Infection of growing swine with porcine reproductive and respiratory syndrome virus and Mycoplasma hyopneumoniae — Effects on growth, serum metabolites, and insulin-like growth factor-I.

Abstract

This study evaluated the influence of concomitant infections with porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae on growth performance, serum metabolite concentrations, and serum insulin-like growth factor-I (IGF-I) in growing pigs. Twenty-two barrows (10 weeks of age) were treated with either an intranasal administration of PRRSV and an intratracheal infusion of M. hyopneumoniae (treatment; n = 8) or a sham inoculation with medium (sham; n = 8), or were not treated (control; n = 6). The sham pigs were matched by body weight and pair-wise fed with treatment pigs. Pigs were weighed on the day of inoculation (day 0) and at 4 weeks postinoculation (day 28). Blood samples were collected prior to inoculation and at weekly intervals for 4 weeks. Pigs in the treatment group exhibited clinical signs consistent with PRRSV infection and M. hyopneumoniae pneumonia. Diagnostic procedures confirmed that treatment pigs were inoculated with PRRSV and M. hyopneumoniae and that sham and control pigs remained free of both pathogens. Average daily gain and feed conversion did not differ among the 3 groups. The IGF-I levels differed (P < 0.05) between control and treatment pigs, even after feed intake returned to similar levels among groups. At day 7, IGF-I concentrations were greater in sham pigs compared with treatment pigs, despite similar feed intake. Sham inoculation and decreased feed intake in sham pigs did not alter serum IGF-I concentrations. Evidently, IGF-I status of pigs affected with disease is influenced by nutritional and nonnutritional factors during the disease process.

Introduction

Slow growth and suboptimal feed efficiency resulting from disease are recognized as economic problems in grow-finish pigs (1). Consequently, management techniques; such as, all-in-all-out animal movement and segregated early weaning, were developed to reduce transmission of pathogens, thereby improving performance. However, disease and growth variation continue to interfere with pig production. Respiratory disease is a major health concern in grow-finish pigs. Several infectious agents have been identified in pigs affected with respiratory disease; however, porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae are considered to be 2 common pathogens (2).

Immunological stress has been shown to be associated with lowered rates of weight gain and feed conversion in pigs (3,4). Immunological stress directs metabolism away from skeletal muscle accretion to provide energy and protein for the immune response (5). The precise mechanisms underlying the changes in metabolism are poorly understood; however, decreased serum insulin-like growth factor–I (IGF-I) concentrations were observed following an immunological challenge in steers (6) and pigs (7). Since serum concentrations of IGF-I are closely associated with animal growth rates and resulting size (8), changes in IGF-I concentrations after an immune challenge presumably contribute to impaired growth.

Most investigators have used the administration of endotoxin to evaluate the effects of an immune challenge on growth in pigs (3,7,9). Endotoxin administration has a short-term effect on growth and IGF-I concentrations (10); thus, it fails to effectively mimic the effects of pathogens and disease in commercially reared pigs. Few studies have determined the collective effects of a challenge with specific pathogens on IGF-I, serum metabolites, and growth performance of swine (3,4,7); therefore, the first objective of this study was to determine the effects of concomitant PRRSV and M. hyopneumoniae infections on growth, serum metabolite concentrations, and serum IGF-I concentrations in grow-finish pigs. The selection of pathogens was based on their significant roles and interactions in a common disease condition (2). The 2nd objective was to develop a disease model, within an experimental setting, that simulates a field situation.

Materials and methods

The North Carolina State University Animal Care and Use Committee approved the experimental protocol used in this study. Twenty-two, large-white crossbred barrows (3 wk of age) were weaned from sows at a university-owned swine farm and were transported to environment-controlled rooms at the laboratory animal facilities at the College of Veterinary Medicine. The farm is a 300-sow, farrow-to-finish facility, which maintains a strict biosecurity protocol. Replacement females are produced on the farm and artificial insemination is routinely used in the breeding program. Boars (< 4 boars/y) are the only replacement animals entering the herd. The presence of PRRSV has not been detected in the herd through serological profiling and regular clinical examinations.

