The Circoviridae family are small, icosahedral, non-enveloped viruses with single-stranded, circular DNA genomes. There are two genera (Gyrovirus and Circovirus) in the family of Circoviridae that are characterised according to their genome sizes and differences in genome organisation. Porcine circovirus types 1 and 2 (PCV1 and PCV2, respectively) belong to the Circovirus genus(Reference Ignjatovic1–Reference Studdert3). PCV1 is non-pathogenic and has not been reported to associate with naturally occurring diseases, while PCV2 has been suggested as the essential pathogen for the post-weaning multi-systemic wasting syndrome (PMWS)(Reference Allan, McNeilly and Kennedy4, Reference Ellis, Hassard and Clark5) and porcine circovirus-associated diseases or porcine circovirus diseases(Reference Segales, Allan and Domingo6, Reference Opriessnig, Meng and Halbur7). The compelling pathological finding is that PCV2 infection damages macrophages, antigen-presenting cells and other immune-related cells(Reference Segales, Rosell and Domingo8). So the PMWS is considered as an immunosuppressive disease, and the interaction between PCV2 and the immune system is suggested as a determining factor in the pathogenesis of the PMWS(Reference Fort, Fernandes and Nofrarias9).
Glutamine, an immunonutrient and modulator, plays a role in the interaction between the carbon metabolism of carbohydrates and proteins, and has also been found to play an important role in the development of fibroblasts, lymphocytes and enterocytes(Reference Cao, Feng and Hoos10–Reference Tapiero, Mathe and Couvreur12). A large body of evidence shows that glutamine has various beneficial effects on immune and intestinal functions. For example, it was found that glutamine functions as a major energy substrate for cells of the immune system(Reference Wu, Field and Marliss13–Reference Wang, Chen and Li15) and that the provision of glutamine enhances the immunity of the host(Reference Li, Yin and Li16–Reference Blachier, Yin and Wu19). Moreover, research demonstrated that the ability of lymphocytes to respond to mitogenic stimulation is impaired after failure to supplement culture media with glutamine. Further studies indicated that glutamine is required in terminally differentiated macrophages for the synthesis of mRNA for producing secretory proteins in immune challenge during pinocytosis or phagocytosis(20). Other compelling evidence for the physiological functions of glutamine is its relationship with intracellular redox status and oxidative stress. Collectively, accumulating evidence suggests that glutamine plays important roles in promoting immune functions and preventing diseases, especially subclinical and immunosuppressive diseases.
The aims of the present study were to evaluate the immune-enhancing effects of dietary l-glutamine supplementation in PCV2-infected mice, and to investigate the potential of clearing PCV2 in experimentally infected mice.
Materials and methods
Animals and feeding
A total of sixty Kunming female mice (body weight 18–22 g) were obtained from the Laboratory Animal Center of Central South University, Hunan, China(Reference Li, Hua and Yang21). Mice were randomly assigned to two treatment groups after 3 d of adaptive feeding: glutamine group (1·0 % glutamine+basal diet, n 30) and control group (1·22 % alanine+basal diet, n 30). Glutamine and alanine were purchased from Beijing Chemclin Biotech(Reference He, Tang and Ren22). Amino acid content in the basal diet was measured using an Automatic Amino Acid Analyzer (L-8900; Hitachi)(Reference Zhang, Yin and He23). This basal diet contained 1·94 % l-glutamate, 1·80 % l-glutamine and 0·91 % l-alanine. Mice were housed in an environmentally controlled pathogen-free condition. All animals were fed ad libitum. The study was carried out in full compliance with the guidelines for animal welfare and was approved by the Animal Care and Use Committee of the Chinese Academy of Sciences (registry no. 011063506)(Reference Yin, Zhang and Huang24).
