The impact of animal age, bacterial coinfection, and isolate pathogenicity on the shedding of Porcine reproductive and respiratory syndrome virus in aerosols from experimentally infected pigs

Abstract

The objective of this study was to evaluate the role of different variables (animal age, bacterial coinfection, and isolate pathogenicity) on the shedding of Porcine reproductive and respiratory syndrome virus (PRRSV) in aerosols. Animals were grouped according to age (2 versus 6 mo) and inoculated with a PRRSV isolate of either low (MN-30100) or high (MN-184) pathogenicity. Selected animals in each group were also inoculated with Mycoplasma hyopneumoniae. The pigs were anesthetized and aerosol samples (1000 breaths/sample) collected on alternating days from 1 to 21 after PRRSV inoculation. The results indicated that animal age (P = 0.09), M. hyopneumoniae coinfection (P = 0.09), and PRRSV isolate pathogenicity (P = 0.15) did not significantly influence the concentration of PRRSV in aerosols. However, inoculation with the PRRSV MN-184 isolate significantly increased the probability of aerosol shedding (P = 0.00005; odds ratio = 3.22). Therefore, the shedding of PRRSV in aerosols may be isolate-dependent.

Since its emergence, porcine reproductive and respiratory syndrome (PRRS) remains 1 of the most difficult and costly diseases to control in the global swine industry (1). A potential challenge to sustainable PRRS control is that commercial production systems frequently mingle pigs from multiple sources of various ages and health status from weaning until market weight is achieved. This practice promotes the circulation of genetically and clinically diverse isolates of Porcine reproductive and respiratory syndrome virus (PRRSV) throughout the population (2). Genetic change induced by replication and shedding of the virus results in highly pathogenic isolates that replicate at a higher rate, elevating concentrations in blood and tissues (3–8). Besides the clinical impact, differences in PRRSV isolate pathogenicity appear to influence shedding and transmission to other animals. In previous studies, differences in transmission to sentinels were observed between pigs infected with a virulent field isolate and those infected with an isolate of lesser pathogenicity (9,10). In addition to isolate pathogenicity, other factors that may affect PRRSV shedding are bacterial coinfection and animal age. Pathogens such as Mycoplasma hyopneumoniae have been shown to increase the duration and severity of PRRSV-induced pneumonia and viremia (11). Younger pigs (6 to 8 wk old versus 6 mo old) are more susceptible to infection, have higher levels of viremia, and excrete virus at higher concentrations (12).

An important component of PRRS control is a thorough understanding of how the virus is excreted from infected animals. Previously published data indicate that PRRSV can be shed in semen, urine, feces, and saliva (13–16); however, information on the shedding of PRRSV in aerosols has not been available. Furthermore, the concurrent influence of isolate pathogenicity, bacterial coinfection, and animal age on aerosol shedding is not understood. Therefore, the objective of this study was to determine the role of multiple variables (animal age, PRRSV isolate pathogenicity, and M. hyopneumoniae coinfection) on the concentration and shedding of PRRSV in aerosols of individual pigs. The study was based on the hypothesis that isolate pathogenicity would significantly affect the shedding of PRRSV in aerosols.

The study was conducted at the University of Minnesota College of Veterinary Medicine isolation facility, in St. Paul, Minnesota, USA. The 50 pigs used in the study were obtained from a PRRSV-negative source; 27 were 2 mo old and weighed 25 kg, and the other 23 were 6 mo old and weighed 120 kg. After the pigs were transported to the isolation facility, blood and nasal swab samples were collected from each pig to ensure that all animals were free from PRRSV and M. hyopneumoniae (17). Throughout the study all pigs were cared for under the guidelines of the University of Minnesota Institutional Animal Care and Use Committee.

The viruses selected for the study were PRRSV MN-30100, an isolate of low pathogenicity, and PRRSV MN-184, an isolate of high pathogenicity. The MN-30100 isolate had been reported to induce mild clinical illness and to replicate at low rates in blood and tissues (8,18). In contrast, the MN-184 isolate was originally obtained from an infected farm experiencing severe reproductive disease and an elevated sow mortality rate. Furthermore, its concentrations in blood and tissues were significantly higher than those of MN-30100 (8,18).

