Materials and Methods

Animals and study site—Procedures were conducted at the University of Minnesota College of Veterinary Medicine isolation facility. Facilities included a series of separately ventilated rooms containing slurry pits to prevent cross-contamination of pathogens among rooms. Twenty-one 2-month-old pigs (body weight, 25 kg) and eighteen 6-month-old pigs (120 kg) were purchased from a source known to be free of PRRSV and M hyopneumoniae on the basis of a 10-year history of diagnostic testing and the absence of clinical signs of infection with either pathogen. Pigs were transported to the isolation facility and acclimatized for 3 days prior to initiation of the study. Blood and swab specimens of nasal secretions were collected for analysis via RT-PCR assay to verify that pigs were free of PRRSV and M hyopneumoniae prior to inclusion in the study.¹³ During the study, pigs were housed and cared for according to University of Minnesota Institutional Animal Care and Use Committee guidelines.

Source of virus—Two PRRSV isolates were selected for use on the basis of clinical signs elicited in pigs after experimental intranasal inoculation. An isolate of low pathogenicity (PRRSV MN-30100) was obtained from a persistently infected sow in a commercial production site.¹⁴ Clinical signs observed after intranasal infection with that isolate included transient depression, lack of appetite for 24 to 48 hours, and mild fever (40° to 41°C).¹³ In contrast, the highly virulent isolate (PRRSV MN-184) originated at a farm in southern Minnesota affected with severe reproductive disease and high sow mortality rates.⁸ In addition to high mortality rates, clinical signs induced by the PRRSV MN-184 isolate after experimental intranasal inoculation included a prolonged period of depression, absence of appetite, and high fever (> 42°C).⁸

PRRSV infection—Pigs were allocated into groups according to age, and groups were housed in separate rooms in the isolation facility. One control pig for each group was housed in a separate room. On day 0, pigs were inoculated intranasally with PRRSV MN-30100 (2-month-old pigs, 5 pigs/group; 6-month-old pigs, 4 pigs/group) or PRRSV MN-184 (2-month-old pigs, 6 pigs/group; 6-month-old pigs, 5 pigs/group). Group size varied between ages and virus types because of space requirements in the isolation facility for differently sized pigs and the anticipated mortality rate in pigs inoculated with the MN-184 strain of virus. Subject pigs received 2 mL of inoculum with a viral concentration of 1 × 10⁴ TCID₅₀/mL. Control pigs were sham-inoculated intranasally with 2 mL of sterile saline (0.9% NaCl) solution.

Successful PRRSV inoculation was determined by results of serum qualitative RT-PCR^a on days 1 to 3 after infection.

PRRSV and M hyopneumoniae coinfection—Pigs infected with M hyopneumoniae and PRRSV were allocated into groups according to age (2-month-old pigs, 6 pigs/group; 6-month-old pigs, 5 pigs/group). One control pig for each group was housed in a separate room. On day 0, pigs were anesthetized with a combination of xylazine hydrochloride^b (dose, 1.5 mg/kg) and a commercially available mixture of tiletamine and zolazepam hydrochloride^c (dose, 8 mg/kg) administered IM.¹⁵ After induction, pigs were intubated and inoculated via intratracheal instillation of a solution containing 10⁵ color-changing units of M hyopneumoniae strain 232/mL in volumes of 10 and 25 mL to 25- and 120-kg pigs, respectively.¹¹ Twenty-one days after bacterial inoculation, pigs were intranasally inoculated with PRRSV MN-30100 or MN-184 according to a described method.¹¹ Control pigs were anesthetized and sham-inoculated with sterile saline solution via the same intratracheal procedure. Nasal swab specimens were collected and tested by use of a nested PCR¹³ on days 7, 15, and 21 to determine successful infection with M hyopneumoniae. Serum samples were collected from each pig to test for serum antibodies against M hyopneumoniae by use of an ELISA^d on days 15 and 21 after bacterial inoculation. Successful PRRSV inoculation was monitored by collection of blood samples on days 1 to 3 after PRRSV infection and by testing via qualitative RT-PCR assay.^16,a

