Bovine and canine acute phase proteins

Acute phase proteins are serum proteins which increase in concentration during the acute phase response to inflammation or infection. The response occurs in all animals, but in different species the response of individual proteins can be significantly different. Of the numerous acute phase proteins which have been identified in humans, a number have been examined in cattle and dogs but usually on an individual basis with little reference to their part in the acute phase response. Biochemical, physiological and clinical investigations into haptoglobin, fibrinogen, ₁-proteinase inhibitor, ceruloplasmin, seromucoid and C-reactive protein of cattle and dogs have therefore been reviewed with the emphasis on their role in this response to tissue damage.     

International standardization of acute phase proteins

Acute phase proteins (APPs) such as haptoglobin, serum amyloid A and pig major acute phase protein are plasma proteins that increase in concentration following infection, inflammation, or trauma. The circulating concentrations of these proteins in pigs and cattle can provide an objective measure of the health status of an animal and are increasingly being used as markers of animal health and welfare. Plasma concentrations of APPs are related to the severity of the underlying condition, and provide a ready means of evaluating both the presence and extent of disease. Haptoglobin, for example, has been used to identify both clinical and subclinical disease in animals, and for objectively monitoring antibiotic therapy in experimentally infected animals. Interpretative benefit can be further enhanced by the "acute phase index", derived from a mathematical formula that uses both positive- and negative-reacting APPs. Research suggests that in the future, assays for APPs will be used routinely to assess animal health, optimize production rates, monitor antibiotic therapy, detect diseases such as mastitis in dairy cows, and assess the health of animals at slaughter. These applications have considerable benefit for human food safety. Before APP assays can be applied in animal production on a worldwide basis, however, the calibration of assay methods must be harmonized to ensure that results obtained in the laboratory or on the farm are universally comparable and of consistent quality. In February 2000 the European Commission Directorate General Research Concerted Action was established to fulfill the task of international standardization of APPs. The Concerted Action Group consists of a network of 14 institutions representing 9 European countries. In this report, the background and goals of the Concerted Action Group, and scientific presentations from the group's first colloquium are described. In addition, the progress of the group to date and the standardization plan for the full 30-month duration of the Concerted Action are summarized.     

Extracellular proteins of Clostridium chauvoei are protective in a mouse model

The anaerobic bacillus Clostridium chauvoei is the causative agent of blackleg, a lethal disease that has an important impact on the sheep and cattle industry worldwide. Immunity to C. chauvoei is considered to be mainly anticellular, and for this reason there is scarce information about the immunogenicity of extracellular proteins. In this work variations in protein profiles, immune response by ELISA and protective capacity of culture supernatants of three C. chauvoei strains, collected at different growth phases, are reported. Sera raised against extracellular antigens also recognised cellular antigens of the same molecular masses. Partially purified cell-free supernatants and those concentrated 10 times by ultrafiltration (C-CFS), obtained at the early stationary phase of growth, induced a strong immunoprotective response, even at low doses, that was more marked for C. chauvoei strain ATCC 10092 (p < or = 0.05). With C-CFS formulations, a clear relationship was observed between IgG titres, protective capacity and concentration of the antigen doses, indicating a specific immune response.     

Serum acute phase proteins and swine health status

The purpose of this study was to investigate the relationship between swine health status and the concentration of the serum acute phase proteins, haptoglobin (HP), and C-reactive protein (CRP). A total of 378 clinically healthy pigs from farms A and B, plus 20 pigs culled from farm A due to poor growth, were used in this experiment. Each pig was examined and blood samples were collected during slaughter. The HP concentration was measured by using an HP-hemoglobin binding assay. The CRP concentration was measured by using a CRP enzyme immunoassay. Gross and histopathological lesions were examined and recorded at slaughter. Representative samples were then collected in order to isolate pathogens. Swine enzootic pneumonia, found in 47.7% of the pigs, was the most common lesion. Other lesions included pleuropneumonia (32.7%), suppurative pneumonia (10.3%), fibrinous pericardititis (4.3%), Ascaris migration in the liver (33.9%), and intestinal serositis (3.0%).     