At 10 wk of age, pigs were assigned by weight to 1 of 3 experimental groups. Pigs (n = 8) in the treatment (TMT) group were inoculated with PRRSV and M. hyopneumoniae. Pigs (n = 8) in the sham (SHM) group were inoculated with medium. The SHM pigs were matched by body weight and pair-wise fed with TMT pigs. Pair feeding was necessary to assess if changes in metabolites and IGF-I concentrations were due solely to diminished feed intake. Pigs (n = 6) in the control (CON) group were not treated with pathogens or medium. To account for the pair-wise feeding protocol, the days of inoculation, blood collection, and other procedures were adjusted accordingly. Pigs were assigned to individual pens (1 m × 2 m) and housed within 3 rooms (1 room/experimental group) upon weaning and throughout the study. Each pen was equipped with a single-hole feeder and 1 water drinker.

From 3 to 9 wk of age, all pigs were fed commercial nursery rations ad libitum, which met or exceeded National Research Council recommendations (11). After reaching 9 wk of age, the pigs in the TMT and CON groups were fed a diet (Table 1) formulated to meet or exceed requirements for all nutrients ad libitum (11). The diet did not contain antibiotics or growth promoting agents. To determine daily feed intake, unconsumed feed was collected and weighed and this value was subtracted from the weight of feed offered. Pigs were weighed on the day of inoculation (day 0) and at 4 wk postinoculation (day 28). General animal health was noted twice daily by monitoring attitude, clinical appearance, and behavior. Blood samples were taken from each pig 14 d prior to inoculation, immediately prior to inoculation, and at weekly intervals for 4 wk thereafter. The blood samples were taken prior to feeding. Necropsies were performed on day 28 and tissue samples of lung, palatine tonsil, and mediastinal lymph nodes were collected for subsequent immunohistochemical (IHC) evaluation for PRRSV (12). Additional samples of lung were collected, placed in polyethylene glycol-based optimal cutting temperature (OCT) medium, and immediately frozen. Then, the samples were shipped to Iowa State University for direct immunofluorescence (IFA) detection of M. hyopneumoniae (13).

Table 1.

To prepare the PRRSV inoculum, confluent flask cultures of monkey kidney cells (MARC-145 cells, American Type Culture Collection) were inoculated with diluted stock virus (500-2000 TCID₅₀/mL). The inoculum was allowed to be absorbed and then the cultures were observed daily for the development of cytopathic effect (CPE). When 60% to 70% of the cell monolayer demonstrated CPE, it was frozen in the flasks. Subsequently, the cell monolayer was thawed and diluted as the stock virus and stored at –70°C. The virus concentration was determined by serial dilution of stock virus to a cell suspension. After a 3-day incubation, the virus dilutions were read for CPE. The number of infected wells was expressed as a ratio of the number of wells inoculated per virus dilution. The Karber method was used to approximate the 50% end point (14). Pigs in the TMT group were inoculated by intranasal administration of 1 mL/naris of PRRSV (10^3-4 TCID₅₀, isolate SD 23983) (15,16). The SHM pigs received 1 mL/naris of Roswell Park Memorial Institute (RPMI) medium.

The M. hyopneumoniae inoculum (LI31 5-13-93, Strain 11) was obtained from Drs. R. Ross and E. Thacker (Iowa State University (ISU), Ames, Iowa). The M. hyopneumoniae inoculum contained a tissue homogenate of M. hyopneumoniae strain 11 (10⁶ organisms/mL) in mycoplasmal Friis medium. Pigs in the TMT group were inoculated with 5 mL of inoculum by intratracheal infusion, as described by Ross and Cox (17). Friis medium was used as the inoculum for the SHM group.

The isolation of PRRSV (18) was conducted on serum samples taken on days −14, 0, and 7 to confirm inoculation and to verify the virus-free status of the SHM and CON groups. Briefly, 100 μL of serum was added to cultured alveolar macrophages and allowed to incubate for 3 to 5 d and then the macrophages were assessed for CPE. Cultures were deemed positive if there was any indication of cell death. At necropsy, lungs were assessed for macroscopic lesions of pneumonia and the proportion of the lung with pneumonia was determined by using a previously established scoring system (17). For pigs with pneumonic lesions, tissues were collected from the area of demarcation between normal and pneumonic tissue. For the SHM and CON groups, tissues from the cranial and accessory lobes of the right lung were submitted to ISU for IFA tests. Mediastinal lymph nodes, tonsil, and pulmonary tissue were submitted to the ISU (Dr. P. Halbur) for IHC evaluation for PRRSV.