The pigs that were to be infected solely with PRRSV were divided into age groups, and each group was housed in a separate isolation room. A control pig was included for each group and was housed in a separate room. On day 0, both 2-mo-old and 6-mo-old pigs were inoculated intranasally with either PRRSV MN-30100 or PRRSV MN-184. All pigs received intranasally 2 mL of inoculum with a median tissue culture dose per milliliter (TCID₅₀/mL) of 10 000 viruses. The control pigs were sham-inoculated intranasally with 2 mL of sterile saline.

The pigs that were to be infected with both M. hyopneumoniae and PRRSV were similarly divided into groups according to age and housed at the isolation facility. On day 0, they were anesthetized with a combination of xylazine hydrochloride (Anased; Lloyd Laboratories, Shenandoah, Iowa, USA), 1.5 mg/kg, and a mixture of tileamine hydrochloride and zolazepam hydrochloride (Telazol; Fort Dodge Animal Health, Overland Park, Kansas, USA), 8 mg/kg, given intramuscularly. The pigs were then intubated and intratracheally inoculated with M. hyopneumoniae 232 (11); inoculum doses of 10 or 25 mL, containing 10⁵ color-changing units [CCU] per milliliter, were used to infect the 25-kg and 120-kg pigs, respectively (11). Then, 21 d later, the pigs were intranasally inoculated with either PRRSV MN-30100 or PRRSV MN-184. The control pigs were anesthetized in the same manner and sham-inoculated intratracheally with sterile saline. Successful M. hyopneumoniae infection was monitored by performing a nested polymerase chain reaction (PCR) assay (17) on nasal swab samples collected on days 7, 15, and 21 after inoculation and by monitoring the development of serum antibodies against M. hyopneumoniae by the Dako enzyme-linked immunosorbent assay (ELISA) (Dako Laboratories, Glostrup, Denmark) on days 15 and 21.

Aerosol and blood samples were collected from each pig on alternating days from days 1 to 21 after PRRSV inoculation. For the collection of aerosols, we devised a device consisting of a conical plastic mask, a breathing valve, and a collection bag. The mask, to be placed over the maxilla and mandible, was sized to provide a tight fit for both ages of pigs. The manually operated breathing valve attached to the mask could be opened and closed approximately every 5 breaths to provide fresh air and minimize the rebreathing of exhaled air. The plan was to collect 1000 breaths from each pig into the collection bag attached to the breathing valve. To collect aerosolized particles, we removed the mask at intervals of 100 breaths and held it vertically, with the bag positioned downward, then covered the opening of the mask with a plastic cap to maximize particle retention. To flush the interior of the mask and facilitate particle concentration into the collection bag, we injected 10 mL of sterile saline through an opening in the cap. A final volume of 100 mL was collected for testing for the presence of PRRSV RNA.

Before initiating the study, we evaluated the aerosol collection method, testing the ability of the collection device to provide samples containing various concentrations of PRRSV. We aerosolized 1 mL of 10-fold dilutions of stock PRRSV (concentration 0.1 to 100 000 TCID₅₀/mL) using a cold mist fogger capable of generating artificial PRRSV aerosols with particle diameters ranging from 0.3 to 3.0 μm (10). The interior of the mask was then rinsed with sterile saline, and a 1-mL aliquot of the rinse fluid for each dilution was tested for the presence of PRRSV RNA.

To initiate the aerosol collection process, we anesthetized the pigs as described earlier. At a suitable plane of anesthesia, the pig’s snout was wiped clean of debris to reduce contamination and the mask positioned. During the collection process, the respiratory rate was measured and blood samples for monitoring the Paco₂ level were collected by jugular venipuncture into a sterile tube containing sodium heparin (Vacutainer; Becton Dickinson, Rutherford, New Jersey, USA). A new mask was used for each dilution of virus to avoid cross-contamination with residual PRRSV. At the end of each trial, the pigs were euthanized on day 21 after PRRSV inoculation with an intravenous injection of sodium pentobarbital, 100 mg/kg.