Cytokines and acute phase proteins associated with acute swine influenza infection in pigs

This study set out to investigate the cytokines and acute phase proteins (APPs) associated with the acute stages of experimentally-induced swine influenza virus (SIV) infection in 3-week-old, colostrum-deprived, caesarean-derived piglets. The piglets were inoculated intratracheally with 10^7.5 50% egg infective dose [EID₅₀] Swine/Belgium/1/98 (H1N1) SIV and were euthanased at time-points between 0 and 120 h post-inoculation (PI). Broncho-alveolar lavage fluid (BALF), lung homogenates and sera were examined for inflammatory mediators by bioassay or ELISA. Interferon (IFN)-α, interleukin (IL)-6, IL-1 and tumour necrosis factor (TNF)-α peaked in BALF 24–30 h PI, when virus titres and the severity of clinical signs were maximal.Whereas IFN-γ and IL-12, but not IL-18, increased in tandem in BALF, serum cytokine concentrations were either undetectable or were up to 100-fold lower. The APP C-reactive protein (CRP) and haptoglobin peaked 24 h later than the cytokines and reached higher levels in serum than in BALF. In contrast, lipopolysaccharide (LPS)-binding protein (LBP) only increased in BALF. Lung virus titres tightly correlated with BALF IFN-α, IL-6, IL-1, TNF-α, IFN-γ and IL-12, as well as with serum IL-6, IFN-α and IFN-γ. Signs of disease correlated with the same cytokines in BALF and serum, as well as with BALF LBP and serum CRP. The findings suggest that IFN-γ and IL-12 play a role in the pathogenesis of SIV and that APPs are induced by cytokines. This influenza infection model may have value in assessing the therapeutic potential of cytokine antagonists.     

Zinc-associated acute pancreatitis in a dog

Zinc-induced haemolytic anaemia is a common phenomenon in dogs in the USA following the ingestion of pennies minted after 1982. A case of acute pancreatitis secondary to zinc toxicosis in a dog is described. Acute pancreatitis has been reported in humans, following the ingestion of liquid zinc chloride, but zinc-associated pancreatitis has not been reported previously in the dog. The mechanism of toxicity is unknown, although the pathophysiology may relate to the role of the pancreas in zinc excretion. Acute pancreatitis as a sequela to zinc toxicosis in the dog represents a complication that may prolong hospitalisation and worsen the prognosis.     

Alterations in bovine platelet function and acute phase proteins induced by Pasteurella haemolytica A1.

Platelet function was assessed by aggregometry in 10 Holstein calves before and after exposure to Pasteurella haemolytica (biotype A, serotype 1) by intrabronchial challenge. At 24 h after exposure the platelets had become more reactive to stimulation with known platelet agonists such as adenosine diphosphate (ADP) and platelet-activating factor (PAF) and the platelet aggregates that formed were more resistant to disaggregation. The activation of platelets was an early response in the challenged calves as platelet function had returned to pretreatment levels 72 h after exposure to the bacteria while the acute phase reactant proteins, haptoglobin and fibrinogen, were approaching their peak values and alpha 2-macroglobulin levels had also risen significantly (P < 0.05) at this time. The plasma levels of these proteins were still elevated and albumin levels were depressed 6 d post-treatment. At post-mortem all calves exhibited pneumonic tissue damage. When P. haemolytica leukotoxin was added directly to bovine platelet suspensions both spontaneous aggregation and an increase in the aggregation response to ADP and PAF stimulation were observed. The morphological appearance of the platelet aggregates exhibited the typical pattern for bovine platelets with 2 distinct zones of cells being visible within each aggregate. One zone contained platelets in which the cytoplasmic granules were still evident and the other zone contained irregularly shaped platelets devoid of granular content. In the latter zone, discrete gaps, or pores, were evident in the plasma membrane of numerous platelets. This pore formation is characteristic of leukotoxin action and is not observed in ADP or PAF induced aggregates.     

Critical differences of acute phase proteins in canine serum samples

The critical difference values for acute phase proteins in canine serum samples were established on a week-to-week basis. Blood samples from 11 apparently clinically healthy dogs were collected once weekly for five consecutive weeks. For each protein the total variance of analytical results was divided into intraindividual variance (S(Intra)(2)), interindividual variance (S(Inter)(2)), and analytical variance (S(Analytical)(2)). The critical difference (d(K)) was then calculated as d(k)=22(S(Intra)(2)+S(Analytical)(2)). The critical difference values were 1.95 g/L for haptoglobin, 4.85 mg/L for C-reactive protein, and 0.016 DeltaAbs/min for ceruloplasmin. When used in conjunction with the corresponding reference interval, critical difference values can be an aid in correctly interpreting acute phase protein results, by determining whether observed differences between two consecutive measurements in individual animals are due to natural variation or due to disease therapy or experimental procedures.     