Sampling protocol—Swab specimens of blood, nasal secretions, and oropharyngeal secretions were collected from each pig on alternating days on days 1 to 21 after the viral inoculation procedures. Blood samples were collected in sterile vacuum tubes^e via jugular venipuncture. Nasal samples were collected by inserting a sterile swab^f 2 cm into each of the nares. Oropharyngeal swabs were collected by drawing a similar sterile swab across the hard and soft portions of the palate for 5 seconds. Swabs were placed into sterile plastic tubes^g containing 2 mL of sterile saline solution. On day 21, post-PRRSV inoculation, pigs were euthanatized with an IV injection of sodium pentobarbital^h at a dose of 100 mg/kg. During necropsy, 1-g specimens of tissue from the lung, tonsil, lateral retropharyngeal lymph node, tracheobroncheal lymph node, and sternal lymph node were collected. Tracheal swabs were collected by inserting a sterile swab 5 cm into the trachea and drawing the swab upwards so that it contacted the luminal surface of the trachea. Samples were stored individually at −20°C for 1 to 2 days prior to testing.

Quantitative RT-PCR procedure—Quantitative assessment of PRRSV in samples was conducted by use of an RT- PCR kit^a at the Minnesota Veterinary Diagnostic Laboratory in a procedure modified from a published protocol.¹⁶ Data were expressed as TCID₅₀ per milliliter (for sera and swab specimens) or TCID₅₀ per gram (for tissues). A standard curve was developed for the quantitative RT-PCR procedure by preparing 10-fold dilutions of PRRSV MN-30100 and PRRSV MN-184, with dilutions ranging from 1 × 10⁻⁶ TCID₅₀/mL to 1 × 10^4.6 TCID₅₀/mL. Samples were assayed in triplicate, and mean RNA concentrations were calculated from those values.

Statistical analysis—Concentrations of PRRSV in specimens from live animals were compared for the variables of viral isolate, concurrent bacterial infection, and animal age by use of a generalized ANOVA.ⁱ Because distributions were often negative, a Kruskal-Wallis 1-way ANOVA was used to assess effects of animal age, concurrent bacterial infection, and isolate pathogenicity on viral concentration in tissues obtained at necropsy. For all comparisons, values of P < 0.05 were considered significant.

Results

All pigs had negative results of tests for PRRSV and M hyopneumoniae prior to initiation of the study. Pigs of all age groups and infection models had clinical signs of viremia 1 to 3 days after PRRSV inoculation. Serum antibodies against M hyopneumoniae were detected by use of ELISA^d by day 21 after bacterial inoculation in all coinfected pigs.

Infection with PRRSV MN-30100—Clinical signs in 2- and 6-month-old pigs infected with PRRSV MN-30100 alone included transient fever (39° to 40°C), inappetance (24 to 48 hours after inoculation), and mild depression. None of those pigs died. In 2-month-old pigs, mean concentrations of PRRSV RNA were 254 TCID₅₀/mL (in blood samples) and 8 TCID₅₀/mL (in both nasal and oropharyngeal swab specimens). In 6-month-old pigs, mean RNA concentrations were 5 TCID₅₀/mL (blood), 4 TCID₅₀/mL (nasal swab specimens), and 8 TCID₅₀/mL (oropharyngeal swab specimens; Table 1). Regarding PRRSV RNA concentrations in tissues, mean values in 2-month-old pigs were 1 TCID₅₀/mL (tracheal swab), 3 TCID₅₀/mL (tonsil and lymph nodes), and 13 TCID₅₀/mL (lung). Mean RNA concentrations in 6-month-old pigs were 1 TCID₅₀/mL (tracheal swab, tonsil), 3 TCID₅₀/mL (lymph nodes), and 27 TCID₅₀/mL (lung; Table 2).

Concurrent infection with PRRSV MN-30100 and M hyopneumoniae—Clinical signs and death rates in 2- and 6-month-old pigs coinfected with PRRSV MN- 30100 and M hyopneumoniae were similar across and within age groups, except that a dry cough was observed by day 21 after inoculation of M hyopneumoniae and continued through the sampling period. In 2-month-old pigs, mean concentrations of PRRSV RNA were 326 TCID₅₀/mL (in blood samples), 6 TCID₅₀/mL (in nasal swab specimens), and 36 TCID₅₀/mL (in oropharyngeal swab specimens). In 6-month old pigs, mean RNA concentrations were 270 TCID₅₀/mL (in blood samples), 20 TCID₅₀/mL (in nasal swab specimens), and 23 TCID₅₀/mL (in oropharyngeal swab specimens; Table 1). Regarding PRRSV RNA concentrations in tissues, mean values in 2-month-old pigs were 240 TCID₅₀/mL (tracheal swab), 49 TCID₅₀/mL (tonsil), 210 TCID₅₀/mL (lymph nodes), and 196 TCID₅₀/mL (lung). Mean RNA concentrations in 6-month-old pigs were 28 TCID₅₀/mL (tracheal swab), 12 TCID₅₀/mL (tonsil), 20 TCID₅₀/mL (lymph nodes), and 36 TCID₅₀/mL (lung; Table 2).

