Abstract
The purpose of this study was to describe the dynamics (shedding and transmission) of Mycoplasma hyopneumoniae infection within a population of swine and to determine the duration of the infection (persistence) through the identification of the agent in bronchial samples. Sixty-three 2-month-old pigs were used in this study. The pigs (n = 28) were experimentally infected by the intratracheal route with M. hyopneumoniae and considered as seeder pigs. The remaining pigs (n = 32) were not inoculated and randomly allocated to 2 different groups: direct contact exposure pigs (n = 12) and indirect contact exposure pigs (n = 20). Blood samples and nasal swabs were collected throughout the study on days 0, 28, 35, 42, 49, 63, 91, and 119 postinfection. To assess the duration of M. hyopneumoniae infection, 9 seeder and 6 contact exposure pigs were slaughtered at days 155 (group 1), 170 (group 2), and 185 (group 3) postinfection. Direct contact pigs showed evidence of infection on day 28 by polymerase chain reaction (PCR) and on day 35 by serology. The indirect contact exposure pigs presented a very delayed and slow seroconversion pattern; they did not present evidence of transmission until 42 d after the infection of seeder pigs. Identification of M. hyopneumoniae in bronchial swabs was confirmed by nested-PCR from days 155 to 185 postinfection. At the last slaughter date, 77.7% and 100% of the seeders and contact exposure pigs, respectively, tested positive. The results of this study reconfirmed direct infection of M. hyopneumoniae and suggest that indirect transmission can occur in a population. Finally, duration of the infection in this study was longer than previously described.
Résumé
Soixante-trois porcs âgés de 2 mois ont été utilisés dans cette étude afin de décrire la dynamique (excrétion et transmission) d’une infection par Mycoplasma hyopneumoniae dans une population porcine et de déterminer la durée de l’infection (persistance) en identifiant l’agent des échantillons provenant des bronches. Des porcs (n = 28) ont été infectés expérimentalement par voie intra-trachéale avec M. hyopneumoniae et étaient considérés comme des propagateurs. Les autres animaux (n = 32) n’ont pas été inoculés et ont été répartis de façon aléatoire en 2 groupes : exposés par contact direct (n = 12) et exposés par contact indirect (n = 20). Des échantillons sanguins et des écouvillons nasaux ont été prélevés tout au long de l’étude aux jours 0, 28, 35, 42, 49, 63, 91, et 119 post-infection (PI). Afin d’évaluer la durée de l’infection par M. hyopneumoniae, 9 animaux propagateurs et 6 animaux exposés par contact direct ont été euthanasiés aux jours 155 (groupe 1), 170 (groupe 2) et 185 (groupe 3) PI. Les animaux exposés par contact direct montraient des évidences d’infection au jour 28 PI par réaction d’amplification en chaîne par la polymérase (PCR) et au jour 35 par sérologie. Les animaux exposés par contact indirect ont montré très tardivement et de façon lente un patron de séroconversion; ils n’ont pas présenté d’évidence de transmission avant 42 j suivant l’infection des animaux propagateurs. L’identification de M. hyopneumoniae à partir d’écouvillons bronchiaux a été confirmée par PCR nichée des jours 155 à 185 PI. Au dernier jour des euthanasies, respectivement, 77,7 et 100 % des animaux propagateurs et des animaux exposés par contact se sont avérés positifs. Les résultats de cette étude confirment de nouveau l’infection par M. hyopneumoniae suite à un contact direct et suggère qu’une transmission indirecte peut se produire dans une population. Finalement, la durée de l’infection dans cette étude a été plus longue que décrite précédemment.
(Traduit par Docteur Serge Messier)
Introduction
Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia and plays an important role in the porcine respiratory disease complex (PRDC) (1). The chronic form of the disease affects up to 80% of pigs around the world and is characterized by high morbidity and low mortality rates (2,3). Mycoplasma hyopneumoniae remains a significant pathogen to the pig industry, despite a trend towards high health production. Changes in production systems, especially all-in/all-out and 3-site production have resulted in changes in the epidemiology of the disease (4,5).
It has been documented that M. hyopneumoniae can be maintained within an infected herd by both vertical and horizontal transmission (3,6). Horizontal transmission is considered a main risk factor in chronically infected populations, where transmission is very likely to occur during the nursery and grow-finishing periods by direct contact between infected and susceptible pigs. This presentation is associated with traditional production systems using continuous flow, or housed in one site; however, in segregated systems the disease is delayed (7).