Preparation of porcine circovirus type 2 stock
A PCV2 infectious clone constructed by self-ligation of the PCV2 genome via the SacII enzyme site was used to generate the virus stock pools required for experimental infections. Briefly, the continuous porcine kidney cell line PK-15 (gift from Professor Yang, China Agricultural University)(Reference Wen, He and Yang25), free of PCV1 and PCV2, was cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 6 % (v/v) fetal calf serum. The cell monolayer was dispersed by treating the cells with trypsin-EDTA, and suspended in RPMI-1640 medium supplemented with 6 % (v/v) fetal calf serum while cells were infected with the PCV2 infectious clone. After 72 h of incubation, the infected cells were frozen and thawed thrice, and the cell mixture was tested by PCR before storing them at − 20°C. PCV2 stocks were titrated on PK-15 cells (gift from Professor Yang, China Agricultural University)(Reference Wen, He and Yang25).
Experimental design
Mice were infected with PCV2 at a dose of 100 TCID50 (50 % tissue culture infection dose) by intraperitoneal injection after 2 weeks of feeding l-glutamine. On 3rd, 5th, 7th, 9th and 11th d post-infection (dpi), six mice from each group were killed to collect liver, spleen, heart, lung, kidney and ovary, and serum was prepared from blood samples collected from the orbital vein. All the samples were stored at − 80°C(Reference Yu, Yin and Yin26, Reference Kong, Zhang and Yin27).
Serum cytokine detection
Serum levels of IL-2, IL-6, IL-10, interferon (IFN)-α, IFN-γ and C-reactive protein (CRP) were measured using ELISA kits in accordance with the manufacturer's instructions (Cusabio Biotech Company Limited)(Reference Ren, Zou and Li28, Reference Wang, Yang and Tang29). Supplied diluent buffer in the kits was used to dilute standards and serum samples. Next, 100 μl of the sample or standard in duplicate were added to the wells of a microtitre plate pre-coated with antibody. Diluent buffer was used as a negative control. The plate was incubated for 2 h at 37°C. After incubation, 100 μl of biotin antibody were added to each well after removing the liquid from each well and incubated for 1 h at 37°C. The wells were washed thrice with 200 μl volumes of wash buffer. Next, 100 μl horseradish peroxidase–avidin were added to each well for 1 h at 37°C(Reference Yang, Li and Kong30). After a final wash, 90 μl of the supplied chromogen were added and incubated for 30 min in the dark at 37°C. The reaction was stopped with 50 μl of the supplied stop solution and absorbance was measured at 450 nm with a spectrophotometer.
DNA extraction and porcine circovirus type 2 quantitative PCR
DNA from samples (liver, 10 mg; spleen, 5 mg; heart, 10 mg; lung, 10 mg; kidney, 10 mg; ovary, 10 mg) was extracted using Tissue Genomic DNA Extraction Kits (Betimes Biotechnology Company, Limited) according to the manufacturer's instructions. DNA from the samples was eluted with 80 μl of elution buffer and stored at − 20°C. PCV2 in the extracted DNA was quantified using real-time PCR. Before quantification of the PCV2 genome in the collected samples, a PCV2 real-time PCR standard was established. Briefly, the PCV2 genome was cloned in the pmD®18-T Vector (TaKaRa) after PCR amplification with primers – forward 5′-CCGCGGGCTGGCTGAACTTTTGAAAG-3′ and reverse 5′-CCGCGGAAATTTCTGACAAACGTTAC-3′ (GenBank accession no. EU095020) – and transformed in TOP10 competent cells (Invitrogen). The plasmid was prepared using a PureLinkTM HiPure Plasmid Midiprep Kit (Invitrogen). The PCV2 plasmid was mixed with mouse DNA extracted from a PCV2 PCR-negative blood sample. Dilutions (10-fold) of this mixture (from 1011 to 102 PCV2 copy numbers/μl) were used as a standard for PCV2 quantification. The PCR was performed using a SYBR Green detection kit (Takara), containing MgCl2, deoxyribonucleoside triphosphate (dNTP) and HotStar Taq polymerase. Then, 1 μl of the template solution was added to a total volume of 10 μl containing 5 μl SYBR Green mix (Takara) and 0·2 μl each of the forward and reverse primers (10 μm). The PCR was performed under the following conditions: (1) pre-denaturation (30 s at 95°C); (2) amplification and quantification, repeated forty cycles (5 s at 95°C, 34 s at 60°C); (3) melting (60–99°C at a heating rate of 0·1°C/s)(Reference Wang, Sh and Zhang31).