Infection with PRRSV MN-184—Clinical signs in pigs infected with PRRSV MN-184 alone were more severe and of longer duration than those observed in pigs infected with PRRSV MN-30100. Clinical signs in pigs infected with PRRSV MN-184 included high rectal temperatures (> 42°C) and profound anorexia and depression throughout the 21-day testing period. One 6-month-old pig died on day 15 after inoculation as a result of respiratory illness. In 2-month-old pigs, mean concentrations of PRRSV RNA were 332 TCID₅₀/mL (in blood samples), 5 TCID₅₀/mL (in nasal swab specimens), and 24 TCID₅₀/mL (in oropharyngeal swab specimens). In 6-month-old pigs, mean RNA concentrations were 472 TCID₅₀/mL (blood), 13 TCID₅₀/mL (nasal swab specimens), and 58 TCID₅₀/mL (oropharyngeal swab specimens; Table 1). Regarding PRRSV RNA concentrations in tissues, mean values in 2-month-old pigs were 12 TCID₅₀/mL (tracheal swab), 51 TCID₅₀/mL (tonsil), 45 TCID₅₀/mL (lymph nodes), and 1150 TCID₅₀/mL (lung). Mean RNA concentrations in 6-month old pigs were 76 TCID₅₀/mL (tracheal swab), 55 TCID₅₀/mL (tonsil), 44 TCID₅₀/mL (lymph node), and 480 TCID₅₀/mL (lung; Table 2).

Concurrent infection with PRRSV MN-184 and M hyopneumoniae—Clinical signs in pigs coinfected with PRRSV MN-184 and M hyopneumoniae were severe. In addition to clinical signs observed in coinfected pigs, labored breathing, severe coughing, and gaunt body condition resulting from anorexia were observed, especially in 2-month-old pigs. Pigs often lay in recumbent positions and were nonresponsive to physical stimulation. Sample collection was suspended on postinoculation day 11 in 6-month-old pigs and on days 11 and 19 in 2-month-old pigs because of the severity of clinical signs. One 2-month-old pig was euthanatized on day 3 after inoculation because of the severity of clinical signs, and one 6-month-old pig died on day 9 after inoculation. Mean concentrations of PRRSV RNA in 2-month old pigs were 10,500 TCID₅₀/mL (in blood samples), 53 TCID₅₀/mL (in nasal swab specimens), and 1,200 TCID₅₀/mL (in oropharyngeal secretions). In 6-month-old pigs, mean RNA concentrations were 890 TCID₅₀/mL (in blood samples), 41 TCID₅₀/mL (in nasal swab specimens), and 50 TCID₅₀/mL (in oropharyngeal swab specimens; Table 1). Regarding PRRSV RNA concentrations in tissues, mean values in 2-month-old pigs were 103 TCID₅₀/mL (tracheal swab), 32 TCID₅₀/mL (tonsil), 52 TCID₅₀/mL (lymph nodes), and 650 TCID₅₀/mL (lung). Mean RNA concentrations in 6-month old pigs were 1 TCID₅₀/mL (tracheal swab), 39 TCID₅₀/mL (tonsil), 104 TCID₅₀/mL (lymph nodes), and 81 TCID₅₀/mL (lung; Table 2).

Statistical analyses indicated that in blood samples, concentrations were significantly higher in pigs infected with PRRSV MN-184 (P = 0.047) and in pigs coinfected with M hyopneumoniae (P = 0.022), regardless of the PRRSV isolate used for infection. Concentrations were significantly (P = 0.045) higher in animals coinfected with M hyopneumoniae, independent of the PRRSV isolate. Animal age did not significantly (P = 0.118) affect virus concentration in oropharyngeal swab specimens.

