Alessia Giordano

The systemic reaction during inflammation: the acute-phase proteins.

The acute-phase response consists in a large number of behavioural, physiologic, biochemical, and nutritional changes involving many organ systems distant from the site, or sites, of inflammation. One of the most investigated, but still not well understood, characteristic of the acute phase is the up-regulation, or down- regulation, of many plasma proteins, known as the acute-phase proteins. The changes in the concentrations of these positive acute-phase proteins and negative acute-phase proteins are due to changes in their liver production. Their increase may vary from 25 percent to 1000 fold, as in the case of C-reactive protein and serum amyloid A. This review summarises the recent advances that have been acquired on the acute-phase proteins, in particular their function in pathologies such as infections or inflammatory lesions.

Changes in some acute phase protein and immunoglobulin concentrations in cats affected by feline infectious peritonitis or exposed to feline coronavirus infection.

Abstract The possible role of some acute phase proteins (APPs) and immunoglobulins in both the pathogenesis and diagnosis of feline infectious peritonitis (FIP) has been investigated. Serum protein electrophoresis and the concentration of haptoglobin (Hp), serum amyloid A (SAA), α1-acid glycoprotein (AGP), IgG and IgM were evaluated in cats exposed to feline coronavirus (FCoV) and in cats with FIP. The highest concentration of APPs was detected in affected cats, confirming the role of these proteins in supporting a clinical diagnosis of FIP. Repeated samplings from both FIP affected and FCoV-exposed cats showed that when FIP appeared in the group, all the cats had increased APP levels. This increase persisted only in cats that developed FIP (in spite of a decrease in α2-globulins) but it was only transient in FCoV-exposed cats, in which a long lasting increase in α2-globulins was observed. These results suggest that changes in the electrophoretic motility of APPs or APPs other than Hp, SAA and AGP might be involved in the pathogenesis of FIP or in protecting cats from the disease.

Validation of thromboelastometry in horses

BACKGROUND Thromboelastometry is used for identifying or monitoring coagulation abnormalities. It has been validated in several species but not in horses and the characteristics of the equine thromboelastogram have not yet been detailed. OBJECTIVES The purpose of this study was to validate a thromboelastometer to be used with equine blood and to define the normal equine thromboelastogram. METHODS A Rotem-gamma thromboelastometer (Pentapharm GmbH, Munich, Germany) was used on 38 citrated blood samples to investigate native coagulation, the intrinsic and extrinsic pathways, the function of fibrinogen (largely dependent on its concentration), and the presence of fibrinolysis. Using classic validation approaches, we evaluated the imprecision of the method and the influence of hemolysis and storage time and temperature. The normal thromboelastogram was defined in both saddle and racing horses (the latter sampled before and after the race). RESULTS For imprecision tests, the analytical variations were <10%. The equine thromboelastogram had a pattern similar to those of other species, but the intrinsic and extrinsic pathways were less and more efficient, respectively. Reference intervals in racing horses, especially after exercise, were different from those of saddle horses, most likely due to a higher RBC mass. Coagulability decreased in hemolyzed samples and significant changes were found between nonrefrigerated and refrigerated blood samples stored for 20 hours. CONCLUSIONS The Rotem-gamma thromboelastometer is a precise instrument for use with equine blood samples. The equine thromboelastogram is similar to that of other species, but reference intervals vary with aptitude and exercise. Hemolysis and refrigeration alter thromboelastometric results.

Performances of different diagnostic tests for feline infectious peritonitis in challenging clinical cases

Objectives: Feline infectious peritonitis (FIP) can be difficult to diagnose. Histopathology is considered the gold standard test but immunohistochemistry (IHC) is mandatory to confirm/exclude the disease. This study aimed to assess the performances of tests carried out in vivo or at postmortem examination in challenging cases in which FIP was confirmed or excluded based on IHC or on adequate follow‐up. Methods: Twelve cases (four without FIP, eight with FIP) were retrospectively studied. Clinical findings, serum protein electrophoresis (SPE), analysis of the effusions (AE), antifeline coronavirus serology, serum concentration of α1‐acid glycoprotein (AGP) and histopathology were classified as consistent, doubtful or non‐consistent with FIP. Sensitivity, specificity and concordance (κ) with the final diagnosis were calculated. Results: Concordance was absent for serology (κ=−0·08) and AE (κ=−0·52), poor for histopathology (κ=0·09), fair for SPE (κ=0·25) and perfect for AGP (κ=1·00). Sensitivity was high for AGP (100%) and low for AE (50%), SPE (37·5%) and histopathology (37·5%). Specificity was high for AGP or histopathology (100%) and low for SPE (50%) and AE (0%). Clinical Significance: IHC must always be performed to confirm FIP. If this is not possible, when histopathology is controversial, elevated AGP concentrations may support the diagnosis of FIP.