Total superoxide dismutase activity detection
Total superoxide dismutase (SOD) activity in serum was determined using spectrophotometric kits at 550 nm (Nanjing Jiancheng Biotechnology Institute) according to the manufacturer's instructions. The results were expressed as units/ml serum.
Statistical analysis
All statistical analyses were performed using SPSS 16.0 software (SPSS, Inc.). Group comparisons were performed using Student's t test, and data from different time points within the glutamine group were performed using the Student–Newman–Keuls method. Data of the detection rate in each tissue were analysed by Fisher's exact test. Differences were considered significant at P< 0·05. Data are expressed as means with their standard errors of the mean(Reference Yin, Huang and Zhong32).
Results
In the present study, a series of cytokines related to the pathogenesis of the PMWS were measured to evaluate the immune-enhancing effects of glutamine supplementation. Meanwhile, the total SOD activity in serum was also analysed to profile the redox status in the body. The results showed that dietary l-glutamine supplementation significantly increased serum IL-2 levels on the 3rd (P< 0·01), 5th (P< 0·01), 7th (P< 0·05) and 9th dpi (P< 0·05), while IL-2 levels on the 7th dpi were much higher (P< 0·05) than those on the 5th and 11th dpi in the glutamine group (Fig. 1). Serum IL-6 levels in the glutamine group were also significantly (P< 0·05) higher than those in the control group on the 3rd dpi, while IL-6 levels on the 3rd dpi were much higher (P< 0·01) than those on the 5th, 7th, 9th and 11th dpi in the glutamine group (Fig. 2). Furthermore, dietary glutamine supplementation significantly increased (P< 0·05) serum IFN-γ levels on the 9th and 11th dpi, while IFN-γ levels on the 9th and 11th dpi were much higher than those on the 3rd, 5th and 7th dpi in the glutamine group (Fig. 5). Serum IL-10 levels in the alanine group were much higher than those in the glutamine group on the 9th and 11th dpi. IL-10 levels on the 3rd dpi were significantly higher (P< 0·05) than those on the 7th, 9th and 11th dpi in the gluatamine group (Fig. 3). No significant difference was observed for serum IFN-α levels (Fig. 4), CRP levels (Fig. 6) and total-SOD activity (Fig. 7) between the glutamine and alanine groups, while, in the glutamine group, IFN-α levels significantly (P< 0·05) decreased on the 5th dpi when compared with those on the 3rd, 9th and 11th dpi (Fig. 4). Meanwhile, CRP levels significantly (P< 0·01) increased on the 9th and 11th dpi compared with those on the 3rd and 5th dpi (Fig. 6).
Serum IL-2 levels in the glutamine (
) and alanine (
) groups. Mice in the glutamine group were fed with the 1·0 % glutamine+basal diet, while mice in the alanine group were fed with the 1·22 % alanine+basal diet. dpi, Days post-infection. * P< 0·05; ** P< 0·01. A colour version of this figure can be found online at journals.cambridge.org/bjn
Serum IL-6 levels in the glutamine () and alanine () groups. Mice in the glutamine group were fed with the 1·0 % glutamine+basal diet, while mice in the alanine group were fed with the 1·22 % alanine+basal diet. dpi, Days post-infection. * P< 0·05. A colour version of this figure can be found online at journals.cambridge.org/bjn
Serum IL-10 levels in the glutamine () and alanine () groups. Mice in the glutamine group were fed with the 1·0 % glutamine+basal diet, while mice in the alanine group were fed with the 1·22 % alanine+basal diet. dpi, Days post-infection. * P< 0·01. A colour version of this figure can be found online at journals.cambridge.org/bjn
Serum interferon (IFN)-α levels in the glutamine () and alanine () groups. Mice in the glutamine group were fed with the 1·0 % glutamine+basal diet, while mice in the alanine group were fed with the 1·22 % alanine+basal diet. dpi, Days post-infection. A colour version of this figure can be found online at journals.cambridge.org/bjn