Serum was analyzed for glucose, cholesterol, triglycerides, urea nitrogen, and IGF-I. Serum metabolite concentrations were determined in the Clinical Pathology Laboratory, College of Veterinary Medicine, by using standard laboratory procedures. Serum IGF-I was determined with established radioimmunoassay methods (7). Recombinant human IGF-I (Monsanto, St. Louis, Missouri) was used for the standards, and rabbit anti-IGF-I serum (National Hormone and Pituitary Program, Harbor-UCLA Medical Center, Torrance, California) was used as the primary antibody. The sensitivity of the assay was 25 ng/mL and the interassay coefficient of variation (CV) was 7.2%; the intra-assay CV's were consistently less than 5%.

Data were analyzed by using a statistical software program (SAS General Linear Model, SAS Users Guide; Statistics (Release 6.03), SAS, Cary, North Carolina). Performance measures were analyzed with a one-way analysis of variance (ANOVA). Similarly, a one-way ANOVA was used for daily feed intake within day. Serum metabolite concentrations were analyzed initially by a repeated measures-split plot design. The model included treatment, time, metabolite, and the interactions. Pig within treatment was used as the error term. A significant time by treatment interaction was evident for 2 of the metabolites. The final analysis for serum metabolite and IGF-I concentrations was a one-way ANOVA and comparisons of means were conducted within day. Due to considerable variation (between animals within group) in serum IGF-I concentrations at day 0, concentrations at days 7 to 28 were converted to percentages relative to concentrations at day 0. Treatment means were compared by Fisher's protected least significant difference test.

Results

The PRRSV infection induced sneezing, hyperpnea, dyspnea, lethargy, cutaneous erythema, and diarrhea in TMT pigs in the 1st week postinoculation. Infection with M. hyopneumoniae was typified by a characteristic cough, which commenced 10 d after inoculation and continued intermittently through to day 28. Necropsy revealed gross lesions in the TMT group that were consistent with pneumonia caused by PRRSV and M. hyopneumoniae infection. Immunohistochemical staining and IFA (Table 2) confirmed PRRSV and M. hyopneumoniae infection, respectively, in TMT pigs. The average percentage of lung that was pneumonic was 14.6% (range 7% to 30%). The CON and SHM groups remained clinically normal throughout the experimental trial, and laboratory procedures failed to detect PRRSV or M. hyopneumoniae infection.

Table 2.

Feed intake was less (P < 0.05) in the TMT group than in the CON group on days 2 through 12, and days 16 and 17 (Figure 1). Average daily gain (ADG) and feed conversion (FC) (feed:gain) did not differ among groups (Table 3).

Figure 1. Daily feed disappearance (mean) in the treatment group of pigs (TMT; n = 8) inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae, pigs sham inoculated (SHM; n = 8) and pair-fed with TMT pigs, and control pigs (CON; n = 6). The slashed bars indicate days in which feed disappearance differed (P < 0.05) between TMT and CON groups. Feed disappearance did not differ between TMT and SHM groups. The standard errors of the means were 0.1, 0.04, and 0.04 for the CON, SHM, and TMT groups, respectively.

Table 3.

Few differences were noted for serum urea nitrogen, triglycerides, or glucose concentrations among groups over the course of the study (Table 4). A trend of increasing serum cholesterol was noted in the TMT group, and, at day 28, serum cholesterol was greater (P < 0.05) in the TMT group than in the SHM group. The percentage points of IGF-I relative to day 0 were greater (P < 0.05) in the CON group than in the TMT group for the 4 wk after inoculation (Figure 2). In addition, the percentage points of IGF-I were less (P < 0 .05) in the TMT group than in the SHM group on day 7.

Table 4.

Figure 2. Serum IGF-I concentrations expressed as a percent (mean + standard error) relative to baseline concentrations at day 0. One group of pigs served as controls (CON; n = 6), and the treatment group (TMT; n = 8) of pigs were inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae. Pigs in the sham group (SHM; n = 8) were inoculated with medium and were pair-wise fed with animals in the TMT group. Inoculations were given on day 0, following the collection of blood samples. Within a day, bars with different superscripts differed by P < 0.05.