Decreased sialylation of the acute phase protein α1-acid glycoprotein in feline infectious peritonitis (FIP)

Abstract Feline infectious peritonitis (FIP) is an immune-mediated disease of domestic and exotic felides infected with feline coronavirus. FIP is characterized by the overexpression of an acute phase protein, the α1-acid glycoprotein (AGP). In humans, AGP is a heavily glycosylated protein that undergoes several modifications of its glycan moiety during acute and chronic inflammatory pathologies. We studied the changes in AGP glycosylation in the course of FIP. Specifically, we focussed our attention on the degree of sialylation, fucosylation and branching. This study presents a purification method for feline AGP (fAGP) from serum, using an ion exchange chromatography strategy. The glycosylation pattern was analyzed in detail by means of interaction of purified fAGP with specific lectins. In particular, Sambucus nigra agglutinin I and Maackia amurensis agglutinin lectins were used to detect sialic acid residues, Aleuria aurantia lectin was used to detect l-fucose residues and Concanavalin A was used to evaluate the branching degree. By this method we showed that fAGP did not present any l-fucose residues on its surface, and that its branching degree was very low, both in normal and in pathological conditions. In contrast, during FIP disease, fAGP underwent several modifications in the sialic acid content, including decreased expression of both α(2–6)-linked and α(2–3)-linked sialic acid (76 and 44%, respectively when compared to non-pathological feline AGP).

Shifts in circulating lymphocyte subsets in cats with feline infectious peritonitis (FIP): pathogenic role and diagnostic relevance

Abstract Cats with feline infectious peritonitis (FIP) are usually lymphopenic and have lymphoid depletion evident in spleen and lymph nodes. In particular, the number of CD4+ lymphocytes in tissues decreases during the evolution of FIP lesions. This decrease is most likely due to increased lymphocyte apoptotic rate. In contrast, cats infected with the Feline Coronavirus (FCoV) develop a follicular hyperplasia in the peripheral lymph nodes. The current study was devised to evaluate the possible pathogenic role of shifts in circulating lymphocyte subsets in FIP. Peripheral blood from cats with FIP was evaluated and compared with peripheral blood from clinically healthy cats living in both FCoV-free and FCoV-endemic catteries. Blood from cats with diseases other than FIP was also examined in order to define the diagnostic relevance of the changes. Lymphocyte subsets were analysed by flow cytometry, using a whole blood indirect immunofluorescence technique and mAbs specific for feline CD5, CD4, CD8, CD21. The results of the current study suggest that cats recently infected with FCoV that do not develop the disease have a transient increase in T cells; cats from groups with high prevalence of FIP have a moderate but persistent decrease in T cell subsets; cats with FIP have a very severe decrease in all the subsets of lymphocytes. Moreover, during FIP many lymphocytes do not express any membrane antigen, most likely due to early apoptosis. Cats with diseases other than FIP also had decreased number of lymphocytes: as a consequence, the diagnostic relevance of these findings is very low. Nevertheless, the lack of flow cytometric changes had a high negative predictive value (NPV), thus allowing to exclude FIP from the list of possible diagnoses in cats with normal cytograms.

Sensitivity of Tru‐cut and fine‐needle aspiration biopsies of liver and kidney for diagnosis of feline infectious peritonitis

Background: The detection of typical lesions and feline coronavirus (FCoV) antigen in tissues is the only conclusive method for making a diagnosis of feline infectious peritonitis (FIP). A positive result using Tru‐cut biopsy (TCB) and fine‐needle aspiration biopsy (FNAB) has high diagnostic specificity, but information about the capacity of these techniques to correctly identify cats with FIP lesions is not available. Objectives: The diagnostic sensitivity of TCB and FNAB for detecting liver and kidney histologic lesions caused by FIP was evaluated. Methods: TCB and FNAB specimens collected mainly at necropsy from 25 cats with FIP were analyzed. Diagnostic sensitivity was calculated on the basis of the number of false‐negative and true‐positive specimens, compared with the number of organs bearing histologic lesions of FIP. Results: Diagnostic sensitivity was higher for hepatic TCB (64%) and FNAB (82%) than for renal (39% and 42%, respectively) procedures. A high percentage of renal cytologic and TCB specimens were inadequate. Combined analysis of TCB and FNAB specimens collected from the same organ increased the diagnostic sensitivity for liver (86%) and kidney (48%). The sensitivity of immunohistochemical/cytochemical analysis was low (11–38% depending on the technique), probably due to variable distribution of feline coronavirus in the lesions. Conclusion: Biopsy of liver and kidney can correctly identify FIP lesions. However, false‐negative results or inadequate samples occur with moderate frequency, especially for immunochemical analysis. Diagnostic sensitivity may be increased when both TCB and FNAB specimens from the same organ are examined.