Regarding concentrations of PRRSV in tissues, 2-month-old pigs had significantly higher concentrations in lymph nodes (P = 0.026), lung (P = 0.019), and tracheal swab specimens (P < 0.001), compared with those in 6-month-old pigs, irrespective of PRRSV isolate. Infection with the PRRSV MN-184 isolate yielded significantly higher concentrations of virus in tonsils (P < 0.001), compared with those in pigs infected with PRRSV MN-30100, whereas pigs concurrently infected with PRRSV MN-184 and M hyopneumoniae had significantly higher concentrations in tonsils (P < 0.001) and lymph nodes (P < 0.001). All samples from control pigs had negative results of RT-PCR and ELISA testing throughout the study.

Discussion

Control of PRRSV infection in pigs in the post-weaning period is dependent on a proper understanding of the effects of certain factors that are commonly encountered in modern production systems on populations of pigs infected with the virus. Our objectives were to evaluate the roles of animal age, concurrent bacterial infection, and pathogenicity of the PRRSV isolate on viral concentrations in individual pigs. Although these variables have been investigated independently, to the authors' knowledge, studies of their combined effect have not been published. Our study was designed to investigate the hypothesis that concentrations of PRRSV would be significantly higher in younger pigs with a poor health status; for purposes of the study, we defined poor health as the state of concurrent infection with PRRSV and M hyopneumoniae.

Our results indicated that the 3 variables evaluated affected PRRSV concentration. Infection with a highly pathogenic isolate (PRRSV MN-184) significantly affected viral concentrations detected in blood and tonsil tissues, whereas concentrations in animals infected with an isolate of lesser pathogenicity (PRRSV MN-30100) were not affected. Age of the animal also played a role, with younger pigs having significantly higher viral concentrations in tissues than older pigs, independent of PRRSV isolate. Concurrent infection with M hyopneumoniae yielded significantly higher PRRSV concentrations in blood and oropharyngeal swab specimens, compared with those in animals infected with PRRSV alone, independent of the isolate. Significantly higher viral concentrations were also observed in lymph nodes and tonsil tissue from pigs coinfected with the highly pathogenic PRRSV isolate and M hyopneumoniae.

These findings were in accordance with results of other studies,¹¹ in which coinfection with PRRSV and M hyopneumoniae exacerbated PRRSV-induced disease and resulted in persistent lung lesions. Pigs with concurrent bacterial-viral infections also had higher mean concentrations of virus in lung tissue than pigs infected with only 1 of those 2 pathogens. Our study expanded that line of investigation by evaluating the role of coinfection with M hyopneumoniae along with the concurrent influences of PRRSV isolate pathogenicity and animal age. This was performed quantitatively across multiple sampling sites in individual pigs, and data were reported as mean virus concentrations rather than as a disease score. Nonetheless, results indicate that infection with M hyopneumoniae in combination with PRRSV infection not only increased the duration and severity of clinical signs but also enhanced the concentrations of viral RNA in various samples.

Our results should be interpreted carefully for several reasons. Only 2 isolates of PRRSV were evaluated; therefore, the applicability of results is limited to pigs infected with PRRSV MN-30100 or PRRSV MN-184. Those isolates were selected on the basis of their representation of a wide spectrum of pathogenicity. Similarly, only a single bacterium was used to assess the effects of concurrent bacterial infection. However, the bacterium was selected on the basis of its association with the porcine respiratory disease complex and on the availability of published data.

The present study was not designed to determine infectious doses of PRRSV. Data were reported as TCID₅₀ per milliliter or TCID₅₀ per gram on the basis of values extrapolated from standard curves.¹⁷ It is possible that higher concentrations of virus may have been detected if another unit of measure, such as RNA copies/mL, had been used or if tonsil scrapings rather than swabs had been used to assess viral concentrations in the oropharynx. However, because of the assay’s commercial availability and the ability to test large volumes of samples and maintain consistency, the quantitative RT-PCR test was used. Results cannot be extrapolated beyond the level of the individual because of the small sample size in each test group. However, this sample size permitted more frequent sampling and, hence, more numerous antemortem (n = 11) and postmortem (6) samples from each pig.

Despite these limitations, results provide pre-liminary data for concentrations of PRRSV recovered from pigs that differed in health status and age. Future studies are warranted to investigate the use of other diagnostic techniques for quantification of infectious dose at the individual animal level and that replicate the present study with a larger number of animals, with the goal of improving PRRS control at the herd level and eventual eradication of the disease.