The concentrations of haptoglobin (Hp), C–reactive protein (CRP) and serum amyloid A (SAA) were measured in wasted pigs, first to evaluate their usefulness in the diagnosis of infectious, wasting diseases in pigs, and second, to evaluate whether their concentrations can distinguish the lymphoid depletion score in the lymph tissues of wasted affected pigs. Fifty–three wasted pigs and seven specific pathogen free (SPF) pigs were postmortem examined. Gross lesions were evaluated and samples for histopathological, immunohistochemical, molecular biology and microbiological analysis were taken. Thirty–one pigs were diagnosed as postweaning multisystemic wasting syndrome (PMWS) and 22 as porcine respiratory disease complex (PRDC). Lymphoid depletion degree in lymph tissues of PMWS and PRDC affected pigs was determined. Serum Hp was significantly higher in pigs with PRDC in comparison with the PMWS affected pigs. Serum CRP concentration was significantly lower in pigs with PRDC than in PMWS affected pigs (P<0.001). CRP and SAA levels increased with the lymphoid depletion score, presenting statistical differences between pigs with no depletion and pigs with low, moderate or severe lymphoid depletion (P<0.05, P<0.05 and P<0.001 for CRP and P<0.01, P<0.01 and P<0.01 for SAA, respectively). Hp was higher in pigs with no or low depletion compared with the pigs suffering severe lymphoid depletion (P<0.001 and P<0.05, respectively).
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ACUTE phase proteins (APPs) are a group of plasmatic proteins whose concentration changes after infection, inflammation or surgical trauma, factors that trigger the acute phase response (APR). Serum concentration of positive APPs, such as haptoglobin (Hp), C–reactive protein (CRP) and serum amyloid A (SAA) increases during APR (Murata and others 2004). APPs have been used to differentiate between viral and bacterial infections in human cases of pneumonia (Virkki and others 2002), and are good markers for evaluating animal health, detecting clinical and subclinical infections (Petersen and others 2002), discriminating between acute and chronic inflammation (Horadagoda and others 1999), evaluating the effect of the treatment against diverse diseases (Lauritzen and others 2003), evaluating animal welfare (Piñeiro and others 2007) as well as assuring hygiene and meat quality (Piñeiro and others 2007, Pallarés and others 2008).
Many conditions and diseases can cause wasting in pigs, which are characterised by thinness, paleness, rough hair, anorexia and decreased growth. To implement appropriate treatment, it is essential to identify the aetiology of the wasting disease. Possible etiologies of wasting are related to the pathogenic action of porcine circovirus type 2 (PCV2), porcine respiratory disease complex (PRDC), chronic digestive diseases, severe parasitism, chronic poisoning, water deprivation or nutritional deficiency (Sorden 2000, Brockmeier and others 2002, Straw and others 2002). Diseases caused by toxins, nutritional deficiencies or parasites are rare in modern swine production farms and diseases associated with enteric pathogens are often accompanied by diarrhoea.
The purpose of this study was to determine the concentrations of APPs in the serum of wasted pigs to evaluate their usefulness in the diagnosis of pig infectious wasting diseases and their relationship with the lymphoid depletion score in the lymph tissues of affected pigs.
Material and methods
The University of Murcia Animal Care Committee reviewed and approved the experimental protocols. All the facilities adhered to the Spanish and European Union Laws regarding the welfare and protection of pigs. The study population was 53 Duroc × (Large White × Landrace) pigs with wasting disease between 18 and 24 weeks of age from three different finishing units of the same pig production company, which shared the origin farm, nutrition, building structure and geographical area. These farms had previous health problems in which postweaning multisystemic wasting syndrome (PMWS) and PRDC cases were diagnosed. All the pigs were reared under the same controlled conditions. Wasted pigs were selected at random, based on the clinical signs which included thinness, paleness, rough hair, anorexia and decreased growth. Seven specific pathogen free (SPF) pigs at the same age from a different SPF farm were used as healthy negative controls. The SPF pigs were negative for porcine reproductive and respiratory syndrome virus (PRRSv), Aujezsky's disease virus, M hyopneumoniae, Actinobacillus pleuropneumoniae, atrophic rinitis and mange.