Serum concentrations of acute phase proteins in dogs with leishmaniasis

The concentrations of haptoglobin, C-reactive protein and ceruloplasmin were measured in symptomatic and asymptomatic dogs naturally infected by Leishmania infantum, and in healthy uninfected dogs to determine the potential value of these proteins for the diagnosis and prognosis of leishmaniasis. The concentrations of the acute phase proteins were significantly higher in the dogs with leishmaniasis than in the control dogs, and the concentration of C-reactive protein was significantly higher in the symptomatic dogs than in the asymptomatic dogs. There were no correlations between the acute phase proteins and the gamma globulins, the albumin/globulin ratio or the titre of anti-leishmanial antibodies.     

蓝塘仔猪IGF-1水平与组织IGF-1、GHR基因的表达

32头不同日龄(出生、3、21、35d)蓝塘仔猪,由前腔静脉采血后剖杀,取肝脏、背最长肌样品。用RIA法测血液、组织中IGF 1浓度,用放射受体法(RBA)检测肝脏、肌肉组织中GHR结合活性,用实时荧光定量PCR法检测IGF 1、GHRmRNA的表达水平。结果表明:(1)血液中IGF 1在出生日显著高于其它时期(P<0 05)。肌肉组织中IGF 1含量高于肝脏组织,肌肉组织IGF 1含量在3、21、35日龄时都显著高于出生日(P<0 05)。(2)肝脏细胞膜GHR结合活性在出生日显著高于3、21日龄(P<0 05),肝脏细胞膜GHR结合活性高于肌肉组织。(3)肝脏组织IGF 1、GHRmRNA的表达量均显著高于肌肉组织(P<0 05)。肝脏IGF 1mRNA的表达在出生日、21日龄时显著高于3、35日龄(P<0 05),GHRmRNA的表达在出生日显著高于其它日龄(P<0 05)。肌肉IGF 1、GHRmRNA的表达在出生日均显著高于其它日龄(P<0 05)。     

Myeloperoxidase-positive acute megakaryoblastic leukemia in a dog

A 16-month-old female spayed Labrador Retriever was referred to the University of Edinburgh for exercise intolerance, inappetence, and severe anemia. A CBC showed severe nonregenerative anemia and moderate numbers of atypical cells with morphologic features most consistent with megakaryoblastic origin. Similar cells were identified in a bone marrow aspirate and accounted for 23% of all nucleated cells. Atypical promegakaryocytes and megakaryocytes were also noted. Myelodysplastic syndrome affecting the megakaryocytic lineage was suspected. Cytologic examination of a fine-needle aspirate of the spleen revealed rare megakaryoblasts similar to those in blood and bone marrow. At necropsy, the bone marrow consisted of atypical megakaryoblasts and megakaryocytes that were also infiltrating spleen, liver, lymph nodes, renal perihilar tissue, and visceral adipose tissue, consistent with acute megakaryoblastic leukemia. Immunohistochemical analysis of splenic sections confirmed megakaryoblastic origin (immunoreactive for CD61 and von Willebrand factor). Some leukemic cells were also immunoreactive for myeloperoxidase (MPO). This aberrant immunophenotype suggested both megakaryocytic and granulocytic/monocytic differentiation of the leukemic cells. To our knowledge, this is the first report of MPO-positive acute megakaryoblastic leukemia in a dog.     

Effects of hyperimmune equine plasma on clinical and cellular responses in a low-dose endotoxaemia model in horses

Endotoxaemia is a major cause of equine morbidity, and plasma from horses immunised against Escherichia coli is used in its treatment. The aim of this study was to determine the effects of hyperimmune plasma on the clinical and leukocyte responses, including production and activity of TNFα, in an in vivo endotoxin challenge model. Pre-treatment with hyperimmune equine plasma had no significant effect on peak total plasma TNFα concentration (occurring 90min after the administration of 30ng/kg LPS). However, the bioavailable (unbound) TNFα measured by bioassay was significantly reduced in plasma-treated horses (1044.44±193.93pg/ml at 90min) compared to saline treated controls (1373.92±107.63pg/ml; P=0.05). Therefore, although pre-treatment with hyperimmune equine plasma did not significantly modify the clinical signs of endotoxaemia in this model, there was some evidence of reduced TNF bioactivity, which may be due to factors in the plasma which bind and reduce the activity of this cytokine.     