Serum interferon (IFN)-γ levels in the glutamine () and alanine () groups. Mice in the glutamine group were fed with the 1·0 % glutamine+basal diet, while mice in the alanine group were fed with the 1·22 % alanine+basal diet. dpi, Days post-infection. * P< 0·05. A colour version of this figure can be found online at journals.cambridge.org/bjn
Serum C-reactive protein (CRP) levels in the glutamine () and alanine () groups. Mice in the glutamine group were fed with the 1·0 % glutamine+basal diet, while mice in the alanine group were fed with the 1·22 % alanine+basal diet. dpi, Days post-infection. A colour version of this figure can be found online at journals.cambridge.org/bjn
Serum total-superoxide dismutase (SOD) activity in the glutamine () and alanine () groups. Mice in the glutamine group were fed with the 1·0 % glutamine+basal diet, while mice in the alanine group were fed with the 1·22 % alanine+basal diet. dpi, Days post-infection. A colour version of this figure can be found online at journals.cambridge.org/bjn
In the present study, to explore the clearance effects of glutamine against PCV2 in the mouse model, PCR and quantitative PCR were used to detect the PCV2 virus load in liver, spleen, heart, lung, kidney and ovary tissue, and serum on the 3rd, 5th, 7th, 9th and 11th dpi. For this purpose, first, PCV2 real-time PCR standard and the DNA template from each sample were prepared. The PCR was then used to screen the template, which showed that most of the samples were negative. Furthermore, using quantitative real-time PCR, we detected the PCV2 virus genome sporadically in liver, spleen, heart, lung, kidney and ovary tissue, and serum on the 3rd, 5th, 7th, 9th and 11th dpi (Table 1 shows the data from the 5th, 7th, 9th and 11th dpi).
Porcine circovirus type 2 (PCV2) virus load in the liver, spleen, heart, lung, kidney and ovary tissue, and serum of control and glutamine-supplemented mice (Mean values with their standard errors for PCV2 log10 genomic copies/g sample or ml serum)
dpi, Days post-infection; 1·0 % Gln, 1·0 % glutamine+gestation diet; 1·22 % Ala, 1·22 % alanine+gestation diet.
Discussion
Glutamine is generally considered as a non-essential amino acid, which is synthesised mainly in the muscle from non-essential amino acids and glucose. However, in a situation of abnormal muscle protein metabolism, the production of endogenous glutamine is probably impaired and becomes a conditionally essential amino acid in disease conditions such as major trauma, major surgery, sepsis, bone marrow transplantation, intense chemotherapy and radiotherapy(Reference Tapiero, Mathe and Couvreur12). So we hypothesised that glutamine content in the feed is not enough for the pig suffering from a viral or bacterial infection similar to arginine(Reference Ren, Yin and Liu33, Reference Tan, Li and Kong34), and dietary l-glutamine supplementation performs various beneficial effects on a PCV2-infected mouse model.
The mechanisms of immunosuppression in PCV2 infection are described by lymphocyte depletion, interfering with antigen presentation and the induction of apoptosis in the immune system for its main target cells that are considered to be monocyte/macrophage lineage cells and other antigen-presenting cells(Reference Kennedy, Moffett and McNeilly35). Meanwhile, recent studies have suggested that lymphocyte-like cells may be important cell populations that support early PCV2 replication, whereas monocytes may be the site for PCV2 persistence in the infected host(Reference Yu, Opriessnig and Kitikoon36). So PCV2 infection could down-regulate IL-2 expression, but up-regulate other pro-inflammatory mediators such IL-8, IL-1 and IL-6(Reference Zoja, Wang and Bettoni37). Other compelling research has indicated that PCV2 infection could impair natural IFN-producing cell activity(Reference Vincent, Carrasco and Guzylack-Piriou38, Reference Vincent, Balmelli and Meehan39). Thus, PCV2 infection may decrease IFN-α levels. The number of IL-10-producing cells is higher in all PMWS animals compared with control pigs not infected with PCV2(Reference Crisci, Ballester and Dominguez40). This could explain why IL-10 is increased in PCV2 infection.