In order to diagnose the cause of the wasting disease, complete postmortem examinations were carried out and samples (lung, heart, kidney, ileum, spleen, tonsil, brain, liver and mediastinic, traqueobronquial, inguinal and mesenteric lymph nodes) were taken for histopathological, immunohistochemical, molecular biology and microbiological analysis.
For the histopathological and immunohistochemical studies, formalin–fixed samples were embedded in paraffin wax and sectioned at 4 μm. Sections were stained with hematoxylin and eosin for the histopathological study. PCV2 and PRRSv immunohistochemical detection was performed. The immunolabelling technique of PRRSv antigen was carried out in tissue sections using a mixture at 50 per cent with the monoclonal antibodies SDOW17 and SR30 (Rural Technologies). For PCV2 antigen immunolabelling, the monoclonal AntiPCV36A9 (Ingenasa) was used. Avidin–Biotin–Peroxidase complex (Vector laboratories) technique was used for the immunohistochemical technique (Hsu and others 1981). All the slides were blindly evaluated by at least two different pathologists.
PCV2 and PRRSv genomes were detected by PCR, as previously published (Donadeu and others 1999, Harms and others 2001). Bacteriological identification was performed by culture following standard operating procedures outlined in the Manual of Clinical Microbiology (Baron and others 1999).
Lymphoid depletion score
Lymphoid depletion was scored in all pigs. The lymphoid depletion in lymph tissues was categorised in grades from 0 to 3. Grade 0 was used for no depletion, grade 1 for low depletion in more than two different lymphoid tissues, grade 2 for moderate lymphoid depletion in more than two lymphoid tissues and grade 3 for severe lymphoid depletion in more than two different tissues. Lymph node enlargement and gross lung lesions were evaluated macroscopically. The lymphoid depletion degree and histopathology features were blindly evaluated by at least two different pathologists.
Serum concentrations of APPs
Whole blood was obtained by venepuncture of the jugular vein in dry tubes. Serum levels of the APP Hp, CRP and SAA were quantified by the spectrophotometric methods using commercial ELISA kits (Tridelta Development). CRP results were expressed in µg/ml, Hp in mg/ml and SAA in µg/ml. All the methods that were used to determine the serum concentrations of Hp, CRP and SAA had been previously validated. All the samples were analysed in the same session to avoid inter assay imprecision (Tecles and others 2007).
A Kolmogorov–Smirnov test was used to assess if APPs followed a normal distribution. To evaluate the differences in the serum concentrations of APPs between PMWS, PRDC and SPF pigs, between the types of lesions, or between the lymphoid depletion scores in wasted pigs, Mann–Whitney's non–parametric U–test was performed. Statistical significance was considered at P<0.05. All analyses were completed using SPSS 15.0.
After diagnostic procedures, among the wasted pigs, 31 pigs were diagnosed as PMWS and 22 as PRDC. PCV2, PRRSv and bacterial diagnosis results for PMWS and PRDC affected pigs are represented in Table 1.
The criteria considered for the diagnosis of PMWS and PRDC were those previously described. PMWS requires a pig to exhibit clinical signs (wasting/weight loss/illthrift/failure to thrive, with or without dyspnea or icterus), histologic lesions (depletion of lymphoid organs/tissues and/or lymphohistiocytic to granulomatous inflammation in more than one lymphoid tissue, or in one lymphoid tissue and at least one other organ system or in two organ systems) and PCV2 infection within characteristic lesions (Sorden 2000, Opriessnig and others 2007). PRDC diagnosis requires a pig to exhibit clinical disease with a failure to gain weight, pneumonia of multiple aetiology, including different viruses (commonly PCV2 or PRRSv) and bacteria (Brockmeier and others 2002).
All the animals diagnosed as PMWS or PRDC had lung lesions including cranioventral suppurative bronchopneumonia (30/53; 57 per cent), chronic pleurisy (five of 53; 9 per cent) embolic pneumonia (one of 53; 2 per cent) and intertitial pneumonia (one of 53; 2 per cent). The percentage of Lung surface affected in the PMWS group varied from 1– to 90 per cent, with an average of 39 per cent and that in the PRDC group varied from 5– to 100 per cent with an average of 31 per cent. No statistical differences were found between the lung lesions in the PMWS and PRDC animals. No lesions were detected in SPF pigs.
