicited a host immune response. Since the half-life of IgM in circulation is less than 40 h (Frommel et al., 1970), there would have been a significant decrease in circulating IgM by two weeks post infection, consistent with the results obtained. The difference in SPA results obtained with the two SPA antigens tested is probably due to differences in growth media component used in the mycoplasma growth medium or preparation of the SPA antigen.
These experiments were restricted because of limited isolation facilities necessary to perform the experiments as well as by only using vaccines/infectious agents currently in use in our facilities. NDV has often been used as a component of mycoplasma disease protocols, with or without IBV (Rodriguez & Kleven, 1980; Bradbury, 1984; Nakamura et al., 1994; Shah-Majid, 1996). The combined disease caused by MG and either or both of these viruses constitutes chronic respiratory disease (Bradbury, 1984; Kleven, 1998). IBV was chosen for this research because the vaccine is used in the non-disease research in our facilities. The use of NDV, a combination of NDV and IBV, or other disease cofactors may enable MG to cause disease by eye drop and other routes of infection without the need for direct placement of MG into the respiratory system. However, the ability to cause disease using intratracheal instillation without the need for an additional vaccine simplifies the infection model, but also leaves an opening for future experiments studying the role of MG in mixed infections.
Another method that has been used to deliver MG to the respiratory system is through the use of aerosols (Rodriguez & Kleven, 1980; Levisohn et al., 1986). Intratracheal instillation was chosen as a similar means of infection without the need for specialized aerosol generation equipment. A specialized aerosol generator is probably required as the typical sprayers used in vaccination and other agricultural settings do not produce respirable particles needed to infect the respiratory system (Purswell et al., 2008). The intratracheal route of infection also opens the possibility of testing precisely applied infectious dosages. However, the utility of studying precisely applied dosages may be limited by the tendency of pullets to cough up a portion of the administered infectious dose during tracheal instillation.
Many MG infection studies have been carried out using young pullets, as young as two weeks of age. This work was performed to follow more closely the vaccination schedule used by commercial producers. Vaccinations were performed at six weeks, because chickens infected with MG prior to four weeks of age may show more severe clinical lesions than those infected at four to six weeks of age (Gaunson et al., 2006). Vaccination at six weeks of age is earlier than recommended by the vaccine manufacturer, who recommends vaccination on or after 9 weeks of age. It is unknown what impact the different ages at vaccination may have on the outcome of vaccination. Infections were not started until after 12 weeks, an age at which minimal impact on egg production would be expected (Branton et al., 1997a). It is unknown how this infection model would impact egg production or egg quality parameters if performed closer to or during lay.
The results of this work demonstrate that MG based disease can be efficiently induced in chickens by the intratracheal route of infection using a virulent MG strain without a requirement for specialized equipment or disease cofactors. In a natural MG infection, co-infection with NDV, IBV, NDV and IBV, or other cofactors may be required to induce pathology.
| Intervet SPA | Air sac lesion | Tracheal lesion | Tracheal mucosa | |||||
|---|---|---|---|---|---|---|---|---|
| Treatment | dpia | Number positiveb | Average scorec | Number positive | Average score | Number positive | Average score | Average thicknessd |
| Control | 7 | 0/4 | 0 | 0/4 | 0 | 0/4 | 0 | 35.2±3.4 |
| 14 | 0/4 | 0 | 0/4 | 0 | 3/4 | 0.33±0.14 | 49.1±7.2 | |
| 21 | 0/4 | 0 | 0/4 | 0 | 2/4 | 0.38±0.18 | 41.6±7.3 | |
| IT media | 7 | 4/4 | 2.0±0 | 0/4 | 0 | 1/4 | 0.50±0 | 35.7±1.8 |
| 14 | 1/4 | 1.0±0 | 0/4 | 0 | 2/4 | 0.25±0 | 37.2±6.0 | |
| 21 | 1/4 | 2.0±0 | 0/4 | 0 | 2/4 | 0.25±0 | 40.7±11.3 | |
| Eye MG | 7 | 3/4 | 2.0±0 | 0/4 | 0 | 1/4 | 0.50±0 | 34.6±1.7 |
| 14 | 4/4 | 2.0±0 | 0/4 | 0 | 4/4 | 0.44±0.13 | 45.2±9.4 | |
| 21 | 4/4 | 2.0±0 | 0/4 | 0 | 4/4 | 0.31±0.13 | 49.6±11.9 | |
| IT MG | 7 | 4/4 | 2.3±0.29 | 4/4 | 3.0±0 | 4/4 | 2.06±0.88 | 97.2±27.9 |
| 14 | 4/4 | 2.5±0 | 4/4 | 2.8±0.5 | 4/4 | 0.94±0.24 | 57.8±14.5 | |
| 21 | 4/4 | 2.4±0.25 | 3/4 | 1.0±0 | 4/4 | 0.94±0.47 | 55.0±15.0 | |
| Eye MG IBV | 7 | 1/4 | 2.0±0 | 0/4 | 0 | 3/4 | 0.50±0.25 | 39.2±2.6 |
| 14 | 4/4 | 2.1±0.25 | 0/4 | 0 | 4/4 | 0.56±0.31 | 48.8±6.0 | |
| 21 | 4/4 | 2.5±0 | 0/4 | 0 | 3/4 | 0.38±0.13 | 41.3±7.5 | |
| IT MG IBV | 7 | 4/4 | 2.0±0 | 3/4 | 2.0±0 | 4/4 | 1.75±0.35 | 79.9±22.0 |
| 14 | 4/4 | 2.1±0.25 | 4/4 | 2.3±0.5 | 4/4 | 1.50±0.71 | 69.9±21.3 | |
| 21 | 4/4 | 2.5±0 | 2/4 | 1±0 | 4/4 | 1.31±1.2 | 84.9±51.5 | |
| IBV | 7 | 0/4 | 0 | 0/4 | 0 | 4/4 | 0.63±0.25 | 44.1±5.6 |
| 14 | 0/4 | 0 | 0/4 | 0 | 4/4 | 0.56±0.13 | 45.8±4.8 | |
| 21 | 0/4 | 0 | 0/4 | 0 | 2/4 | 0.25±0 | 37.2±7.4 | |
| aDays post infection. bNumber of positive (non-zero) chickens/total chickens. cAverage of positive (non-zero) scores±standard deviation. dAverage of four scores±standard deviation. | ||||||||
| Intervet SPA | Air sac lesion | Tracheal lesion | Tracheal mucosa | ||||
|---|---|---|---|---|---|---|---|
| Treatment | Number positivea | Average scoreb | Number positive | Average score | Number positive | Average score | Average thicknessc |
| Control | 4/4 | 2.0±0d | 0/4 | 0 | 4/4 | 0.33±0.06 | 58.4±5.3 |
| IT MG | 4/4 | 2.3±0.29 | 4/4 | 3±0 | 4/4 | 1.41±1.2 | 95.8±38.5 |
| F, IT MG | 4/4 | 2.5±0 | 0/4 | 0 | 4/4 | 0.50±0.18 | 59.8±15.9 |
| F MG | 4/4 | 2.4±0.25 | 1/4 | 2±0 | 4/4 | 0.69±0.63 | 65.8±12.0 |
| aNumber of positive (non-zero) chickens/total chickens. bAverage of positive (non-zero) scores±standard deviation. cAverage of four scores±standard deviation. dSerum from the control group was serum plate agglutination (SPA)-negative when tested with Charles River Laboratories SPA antigen. | |||||||
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