At this time, little information is available on the dynamics (shedding and transmission) of M. hyopneumoniae infection in a population of pigs. The organism has been isolated (8) and detected by polymerase chain reaction (PCR) (9–11) in nasal samples from naturally and experimentally infected pigs. However, this only documents the presence of the agent in the nasal cavity, but does not prove whether those animals are potential shedders of M. hyopneumoniae. Clark et al (12) demonstrated that infected seeder pigs were able to infect susceptible pigs by direct contact exposure. Other studies have described that transmission of the agent can occur among pen mates, but the onset of shedding and transmission seems to be variable (5,13,14). There are, therefore, questions regarding the role of infected animals and the speed at which transmission occurs within a large population of pigs.
Furthermore, little information on the duration of M. hyopneumoniae infection (persistence) is available at this time. Studies regarding the duration of the disease in experimentally infected pigs have indicated that clinical signs (coughing) can disappear around 10 wk postinfection and that pigs can show recovering lung lesions 8 to 12 wk after exposure (15,16). Mycoplasma hyopneumoniae has been detected in lungs by using cultivation and PCR techniques on day 85 postinfection (16,17). Kobish et al (15) found infected tissues by using immunofluorescence up to 119 d post experimental infection. However, the total length of M. hyopneumoniae infection is still unknown and this information, together with a better understanding of the infection dynamics within a population can be critical in designing control and eradication programs. Therefore, the objectives of this study were to describe the dynamics (shedding and transmission) of M. hyopneumoniae infection within a large population of pigs (experiment 1) and to determine the duration of the infection (persistence) by the identification of the agent in bronchial samples at 155, 170, and 185 d after infection (experiment 2).
Materials and methods
Source of animals and housing
Sixty-three 2-month-old gilts were obtained from a source known to be negative for M. hyopneumoniae on the basis of diagnostic data and the absence of clinical signs. The pigs were housed in a mechanically ventilated finishing barn, containing 11 pens. Inlets were distributed along the length of the building, so that all pens received approximately the same air exchange rates. The dimensions of the pens were 10 × 2.5 m, and all of them had partially slatted floors. Pen partitions were a combination of solid walls and vertical rod (open) gating. The ventilation system consisted of 20 inlets and 7 exhaust fans (6100 cfm). The animals were placed in 6 pens (1, 3, 5, 7, 9, and 11), 10 pigs per pen allowing 1 m2 of space per pig. During the length of this study, animals were cared under the guidelines of the University of Minnesota Institutional Animal Care and Use Committee guidelines.
Experiment 1: Assessment of the shedding and transmission of M. hyopneumoniae in a population of pigs
On day 0 of the study, 28 of the 63 pigs were experimentally infected by the intratracheal route with 10 mL of M. hyopneumoniae strain 232. A total dose of 105 color-changing units (CCU) per mL was administered to each pig. These animals (seeder pigs) were located in each of pens 3, 5, 7, and 9 and identified with green ear tags. The remaining 32 animals were not inoculated and randomly allocated to 2 different groups: direct contact exposure pigs (n = 12) and indirect contact exposure pigs (n = 20). The direct contact exposure pigs were placed in the same 4 pens where the seeder pigs were located, 3 animals in each pen. These animals were identified with white ear tags. The indirect contact exposure pigs (yellow ear tags) were placed in the corner pens 1 and 11 (10 animals per pen), leaving 1 empty pen between these animals and the seeder pigs (Table I). There were no significant differences in the location of the 3 groups in relation to air inlets and exhaust fans, with all groups being exposed to the same environmental conditions. Special care was taken to ensure that the animal caretakers did not enter pens 1, 2, 10, and 11 until transmission was documented in the indirect contact exposure pigs. Three negative pigs housed in an independent room served as negative controls.
Table I.