Adiponectin and leptin are related to fat mass in horses

Plasma concentrations of adiponectin and leptin were measured in 23 mature Standardbred mares (age: 10 ± 3 years) and 12 weanling fillies (10 Quarter Horse/Belgian crossbreds and two Quarter Horses; aged: 4 ± 3 months) to test the hypothesis that adipocytokines are proportional to adiposity in horses. Rump fat thickness was measured using B-mode ultrasound and percent body fat (% fat) calculated using a published formula for the estimation of fatness in horses. Plasma adiponectin and leptin were determined using radioimmunoassay. In the absence of purified equine adiponectin or leptin, results were expressed as human equivalents (HE) of immunoreactive (ir) adipocytokines.Plasma ir-leptin HE concentration was positively correlated (r = 0.543; P < 0.001) with percent body fat and (r = 0.556; P < 0.001) to fat mass in all horses. The plasma ir-leptin HE concentration was lower (P = 0.03) in weanlings (1.90 ± 0.34 ng/mL HE) than in mature mares (3.47 ± 0.50 ng/mL HE). The ratio of ir-adiponectin HE to ir-leptin HE was negatively correlated (r = −0.621; P < 0.001) to percent fat and (r = −631; P < 0.001) to fat mass in all horses. The ratio of ir-adiponectin HE to ir-leptin HE was higher (P < 0.001) in weanlings (3.04 ± 0.51) than mature mares (1.03 ± 0.13). These data suggest that leptin is proportional while adiponectin is inversely proportional to adiposity in horses.     

Hepatic lipidosis: Liver characteristics and acute phase proteins in affected turkeys

The hepatic lipidosis (HL) in fattening turkeys is a disease has been known for a long time, but the cause and pathogenesis is still not clarified. A recent study reported unexplained high levels of iron in liver tissue of fattening turkeys suffering from HL. In this study, the iron status, possible infectious or inflammatory influences in form of an acute phase reaction and the analysis of fatty acid pattern in liver tissue of turkeys affected by HL were examined. Three cases of HL on three different fattening turkey farms were investigated during the outbreak of the disease. Clinically affected and non-affected animals were subjected to a pathological examination, where the diagnosis HL or non-affected was made. In total, 70 birds were examined (40 with HL, 30 without HL) and blood and liver samples were taken. Additionally, samples from 15 slaughtered birds were taken as a further control group. In liver tissue, the iron content and the content of long-chain fatty acids were determined; in blood samples, ferritin and transferrin were measured. The iron content in liver tissue was more than three times higher for animals with HL than among non-affected animals and the control group. The transferrin levels were lowest for animals with HL, highest in the control group and in between for non-affected animals. The fatty acid pattern in liver tissue of affected animals indicated a shift from polyunsaturated fatty acids to saturated fatty acids and monounsaturated fatty acids compared to the control group and the non-affected animals. Overall, the non-affected animals of a flock affected by HL were similar to the healthy animals of the abattoir. The low acute phase protein levels for animals with HL together with high iron contents could indicate a previous malnutrition/starvation period and/or severe liver damage for those animals suffering from HL.     

Effect of acute sublethal endotoxaemia on in vitro digital vascular reactivity in horses

Endotoxaemia is a syndrome linked to the development of equine laminitis; however, the relationship between them is uncertain. The aim of this experiment was to evaluate the effect of an experimental acute sublethal endotoxaemia model on in vitro equine palmar digital vascular reactivity. Rings of arteries and veins of each forelimb were obtained from 11 clinically healthy horses submitted to two surgical procedures, 3 weeks apart. Before the second surgery, 0.25 microg/kg of lipopolysaccharide from Escherichia coli O55:B5 in saline, was administered i.v. in 30 min. After 3 h, the vessels were harvested and submitted to in vitro vascular reactivity experiments and histopathology. The response to depolarizing Krebs solution (DKS, 40 mm), phenylephrine (PHE), acetylcholine (ACh) and sodium nitroprusside (SNP) were evaluated. All horses showed colic pain and watery diarrhoea, tachycardia, tachypnea, hyperthermia and leucopenia. Concentration-response curve (CRC) to PHE was shifted to the left in arteries rings from endotoxemic horses without any effect on vein rings. The CRC to ACh was shifted to the right with a reduction in the maximal response. The response to SNP and DKS was similar between groups. There was no evidence of histopathological effects. The increased response to PHE in digital arteries together with a reduction of the endothelium-dependent response to ACh in arteries and veins, confirm the existing reports where endotoxaemia was found to modify the digital vascular reactivity during the acute phase. As the digital endothelial function is impaired, there may be an increased potential to develop a digital prothrombotic state with a reduced vasodilatory capacity.     