Lymphoid depletion score
The lymphoid depletion analysis in PMWS affected pigs showed that 35 per cent (11/31) of them had low lymphoid depletion, 26 per cent (eight of 31) had moderate depletion and 39 per cent (12/31) of them had severe lymphoid depletion. 59 per cent (13/22) of PRDC affected pigs had no depletion, 27 per cent (six of 22) had low depletion, 10 per cent (two of 22) had moderate depletion and 4 per cent (one of 22) had high lymphoid depletion. All the SPF pigs had no lymphoid depletion (Table 2).
Serum concentrations of APPs
The statistical analysis showed that APPs did not follow a normal distribution. Serum concentrations of Hp, CRP and SAA in all groups are shown in Fig 1. Serum Hp was significantly higher in pigs with PMWS or PRDC than in the SPF pigs (P<0.001 and P<0.001, respectively). The PRDC affected pigs had higher serum Hp levels than the PMWS affected pigs (P<0.001) (Mean PMWS: 4.92; mean PRDC: 232.32; mean SPF: 0.83).
Serum CRP was significantly higher in pigs with PMWS than in the SPF pigs (P<0.03). No significant differences were detected for CRP between the SPF and PRDC groups. PRDC affected pigs had lower serum CRP levels than PMWS affected pigs (P<0.001) (Mean PMWS: 89.13; mean PRDC: 54.61; mean SPF: 31.09).
Serum SAA was significantly higher in pigs with PMWS than in the SPF pigs (P<0.001). There were no significant differences of SAA between SPF and PRDC groups (mean PMWS: 89.13; mean PRDC: 152.45; mean SPF: 25.78).
Hp, CRP and SAA concentrations depending on the lymphocyte depletion score are represented in Table 3. CRP and SAA levels presented statistical differences between pigs with no depletion and pigs with low, moderate or high depletion (P<0.05, P<0.05 and P<0.001 for CRP and P<0.01, P<0.01 and P<0.01 for SAA, respectively). Hp levels presented statistical differences between pigs with no depletion and pigs with severe lymphoid depletion (P<0.001), and between pigs with low and severe depletion (P<0.05).
In the present study, a marked APR in pigs with wasting disease could be shown. Hp, CRP and SAA are useful markers of clinical disease in pigs (Pallarés and others 2008), and they can be used to monitor the evolution of experimental infections with PCV2 (Stevenson and others 2006), in field cases of A pleuropneumoniae, PCV2, Aujeszky's disease virus and M hyopneumoniae (Petersen and others 2002) in pigs with diarrhoea, arthritis or cannibalism lesions (Petersen and others 2002, Piñeiro and others 2007). All the pigs used in the present study were reared under the same controlled conditions and the diagnostic assays performed did not reveal any other injuries apart from those inherent to PMWS and PRDC. The results obtained are consistent with the previous studies that demonstrated APPs levels to be significantly higher in animals diagnosed as PMWS compared with healthy animals (Segalés and others 2005). The absence of statistically significant differences for CRP and SAA between SPF animals and PRDC may be explained by the chronicity of the disease in the studied animals coupled with the fact that these APPs increase more rapidly and return faster to their normal values (Eckersall 2000). Most of the pathogens causing PRDC induce an APR, but after day 15 postinfection, the concentrations of CRP and SAA decrease to normal values (Heegaard and others 2011), which could explain why in the present study there were no significant differences for CRP and SAA between SPF animals and PRDC pigs.
In a previous publication, it was recommended that at least two APPs (a fast one such as CRP or SAA and a slow one such as Hp) should be used to differentiate between pathologic states or temporal evolution of the disease (Ceron and others 2005). In this study, the authors selected SAA, CRP and Hp because they have shown their usefulness in swine (Pallarés and others 2008). According to the results, low Hp and high CRP serum concentrations should increase the suspicion that wasted pigs are suffering PMWS (P<0.001 and P<0.001, respectively).