Distribution of pigs into 3 groups (infection model): seeders pigs, direct contact exposure pigs, and indirect contact exposure pigs
| Pen number | Seeder pigs | Direct contact exposure | Indirect contact exposure |
|---|---|---|---|
| 1 | — | — | 10 |
| 2 | Empty | ||
| 3 | 7 | 3 | — |
| 4 | Empty | ||
| 5 | 7 | 3 | — |
| 6 | Empty | ||
| 7 | 7 | 3 | — |
| 8 | Empty | ||
| 9 | 7 | 3 | — |
| 10 | Empty | ||
| 11 | — | — | 10 |
| Total (n) | 28 | 12 | 20 |
Blood samples and nasal swabs were collected from 3 of the 7 seeder pigs in each pen (n = 12), all the direct contact exposure pigs (n = 12), and all the indirect contact exposure pigs (n = 20). These animals were tested throughout the study on days 0, 28, 35, 42, 49, 63, 91, and 119 postinfection. The same monitoring schedule was performed with the negative control pigs. Additionally, on days 0, 14, 21, 28, 35, 42, 49, 63, 91, 119, and 155 postinfection, coughing was noted in each group for 30 min in the morning. This procedure was performed in order to record the onset and duration of clinical signs (coughing). The onset of coughing was determined when at least 1 pig from each group coughed during the observation period.
Experiment 2: Assessment of the duration of M. hyopneumoniae infection (persistence) in a population of pigs
To assess the duration of M. hyopneumoniae infection, animals utilized in experiment 1 were randomly allocated into 3 different groups (1, 2, and 3), with 15 pigs per group. This sample size of 15 pigs per slaughter group was capable of detecting at least 1 M. hyopneumoniae positive animal, assuming an estimated prevalence of 20% with 95% confidence (18). Each group constituted 9 seeder pigs (experimentally infected) and 6 contact exposure (either direct or indirect) pigs that were considered naturally infected. On day 70 of the study, 6 direct contact exposure and 8 indirect contact exposure pigs were sent to the slaughterhouse as part of another study. Following this, group 1 was slaughtered at 155 d, group 2 at 170 d, and group 3 at 185 d postinfection. Bronchial samples were collected from each lung using rayon swabs, taking special care to avoid cross contamination between animals. Samples were kept on ice and then sent to the laboratory. Average lung lesion score was then measured as described before (19). Negative animals were also sent to slaughter and bronchial swabs were then collected.
Diagnostic testing
Blood samples were tested for M. hyopneumoniae antibodies using an enzyme-linked immunosorbent assay (ELISA) (DAKO ELISA; DAKO Laboratories, Denmark) (20). This technique has a high sensitivity and specificity to detect specific antibodies against the agent (21). Nasal swabs were tested to detect M. hyopneumoniae using a nested-PCR technique previously described (9). This PCR technique has been validated under both field and experimental conditions (22,23) and it has been described as a reliable tool in profiling populations for M. hyopneumoniae infection (24).
Results
Infection dynamics (shedding and transmission)
Table II summarizes the diagnostic data from seeder, direct contact exposure, and indirect contact exposure pigs. All tested animals were negative by nested-PCR and serology on day 0 of the study. Mycoplasma hyopneumoniae was identified in 5 of the 12 seeder animals (nasal swabs) by nested-PCR, following the inoculation of the seeder pigs on day 28 postinfection, confirming a successful experimental infection. This was also supported by the serologic results, which showed that 6 of 12 seeder animals were positive at this time. Detection of the agent from nasal swabs of the seeder pigs showed a peak in the number of positive animals on day 42 postinfection. However, identification of the agent by nested-PCR from nasal swabs throughout the study was irregular. Conversely, the serologic profile indicated a progressive infection and all tested pigs were positive by day 63.
Table II.