IL-2 is secreted by activated T lymphocytes, especially by activated CD4+ T-helper cells and CD8+ T-helper cells(Reference Keene and Forman41), and named T-cell growth factor as it stimulates T cell proliferation and differentiation(Reference Morgan, Ruscetti and Gallo42, Reference Smith43). IL-2 could promote the activity of immune cells to kill cancer cells, abnormal cells infected by virus and bacteria(Reference Flexner, Hugin and Moss44, Reference Allen, Weir and Martin45). In the present study, dietary l-glutamine supplementation significantly increased serum IL-2 levels on the 3rd, 5th, 7th and 11th dpi. IL-6 plays a very complex role in biological events, including immune responses, haematopoiesis, and regulation of the endocrine and nervous systems(Reference Biffl, Moore and Moore46, Reference Naugler and Karin47). Here, we found that serum IL-6 levels in the glutamine group was significantly higher than those in the control group on the 3rd dpi. The prime function of IL-10 is to inhibit many functions of natural killer cells, T cells, and macrophage and dendritic cells, and to reduce the production of inflammatory cytokines(Reference Trinchieri48, Reference O'Garra, Barrat and Castro49). There is an indication of a transient correlation between IL-10 levels and the viral load of PCV2 in pigs(Reference Darwich, Segales and Resendes50). In the present study, serum IL-10 levels were significantly decreased (P< 0·01) in the glutamine group on the 9th and 11th dpi. IFN-γ is a multifunctional protein first observed as an anti-viral activity in cultures of Sindbis virus-infected human leucocytes stimulated by phytohaemagglutinin (PHA). Later work indicated that it induces anti-viral, anti-proliferative and immunomodulatory effects on target cells(Reference Farrar and Schreiber51). A significantly higher serum IFN-γ level was observed in the glutamine group on the 9th and 11th dpi. IFN-α is a member of the type I IFN family, which is active as an anti-viral and immunomodulatory cytokine. CRP is a product of hepatocytes as a non-specific response to tissue damage. However, no significant difference in serum IFN-α and CRP levels was found between the glutamine and control groups. SOD is an important antioxidant enzyme that could increase the clearance of the superoxide radical. It has already been reported that administration of SOD is effective in decreasing tissue inflammation and injury in experimental models of ischaemia and reperfusion, chronic gut inflammation, arthritis and immune complex-induced pulmonary disease(Reference Conner and Grisham52). Evidence indicates that glutamine supplementation increases antioxidant defence ability as glutamine up-regulates the synthesis of glutathione and antioxidative gene expression(Reference Lora, Alonso and Segura53, Reference Wu, Fang and Yang54). Unfortunately, no significant difference was observed for total-SOD activity between the glutamine and alanine groups.
Collectively, dietary 1·0 % l-glutamine supplementation had a beneficial effect on the cytokine profile in the PCV2-infected mouse model. Cytokines are a large family of proteins and important players in innate and adaptive immune systems, which are produced by leucocytes and other cells. Glutamine displays a number of pharmacological actions and is a fuel for all rapidly differentiated and/or activated cells, including phagocytes, lymphocytes and other immune-related cells(Reference Gras, Porcheray and Samah55, Reference Spittler, Winkler and Gotzinger56). Thus, it is not unexpected that serum cytokine levels would not be ameliorated after glutamine supplementation. In fact, another interesting experiment showed that maximal IL-1 and TNF-α production by cultured murine macrophages, and maximal production of IL-6 and IL-8 by cultured human monocytes require a sufficient supply of glutamine(Reference Wells, Kew and Yaqoob57). However, the other mechanisms by which glutamine regulates the cytokine profile need further study.