Discussion

Several infectious agents pose significant health threats to growing pigs. Porcine reproductive and respiratory syndrome virus and M. hyopneumoniae are common pathogens and have been associated with impaired growth and economic losses (1,19,20,21). It is thought that the activation of the immune system in response to challenge by disease pathogens repartitions nutrients from muscle accretion toward metabolic responses that support the immune system. Consequently, growth in affected pigs becomes more variable rather than attaining the uniform size, which is desirable for market pigs. Disease interactions seen (or suspected) under field conditions have largely not been reproduced experimentally (22). Most previous studies attempted to use endotoxin to reproduce some of the clinical signs associated with disease as observed in commercial farms (3,7,9). However, the transient effects of endotoxin treatment may not mimic the clinical disease associated with PRRSV and M. hyopneumoniae infection in grow-finish pigs.

Clinical signs associated with PRRSV include lethargy, mild to moderate dyspnea, nasal discharge, and coughing (15,16). A characteristic cough is typical in pigs with mycoplasmal pneumonia. In the present study, pigs in the TMT group exhibited clinical signs consistent with PRRSV infection and M. hyopneumoniae pneumonia. In addition, diagnostic assessments confirmed the presence of PRRSV and M. hyopneumoniae. Both CON and SHM groups remained serologically negative throughout the trial, and diagnostic tests did not detect either pathogen. Thus, this investigation successfully reproduced concurrent PRRSV and M. hyopneumoniae infections, as previously described (2).

Another objective of this study was to assess the effects of PRRSV and M. hyopneumoniae infections on growth performance, serum metabolites, and serum IGF-I concentrations. Feed intake for the TMT group was less than that for the CON group for several days after inoculation with the pathogens; however, feed intake was similar between groups during the latter phase of the study. Anorexia is often associated with activation of the immune system and infection with pathogens; thus, it was not surprising that the TMT pigs had diminished feed consumption. In contrast, ADG and FC were not different between the TMT and SHM groups. This finding is supported by the observation that defined stress, under specific stress-free conditions, ADG or FC are not altered (23). Perhaps the severity of the lesions was insufficient to compromise ADG and FC in pigs housed in the ideal conditions. In addition, the time course of the experiment may not have been adequate to detect the detrimental changes in ADG and FC in the infected pigs. Conversely, the detrimental effects of the 2 pathogens on ADG and FC may be transient in an experimental setting and by 21–28 d postinoculation, pig performance is no longer compromised.

Few differences were noted for serum urea nitrogen, triglycerides, or glucose among the experimental groups over the course of the study. Hypertriglyceridemia and cholesterolemia are responses to minimize the effect of endotoxin and viral challenges by binding these agents and preventing their stimulation of cytokine production by macrophages (24). However, the results of the present study failed to reveal significant alterations in triglycerides, cholesterol, or glucose in TMT pigs. This failure to detect hypertriglyceridemia, cholesterolemia, or hyperglycemia is not unique to this study. Previous investigations also were unable to document changes in lipid metabolism after endotoxin challenge in pigs (4,25). An increase in plasma urea nitrogen, indicative of increased protein metabolism, was not observed in the present study. With endotoxin administration, changes in plasma urea nitrogen occurred within 24 h (25). If infection with PRRSV and M. hyopneumoniae alters protein metabolism, the changes were not evident at 7 d postinoculation in the present study.

The structure of IGF-I is similar to pro-insulin and produces insulin-like effects. In the growing pig, IGF-I is present in increasing concentrations and is affected by changes in metabolic demand. Cytokines, produced as part of the immune response, were shown to suppress the secretion of growth promoting hormones (26). Depression in IGF-I levels was observed in the SHM group and is related to the fact that serum IGF-I concentrations are affected by feed intake over the short term (27,28). Serum IGF-I concentrations were consistently less in the TMT group than in the CON group and, at day 7, less than in the SHM group. It was reported that endotoxin-treated pigs have IGF-I concentrations that were considerably lower compared with control animals and feed intake was not the only factor contributing to diminished IGF-I (7). In the present study, feed intake for the TMT group returned to levels that were similar to those of CON pigs after day 11. However, IGF-I depression in the TMT group continued through to day 28. It is evident that the IGF-I status of pigs, affected with disease, is influenced by nutritional and possibly nonnutritional factors during the disease process.

In conclusion, a disease model using PRRSV and M. hyopneumoniae was established under experimental conditions. With the appropriate modifications; such as, increasing the severity of the clinical disease, this model should prove useful to evaluate the interactions between disease and growth in the pig. This study suggested that disease, independent of feed intake, may suppress serum IGF-I. The duration of the IGF-I suppression, its affects on growth, and alterations in metabolism warrant further research.