CRP was found to be a useful indicator of lymphoid depletion, differentiating animals with no depletion and pigs with low, moderate or severe depletion (P<0.05, P<0.05 and P<0.001, respectively). The higher levels of CRP in the PMWS affected pigs than in the PRDC affected pigs could be explained because in addition to liver production, CRP has been reported to be expressed by macrophages (Yasojima and others 2001), and inflammatory infiltration by histiocytes in any organ (typically lung and/or lymphoid tissue and less often kidney, liver, intestine and pancreas tissue) with lymphocyte depletion of follicular and interfollicular areas as a constant and distinctive lesion of PMWS affected pigs (Segalés and others 2004). The results obtained show that with increasing severity of lymphoid depletion, the concentration of CRP increases while that of Hp decreases. It has been reported that elevated levels of CRP diminish lymphocyte–mediated immune responses proportional to its concentration (Mortensen and others 1977). In this way, elevated levels of CRP may modulate cell–mediated immune responses and may contribute to the immune impairment caused by PCV2.
SAA differentiates between pigs with no lymphoid depletion and pigs with low, moderate or severe lymphoid depletion (P<0.01, P<0.01 and P<0.01, respectively). SAA concentrations were higher in low or moderate lymphoid depleted pigs than in non–lymphoid depleted pigs, which could be explained because SAA can increase monocyte and macrophage cytokine production contributing to the creation of the pro–inflammatory state associated with lymphoid depletion in PCV2 infected pigs (Song and others 2009).
Two factors could explain the decrease in Hp in PMWS affected pigs. First, Hp captures hemoglobin in order to prevent both iron loss and hemoglobin–mediated renal parenchymal injury during hemolysis. Decreased serum Hp concentrations have been reported in cases of iron deficiency and hemolysis (Ravel and others 1995). PMWS affected pigs have pallor and microcytic hypochromic anemia with a decrease in serum iron (Darwich and others 2004), which could explain why Hp serum concentration in pigs with severe lymphoid depletion caused by PMWS was lower than in pigs with no or low depletion. Second, APPs synthesis in the APR is triggered by tumor necrosis factor(TNF)–α, interleukin (IL)–1 and IL–6, demonstrating that T CD4+ lymphocytes can produce IL–6 and TNF–α (Kharkevitch and others 1994). PMWS affected pigs have a diminished number of T cells in the follicular areas, mainly affecting the CD4+ cells (Darwich and others 2004). It is known that human immunodeficiency virus (HIV) also leads to CD4+ T cell depletion, and it has been reported that that many HIV infected patients show a reduction in the serum Hp (Yamamoto and others 2003). The reduction of Hp concentrations in these two lymphoid depleting diseases could suggest that, the greater the severity of lymphoid depletion, the lower the levels of IL–6 and TNF–α, producing a reduction in Hp synthesis as a final result.
PMWS and PRDC were the main diseases diagnosed in the present study. Postmortem examinations, histopathological, immunohistochemical, molecular biology and microbiological techniques ruled out other wasting diseases, as for example, proliferative enteropathy or gastric ulcers. No difference in serum APPs concentration was found depending on the types of lung lesions, which is in line with the results of Pallarés and others (2008). The wasted pigs involved in this study and diagnosed as PMWS or PRDC cases had similar clinical signs, all of them presented similar lung lesions and lymph nodes enlargement. Differences between the diagnosis of PMWS and PRDC were based on the histological lesions associated with the virus, that the former had in more than one organ, while the latter was limited to lung injury (Sorden 2000, Opriessnig and others 2007). The clinical similarities between PMWS and PRDC indicate that, in field conditions, objective criteria such as APPs concentration could provide information for veterinary practitioners to help them to differentiate between both diseases in order to apply opportune managing and therapeutic actions. There are other diseases than those found in the present study, that may cause wasting in pigs. This fact indicates that more research will be required to implement the clinical application of APPs in these other cases.
To the authors knowledge, these are the first data in the literature about serum APPs' concentrations and its use for differential diagnosis of wasting disease in pigs. From the results obtained in this study, it can be concluded that a blood sample would provide information that could be useful for the differential diagnosis of PMWS and chronic PRDC, based on the Hp and CRP serum values. CRP and SAA could be used as a complementary tool to monitor the existence of lymphoid depletion in experimental viral infections, and Hp could provide information about the severity of this depletion. Further research is needed to clarify the role of APPs in lymphoid depleting diseases.
The authors thank A. Sánchez de la Vega for his technical assistance. Juan J. Quereda was supported by a doctoral grant from the Spanish Ministry of Education and Science (AP–2005–3468). This work was funded by Fundación Séneca, Región de Murcia, Spain (AGR/14/FS/02 and 03036/PI/05).
Provenance not commissioned; externally peer reviewed