Evaluation of equine hemograms using the ADVIA 120 as compared with an impedance counter and manual differential count.

BACKGROUND The ADVIA 120 is an automated laser cell counter widely used in veterinary medicine. Although specific software for equine samples is available and validated, only a few reports have been published comparing the ADVIA 120 with other methods for equine hemogram evaluation. OBJECTIVES The purpose of this study was to compare the hematologic values and reference intervals obtained on the ADVIA 120 with those obtained on an impedance cell counter and manual differential counts in healthy horses. METHODS EDTA-anticoagulated blood samples were obtained from 114 clinically healthy horses of various breeds, both sexes, and 2-6 years of age. Samples were stored for up to 12 hours at 4 degrees C and then analyzed on the ADVIA 120 and the Hemat 8. A 100-cell to 200-cell differential leukocyte count was performed by 3 independent observers on May-Grünwald-Giemsa-stained smears. Intra-assay precision of the ADVIA 120 was determined by analyzing 5 replicates each of 10 of the blood samples. RESULTS Results from the ADVIA were significantly higher than those from the impedance counter for RBC count, total WBC count, hemoglobin concentration, red cell distribution width, MCH, and MCHC, and significantly lower for HCT and platelet count. Significantly higher neutrophil and basophil counts and significantly lower lymphocyte counts were obtained with the ADVIA 120 compared with manual counts. Based on Passing-Bablok regression analysis, RBC and platelet counts were in good agreement between the 2 analyzers; a constant and proportional bias was present for other values. Coefficients of variation for erythrocyte parameters on the ADVIA were <1%, but were higher for platelet (6%), total WBC (2%), differential WBC (4%-30%), and reticulocyte (75%) counts. CONCLUSIONS Results obtained with equine samples on the ADVIA 120 were comparable with those obtained on an impedance counter; reference intervals differed statistically but overlapped. The ADVIA had poor precision for reticulocyte and differential leukocyte counts such that the latter should always be verified on smears.

Serum α1-acid glycoprotein (AGP) concentration in non-symptomatic cats with feline coronavirus (FCoV) infection

Previous studies have demonstrated that the concentration of α1-acid glycoprotein (AGP) transiently increases in asymptomatic cats infected with feline coronavirus (FCoV). In order to establish whether these fluctuations depend on the FCoV status, the serum concentration of AGP and anti-FCoV antibody titres and/or faecal shedding of FCoVs in clinically healthy cats from catteries with different levels of prevalence of FCoV infection were monitored over time. Serum AGP concentrations fluctuated over time in clinically healthy cats from the cattery with the highest prevalence of feline infectious peritonitis (FIP) and significantly increased just before an outbreak of FIP. Further studies are required to clarify whether the observed increase of AGP concentration is a consequence of the increased viral burden or a protective response against mutated viral strains. Nevertheless, the results of the present study suggest that AGP might be useful in monitoring FCoV–host interactions in FCoV-endemic catteries.

Thromboelastometric Profiles of Horses Affected by Exercise-Induced Pulmonary Hemorrhages

Exercise-induced pulmonary hemorrhage (EIPH) commonly occurs in race horses. Thromboelastometry (TEM) investigates the whole hemostatic process by evaluating the viscoelastic properties of the blood clot from its formation to fibrinolysis. The aim of this study was to assess whether horses with EIPH have abnormal thromboelastometric profiles. Intrinsic and extrinsic pathways, fibrinogen activity and fibrinolysis were investigated by TEM before and after the race in negative controls and in horses on which EIPH was confirmed by bronchoscopy. Compared with controls, horses with EIPH had an increased coagulability in both pre- and postrace samplings, especially for the intrinsic pathway and for the fibinrolytic activity. These results suggest that coagulation is preactivated in horses prone to develop EIPH, possibly due to recent or recurrent hemorrhage.