The PRRSV concentrations in aerosols and blood were determined by means of a TaqMan-based quantitative (real-time) reverse transcriptase (RT)-PCR assay for the PRRSV open reading frame (ORF) 6 gene at the Minnesota Veterinary Diagnostic Laboratory, University of Minnesota, St. Paul. The procedure was a modified version of a PCR protocol previously described (19). The assay measured the concentration of PRRSV RNA in the pooled saline rinse fluid; the data were expressed in units of TCID₅₀/mL. Concentrations ranging from 0.1 to 100 000 TCID₅₀/mL were detected during laboratory evaluation of the collection mask with the 10-fold dilutions of PRRSV MN-30100 and PRRSV MN-184, and a standard curve was developed. Each sample was run in triplicate and the mean RNA concentration calculated from these values. Results were compared across the variables of isolate pathogenicity, bacterial coinfection, and animal age. A generalized analysis of variance (ANOVA) and logistic regression were used to test for differences between variables. Differences with a P-value of less than 0.05 were considered significant.

The mean PRRSV RNA concentrations for each study group are summarized in Table I, along with the peak concentrations observed for individual pigs in each group. Among the pigs inoculated solely with PRRSV MN-30100, we observed a transient elevation of rectal temperature (to 39°C to 40°C), anorexia (at 24 to 28 h after inoculation), and mild depression in both 2-mo-olds and 6-mo-olds; no deaths occurred. Among the 2-mo-olds, PRRSV RNA was detected in aerosols intermittently in 20% to 40% on days 3 to 7, 15, and 19 after inoculation (Table II), with a peak concentration of 1.0 TCID₅₀/mL. Among the 6-mo-olds, shedding was observed in 25% to 100% over days 3 to 9 after inoculation, with a peak concentration of 0.5 TCID₅₀/mL. The mean blood concentrations of PRRSV RNA were 2.54 TCID₅₀/mL in the 2-mo-old pigs and 5.0 TCID₅₀/mL in the 6-mo-old pigs.

Table I.

Concentrations of Porcine reproductive and respiratory syndrome virus (PRRSV) RNA detected in aerosol samples from pigs inoculated with PRRSV alone or after prior inoculation with Mycoplasma hyopneumoniae

	Concentration of PRRSV RNA (TCID50/mL)
Agent(s) inoculated; animal age (mo)	Peaka	Mean over sampling period	Standard deviation
PRRSV MN-30100
2	1.0	0.05	0.10
6	0.5	0.05	0.09
PRRSV MN-30100 + M. hyopneumoniae
2	0.1	0.007	0.01
6	2.0	0.005	0.02
PRRSV MN-184
2	35	0.6	1.7
6	5	0.2	0.3
PRRSV MN-184 + M. hyopneumoniae
2	1330	40	125
6	6	0.2	0.4

TCID₅₀/mL — median tissue culture dose per milliliter

^aFor an individual animal in each group

Table II.

Proportions of pigs shedding PRRSV by aerosol on each collection day after PRRSV inoculation

	Collection day; number of pigs shedding/number of pigs in the group
Agent(s) inoculated; animal age (mo)	1	3	5	7	9	11	13	15	17	19	21
PRRSV MN-30100
2	0/5	1/5	2/5	1/5	0/5	0/5	0/5	1/5	0/5	1/5	0/5
6	0/4	1/4	4/4	2/4	1/4	0/4	0/4	0/4	0/4	0/4	0/4
PRRSV MN-30100 + M. hyopneumoniae
2	0/4	0/4	0/4	1/4	1/4	1/4	0/4	0/4	0/4	0/4	0/4
6	0/4	0/4	0/4	0/4	0/4	1/4	0/4	0/4	0/4	0/4	0/4
PRRSV MN-184
2	0/6	0/6	3/6	0/6	1/6	1/6	2/6	2/6	1/6	1/6	3/6
6	0/5	3/5	2/5	0/5	0/5	1/5	1/5	1/4	1/4	1/4	0/4
PRRSV MN-184 + M. hyopneumoniae
2	1/6	0/6	4/5	4/5	2/5	NC	4/5	2/5	5/5	NC	1/5
6	0/5	3/5	5/5	1/5	1/4	NC	0/4	0/4	1/4	0/4	0/4