Serial evaluation of thoracic radiographs and acute phase proteins in dogs with pneumonia

BackgroundAcute phase proteins (APP) may guide treatment of pneumonia in dogs but correlations with radiographic abnormalities are poorly characterized.ObjectivesDevelop a thoracic radiographic severity scoring system (TRSS), assess correlation of radiographic changes with APP concentrations, and compare time to APP and radiograph normalization with duration of antimicrobials treatment.AnimalsSixteen client‐owned dogs, 12 with aspiration pneumonia, and 4 with community‐acquired pneumonia.MethodsConcentrations of C‐reactive protein (CRP), serum amyloid A (SAA), and haptoglobin were measured on days 1, 3, 7, 14, 28, and 60 and orthogonal 2‐view thoracic radiographs were obtained on days 1, 7, 14, 28, and 60. Treatment was clinician‐guided and blinded to APP concentrations. Radiographic severity scores were assigned by blinded, randomized retrospective review by 2 board‐certified radiologists with arbitration by a third radiologist.ResultsMedian (interquartile range [IQR]) time to normalization of CRP (7 days [7‐14]) and SAA concentrations (7 days [7‐14]) were shorter than antimicrobial treatment duration (17.5 days [14.5‐33.5]; P = .001 and .002, respectively) and TRSS normalization (14 days [8.8‐52], P = .02 and .02, respectively). The CRP and SAA concentrations were positively correlated with TRSS (CRP r _s , 0.643; SAA r _s , 0.634; both P < .0001). Both CRP and SAA identified normal thoracic radiographs area under the curve (AUC) 0.873 and 0.817, respectively, both P < .0001. Interobserver agreement for TRSS assignment was moderate (κ, .499; P < .0001).Conclusion and Clinical ImportanceConcentrations of CRP and SAA normalized before radiographic resolution and before clinicians discontinued antimicrobial treatment. The CRP and SAA concentrations may guide duration of antimicrobial treatment for dogs with pneumonia.     

Increased GH secretion in scrapie,a prion-associated neurodegenerative disease,is not due to suppressed IGF-1 negative feedback

GH secretion is increased in scrapie-diseased sheep. Although the role of the somatotropic axis as a neurotrophic and neuroprotective factor is well documented, no studies have been carried out on the mechanisms and functional significance of somatotropic perturbation in the pathophysiology of prion-associated neurodegenerative disease. The goal of this study was to test the hypothesis that increased GH secretion observed in a natural animal prion disease, scrapie, might reflect a general lack of action of IGF-1 and, more particularly, a suppressed IGF-1 negative feedback. The effect of human recombinant IGF-1 (rhIGF-1) on spontaneous and GHRH-induced secretions was studied in so-called “scrapie-resistant” and “scrapie sensitive” rams in vivo and in vitro on pituitary dissociated cells from both groups. The effect of rhIGF-1 infusion on spontaneous and GHRH-induced GH secretions was evaluated during the preclinical and clinical stages of the disease in vivo. Our results indicated that rhIGF-1 suppressed spontaneous GH secretion but not GHRH-induced secretion in vivo. RhIGF-1 had no effect on spontaneous and GHRH-induced GH secretion from dissociated pituitary cells. Clinical scrapie was associated with a significantly greater rhIGF-1-induced inhibition of GH spontaneous secretion (mean ± S.E.M. inhibition of GH secretion: 31 ± 8% vs. 45 ± 4% in control and scrapie-affected rams, respectively). It can be concluded that the increase in GH secretion in scrapie-affected animals does not reflect a global lack of action of IGF-1. Further investigations are required to determine if other IGF-1 effects and more particularly neuroprotective mechanisms are altered in prion-associated neurodegenerative diseases.