Diagnostic data from seeder, direct contact exposure, and indirect contact exposure pigs
| D 0
|
D28
|
D35
|
D42
|
D49
|
D63
|
D91
|
D119
|
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Ser | PCR | Ser | PCR | Ser | PCR | Ser | PCR | Ser | PCR | Ser | PCR | Ser | PCR | Ser | PCR | |
| Seeders | 0/12a | 0/12 | 5/12 | 5/12 | 7/11 | 6/11 | 7/10 | 9/10 | 8/10 | 7/10 | 10/10 | 3/10 | 10/10 | 6/10 | 10/10 | NT |
| Direct | 0/12 | 0/12 | 0/12 | 4/12 | 1/12 | 4/12 | 2/12 | 10/12 | 7/12 | 8/12 | 12/12 | 6/12 | 6/6 | 2/6 | 6/6 | NT |
| Indirect | 0/20 | 0/20 | 0/20 | 0/20 | 0/20 | 0/20 | 0/20 | 8/20 | 1/20 | 6/20 | 11/20 | 10/20 | 10/12 | 0/12 | 12/12 | NT |
| Negative C | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | 0/3 | NT |
D — Days postinfection; Ser — Serology; PCR — Nested-PCR from nasal swabs; Seeders — Seeder pigs; Direct — Direct contact exposure pigs; Indirect — Indirect contact exposure pigs; Negative C — Negative controls pigs; NT — not tested
Number positive/number tested
Additionally, on day 28 after infection of the seeder pigs, there was evidence of transmission to the direct contact exposure pigs, with 4 of 12 of these animals testing positive by nested-PCR. Subsequent PCR testing also indicated a peak in the detection of positive animals on day 42 of the study. However, the serologic profiles showed a delay as compared with the seeder animals, until day 35, when the first pig became seropositive. All tested pigs were positive by day 63, similar to the experimentally infected pigs. In contrast, the indirect contact exposure pigs did not present evidence of transmission until day 42 after infection of the seeder pigs. Longitudinal assessment showed an inconsistent pattern with regards to the identification of the agent in nasal cavities. On day 49, 1 out of 20 indirect contact exposure pigs was positive by serology; complete seroconversion in these animals was not detected until day 119 after the original infection. Serologic profiles from the 3 groups showed differences in the onset of seroconversion, which was delayed in both direct contact exposure and indirect contact exposure pigs, this being more noticeable in the indirect contact pigs (Figure 1). Finally, the onset of clinical signs (coughing) was observed on day 14 postinfection for the seeder pigs, on day 28 for the direct contact exposure pigs, and on day 42 for the indirect contact exposure animals (Table III).
Figure 1.
Serological response (percentage of positives) from seeders, direct contact exposure and indirect contact exposure pigs. Mycoplasma hyopneumoniae antibodies measured by enzyme-linked immunosorbent assay (ELISA)
Table III.
Onset and duration of coughing within the 3 groups
| Days pi | Seeder pigs | Direct contact exposure | Indirect contact exposure |
|---|---|---|---|
| 0 | − | − | − |
| 14 | + | − | − |
| 21 | + | − | − |
| 28 | + | + | − |
| 35 | + | + | − |
| 42 | + | + | + |
| 49 | + | + | + |
| 63 | + | + | + |
| 91 | − | − | + |
| 119 | − | − | + |
| 155 | − | − | − |
+ — at least 1 pig coughing in the group; − — no pigs coughing in the group; pi — postinfection
Duration of M. hyopneumoniae infection (persistence)
Diagnostic data from samples of the 3 different slaughter dates from both seeder pigs and contact exposure animals are shown in Table IV. All bronchial swabs from the negative control pigs tested negative and no lung lesions were observed in these pigs. The approximate ages of the inoculated and contact animals at slaughter were 211, 226, and 241 d for groups 1, 2, and 3, respectively. Presence of M. hyopneumoniae in bronchial swabs was confirmed by nested-PCR from days 155 to 185 postinfection. On day 155, 75% of the seeder pigs (experimentally infected) and 33.3% of the contact exposure pigs (both direct and indirect exposure) tested positive. On day 170, 100% of both seeders and contact exposure animals tested positive. In the last slaughter date (185 d postinfection) 77.7% and 100% of the seeders and contact exposure pigs, respectively, were positive. Lung lesions suggestive of recovery were observed at slaughter; with the more extensive lesions in the contact exposure animals (Table IV).
Table IV.