NC — no collection because of severe respiratory illness

Both age groups of pigs inoculated with PRRSV MN-30100 and M. hyopneumoniae had clinical signs similar to those produced by PRRSV MN-30100 alone, in addition to a dry coughing observed by day 21 after inoculation with M. hyopneumoniae that continued throughout the sampling period. None of the pigs died. The concentrations and frequency of detection of PRRSV RNA in aerosols from both ages of this experimental group of pigs were low, despite extensive coughing during the sample collection procedure. Aerosol shedding of PRRSV was observed in 25% of the 2-mo-old pigs on days 7 to 11 after inoculation, and the concentration peaked at 0.1 TCID₅₀/mL. In contrast, aerosol shedding was detected in only 1 of the older pigs, on day 11 after inoculation, and a concentration of 2.0 TCID₅₀/mL was recorded that day for that pig. The mean blood concentrations of PRRSV RNA were 326 TCID₅₀/mL in the 2-mo-old pigs and 270 TCID₅₀/mL in the 6-mo-old pigs.

Clinical signs in the pigs inoculated solely with PRRSV MN-184 were more severe and longer lasting than the signs in the pigs inoculated with PRRSV MN-30100 and included rectal temperatures above 42°C, severe anorexia, and severe depression throughout the 21-d sampling period. One 6-mo-old pig died from respiratory illness on day 15 after inoculation. We detected PRRSV MN-184 RNA in aerosols from 17% to 50% of the 2-mo-old pigs on day 5 and days 9 to 21 after inoculation and in 20% to 60% of the 6-mo-old pigs on days 3, 4, and 11 to 19, with peak concentrations of 35 and 5 TCID₅₀/mL in the 2-mo-olds and 6-mo-olds, respectively. The mean blood concentrations of PRRSV RNA were 332 TCID₅₀/mL in the 2-mo-old pigs and 472 TCID₅₀/mL in the 6-mo-old pigs.

Inoculation with both PRRSV MN-184 and M. hyopneumoniae resulted in greater clinical disease. In addition to the previously mentioned signs and diagnostic features after inoculation with both PRRSV and M. hyopneumoniae, we observed labored breathing, severe coughing, and gauntness due to prolonged anorexia, particularly in the 2-mo-olds. The pigs were often found recumbent and were often nonresponsive to physical stimulation. Severe apnea and dyspnea after anesthesia resulted in the suspension of sample collection on day 11 after inoculation in the 6-mo-old pigs and days 11 and 19 in the 2-mo-old pigs. Because of severe respiratory disease, a 2-mo-old pig was humanely destroyed on day 3, and a 6-mo-old pig died on day 9. We detected PRRSV RNA from days 1 to 21 after inoculation in 17% to 100% of the 2-mo-old pigs, with a peak concentration of 1330 TCID₅₀/mL. Large quantities of virus were detected in the aerosols of 2-mo-old pigs in this experimental group; concentrations of 1330 and 740 TCID₅₀/mL were detected in 2 individual pigs on days 9 and 17 after inoculation. Among the 6-mo-old pigs, aerosol shedding was detected in 20% to 60% on days 3 to 9 and 17 after inoculation. However, in contrast to the 2-mo-old pigs, the peak concentration was only 6 TCID₅₀/mL. The mean blood concentrations of PRRSV RNA were 10 500 TCID₅₀/mL in the 2-mo-old pigs and 890 TCID₅₀/mL in the 6-mo-old pigs.

The patterns of shedding and the mean concentrations of PRRSV RNA across all pigs from each group over time are summarized in Tables II and III, respectively. All samples collected from the control pigs remained negative by RT-PCR and ELISA throughout the study. The pigs inoculated with both PRRSV and M. hyopneumoniae were positive for antibodies against the latter and also showed evidence of M. hyopneumoniae DNA in nasal swab samples by day 21 after inoculation. The mean respiratory rate across all groups was in the normal range of 20 to 40 breaths/min. The mean Paco₂ level was also in the normal range, at 43.3 (standard deviation 6.3) mm Hg.

Table III.