In the present study, to explore the clearance effects of glutamine against PCV2 in a mouse model, PCR and quantitative PCR were applied to detect the PCV2 virus load in liver, spleen, heart, lung, kidney and ovary tissue, and serum on the 3rd, 5th, 7th, 9th and 11th dpi. Unfortunately, the PCV2 virus genome was detected sporadically. In fact, this irregularity also existed in other experiments using PCV2 experimentally infected mice(Reference Ren, Luo and Wu58). Although there has been a promising progress in PCV2 research, little information is known about PCV2 performance in species other than swine. There are only a few studies about PCV2 performance in mice, but their results are mixed. Kiupel et al. (Reference Kiupel, Stevenson and Choi59, Reference Kiupel, Stevenson and Galbreath60) showed that PCV2 replicates in 8-week-old BALB/c mice inoculated with PCV2, and PCV2 is detected by in situ hybridisation and PCR in mice on 7, 14, 28 and 42 dpi. In agreement with Kiupel, Li et al. (Reference Li, Yuan and Zhang61) also found that PCV2 replication, seroconversion and microscopic lesions are found in inoculated Kunming mice, and that Kunming mice could be infected by the PCV2 virus and used as a PCV2-infected experimental model. However, Quintana et al. (Reference Quintana, Balasch and Segales62) indicated that porcine circoviruses do not cause any disease or microscopic lesions in inoculated mice during the experimental period, and intraperitoneally inoculated mice might have harboured PCV2 in the circulation without the evidence of viral replication. Meanwhile, Opriessnig et al. (Reference Opriessnig, Patterson and Jones63) also reported that the PCV2 DNA is detected by PCR in 93 % (100/108) of tissues and 42·6 % (46 out of 108) of serum samples from PCV2-inoculated mice from days 12 to 37 and the mouse model probably has limited utility to advance the understanding of the pathogenesis of PCV2-associated lesions, but mice could be potentially important in the epidemiology of PCV2. The main reason is that the virus stock (i.e. passage, origin and dose) used in these studies differed.
The speculative reasons that the PCV2 virus genome was detected sporadically are as follows. (1) Although the virus stock is different, the dose that we used was not enough. Li et al. (Reference Li, Yuan and Zhang61) inoculated mice orally and intramuscularly with 0·1 ml PCV2 (106·2 TCID50/ml). (2) The time limited our observation. Bolin et al. (Reference Bolin, Stoffregen and Nayar64) reported that PCV2 DNA could be detected by PCR from brain, lymphoid tissue, bone marrow, kidney, ileum, liver, heart, lung, spleen, thymus, tonsil and pancreas from 20 to 28 dpi in caesarean-derived, colostrum-deprived pigs inoculated with PCV2 intranasally and subcutaneously. In fact, we found that PCV2 DNA could be detected in serum from 14 dpi in our other studies when we incubated mice with 100 TCID50. (3) Amino acid supplementation in the present study could be sufficient to clear the virus. Questions about this phenomenon also existing in the control group of the present study will be generated. In the present study, alanine was chosen as a N control in such a model; however, research has found that alanine also had some effect on immune function(Reference Lewis and Langkamp-Henken65). Meanwhile, alanine also plays a role in the inhibition of pyruvate kinase and hepatic autophagy, gluconeogenesis, transamination and the glucose–alanine cycle(Reference Wu66, Reference Kudsk67).
In conclusion, this is the first report that dietary l-glutamine supplementation enhances immune function in PCV2-infected mice, and may clear PCV2 in experimentally infected mice; however, its effect on swine and its mechanism need further investigation.
Acknowledgements
The present study was jointly supported by the National Basic Research Project (2012CB124704 and 2013CB127301), the NSFC (31110103909, 30901041, 31001016 and 31101729), the State Key Laboratory of Food Science and Technology, the Nanchang University project (SKLF-TS-201108, SKLF-KF-201005 and SKLF-KF-201216) and Hunan Provincial Science and Technology Department (2011NK2011 and 2012NK4048). Y. Y. was in charge of the whole trial. W. R. conducted the experiment and wrote the paper. Y. L., G. L., X. Y. and W. L. helped W. R. with the analysis of the data. H. S. helped Y. Y. with the design and review of the whole experimental design. The authors declare that they have no conflicts of interest.