Proportion of pigs tested positive by nested-polymerase chain reaction (PCR) (bronchial swab) and lung lesion scores in seeder and contact exposure pigs
| Seeder pigs
|
Contact exposure pigs
|
||||
|---|---|---|---|---|---|
| Days pi | PCR | Ave lung lesion | PCR | Ave lung lesion | |
| Group 1 | 155 | 6/8a (75 %) | 0.25 | 2/6 (33.3 %) | 4.8 |
| Group 2 | 170 | 9/9 (100 %) | 3.66 | 6/6 (100 %) | 6.16 |
| Group 3 | 185 | 7/9 (77.7 %) | 1.55 | 6/6 (100 %) | 5.33 |
Number positive/number tested; pi — postinfection; PCR — Nested-PCR from bronchial swabs; Ave lung lesion — Average lung lesion score. This measure was calculated over all inspected lungs in each group. The percentage of the affected area (lung lesion score) was calculated and recorded for each inspected lung as described before (19)
Discussion
The objectives of this study were to assess shedding and transmission, as well as duration of infection (persistence), of M. hyopneumoniae in an experimental population of pigs. Results from experiment 1 suggest that seeder pigs were active M. hyopneumoniae shedders, since the microorganism was found in their nasal samples and infection occurred in the direct contact exposure pigs. The onset of clinical signs and seroconversion of the seeder pigs was similar to that described in previous studies (1,15,16). As described before by several authors (5,12–14), transmission between pen mates was also observed in this study. Direct contact pigs showed evidence of infection on day 28 by PCR and on day 35 by serology. Previous papers have suggested that seroconversion in natural infections takes about 28 to 35 d (20,25). Therefore, it appears that the onset of shedding in seeder pigs may have occurred between days 7 and 14 postinfection. However, a limitation of this study was its inability to identify the exact time of infection, because the first testing was not performed until day 28 postinfection. The range between seroconversion of the first animal and that of the complete group was 28 d. The same range was also observed in a previous study using an infection model with seeder and direct contact exposure pigs, suggesting that a slow process of infection had taken place (7). However, under natural conditions, the infection process could be even slower. Morris et al (26), in a study of natural transmission by direct contact, reported that seroconversion of negative animals was not detected until 21 d post exposure in older seropositive pigs and 77 d later, only 19% of the tested pigs had seroconverted. One explanation for the difference from that paper and the present results could be that the excretion level of naturally infected shedders may be lower and more variable, whereas in our study each of the shedder pigs was experimentally infected at the same time with a high infection dose. Furthermore, although group size was strength of this study, the number of pigs involved is still much smaller than that observed under commercial conditions.
Results of the study also suggest that indirect transmission within a population occurred, since infection of the indirect contact exposure pigs was evidenced by both identification of the agent and identification of specific antibodies. This information is in agreement with the results of Leon et al (27). These authors, using a seroepidemiological approach under field conditions suggested that in addition to direct contact transmission, indirect or airborne transmission seems to play a role in the spread of the agent within a population. Indirect contact exposure animals presented a very delayed and slow seroconversion pattern, with a range between the first positive animal and the complete group seroconverting of more than 42 d. This suggests that the spread of the agent in this group was very slow. Since onset of disease and spread of the agent may be dependent on the level of colonization of the agent on the surface of the airway (3), the indirect contact pigs may have been exposed to a lower infectious dose than that of the seeders or the direct contact animals.
Regarding the duration of the infection (persistence), under the conditions of the study, 77.7% of experimentally infected and 100% of contact infected pigs were positive for M. hyopneumoniae for up to 185 d postinfection. In contrast to our results, a previous study detected a low proportion (29%) of PCR-positive animals at 85 d postinfection (16). An explanation for this difference could be that the nested-PCR technique used here was more sensitive than the one used in the previous study (9) or a difference in age and number of pigs used. While these results are interesting, conclusions at this time regarding the impact that this prolonged persistence has on control of the disease in conventional populations cannot be made, as this study did not look at the ability of the infected animals to transmit the organism to sentinels. More attention should be focused on this specific point, as this epidemiological characteristic of prolonged infection could be the key factor in the control and eradication of the agent in large production systems, especially in the sow herd, where introduction of replacements is a constant process (28). Results also suggest that the cutoff age used in some eradication programs (29) may be insufficient in specific cases, especially in production systems where gilts are raised offsite, resulting in a delayed time of infection, with animals potentially infected even after the 10 mo of age cutoff that is used in eradication programs.
To conclude, this study confirmed direct contact transmission within a population and suggests that this process may take several weeks to complete. The study likewise confirmed that indirect transmission also plays a role in the infection dynamics within a population and that animals infected by this route can present a more delayed onset of detection of the agent in nasal cavities, clinical signs, and seroconversion. Vica et al (30) suggested that other factors, such as differences among strains, can determine variations in the infection pattern of M. hyopneumoniae. The information generated in the present study regarding the dynamics of infection suggests that differences in the route of infection could also be a factor affecting the spread pattern. Results from this study also indicate that the duration of the infection may be longer than previously described. Future studies will be necessary to better define the duration of the infection, the infectivity of these asymptomatic carriers following exposure to naïve pigs, and the impact of various intervention strategies (vaccination, medication) on clearance of the organism from infected animals.
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