Mean concentrations of PRRSV RNA detected in aerosol samples over time

	Collection day; concentration of PRRSV RNA (TCID50/mL)
Agent(s) inoculated; animal age (mo)	1	3	5	7	9	11	13	15	17	19	21
PRRSV MN-30100
2	0	0.04	0.4	0.02	0	0	0	0.06	0	0.02	0
6	0	0.1	0.3	0.05	0.1	0	0	0	0	0	0
PRRSV MN-30100 + M. hyopneumoniae
2	0	0	0	0.03	0.03	0.03	0	0	0	0	0
6	0	0	0	0	0	0.05	0	0	0	0	0
PRRSV MN-184
2	0	0	0.1	0	0.02	0.02	0.03	6.0	0.02	0.2	1.0
6	0	0.2	0.04	0	0	0.02	1.04	0.3	0.1	0.1	0
PRRSV MN-184 + M. hyopneumoniae
2	0.4	0	10.0	3.3	266	NC	0.4	0.1	152	NC	4.0
6	0	1.14	1.0	0.03	0.1	NC	0	0	0.03	0	0

With ANOVA, the difference in the mean concentrations of PRRSV RNA in aerosols from pigs infected with PRRSV MN-30100 or PRRSV MN-184 was not significant (P = 0.15). Neither animal age (P = 0.09) nor inoculation with M. hyopneumoniae in addition to PRRSV (P = 0.09) significantly affected the aerosol concentration of PRRSV MN-30100 or PRRSV MN-184. In contrast, logistic regression showed that inoculation with PRRSV MN-184 resulted in a significantly higher likelihood of aerosol shedding (P = 0.00005, odds ratio = 3.22) than did inoculation with PRRSV MN-30100. The effects of age (P = 0.8) and inoculation with M. hyopneumoniae in addition to PRRSV (P = 0.05) on the frequency of shedding were not significant. The mean blood concentrations of PRRSV RNA were significantly higher in the pigs inoculated with PRRSV MN-184 (P = 0.047) and in those also inoculated with M. hyopneumoniae, independent of the PRRSV isolate used (P = 0.02), than in the other experimental groups. Again, the impact of animal age on RNA concentration was not significant (P = 0.09).

The objective of this study was to evaluate the role of animal age, bacterial coinfection, and PRRSV isolate pathogenicity on the concentration and shedding of PRRSV by aerosol. Under the conditions of this study, isolate pathogenicity appeared to significantly influence the frequency of PRRSV shedding in aerosols. During the sampling period, differences were observed in both the proportion of pigs shedding and the pattern of aerosol shedding across the 2 isolates. Although a small number of pigs inoculated with PRRSV MN-30100 shed intermittently throughout the sampling period, more consistent shedding was observed in a larger number of pigs inoculated with PRRSV MN-184. In contrast, isolate pathogenicity did not appear to significantly affect virus concentration in aerosols. Across both isolates, the overall levels of PRRSV RNA detected in aerosol samples were low, and numerous samples were negative. This may explain why experimental reproduction of this route of transmission of PRRSV has been so difficult. In other words, it is possible that pigs do not shed large quantities of PRRSV in aerosols. This suggests that, besides isolate pathogenicity, population size may be an important risk factor for PRRSV aerosol transmission in the field.

The rudimentary design of the collection device may have influenced the results; however, its sensitivity was validated before the start of the study. Although re-evaluation with more sophisticated equipment is an option, initial reports indicate that devices such as cyclonic impingers may lack the sensitivity required to accurately detect and quantify the inherently low levels of PRRSV in aerosols (Joe Hermann, Iowa State University, Ames, Iowa, USA: personal communication, 2005).

In conclusion, the results of this study suggest that PRRSV isolate pathogenicity affects the shedding of PRRSV in aerosols. Future laboratory studies should focus on determining the infectious dose and half-life of PRRSV in aerosols. Studies involving large populations may be necessary to better understand the frequency of aerosol shedding and the concentration of PRRSV shed in this manner from infected pigs in the field. Once this information is available, swine producers and practitioners may be able to develop strategies to reduce the risk at the farm level, thereby enhancing PRRS control and eradication programs at the regional level.