Tina H. Pihl

Serum amyloid A and haptoglobin concentrations in serum and peritoneal fluid of healthy horses and horses with acute abdominal pain

BACKGROUND Peritoneal fluid (PF) analysis is a valuable diagnostic tool in equine medicine. Markers such as serum amyloid A (SAA) and haptoglobin (Hp) could facilitate the diagnosis of inflammatory abdominal conditions. OBJECTIVES The objectives were to (1) establish reference intervals (RI) for SAA and Hp in serum and PF in healthy horses, (2) compare SAA and Hp concentrations between healthy horses and horses with colic, and (3) to assess the correlation between serum and PF concentrations. METHODS Serum amyloid A and Hp concentrations were determined by automated assays in prospectively enrolled healthy reference horses and horses with colic. RIs were calculated, group concentrations were compared by Students t-test, and Pearsons correlation for serum and PF concentrations were determined. RESULTS In healthy horses (n = 62) the measurements for SAA were below the detection limit (0.5 mg/L) in 94% of serum samples and 98% of PF samples. Horses with colic (n = 61) had statistically significantly increased SAA concentrations in serum (P < .0001) and PF (P = .0013). While PF Hp concentrations were increased in horses with colic the serum concentrations of Hp were decreased (P < .0001). There was a strong correlation between paired serum and PF SAA concentrations (n = 94, R = .72, P < .0001), whereas the correlation between paired serum and PF Hp was weak (n = 94, R = .22, P = .0382). Finally, horses with colic tended to have serum SAA and PF Hp concentrations above the RIs. CONCLUSIONS With the apparent difference between healthy horses and horses with colic and the presently established RIs, serum SAA and PF Hp concentrations represent potential valuable diagnostic markers for inflammatory abdominal conditions in that species.

Influence of Disease Process and Duration on Acute Phase Proteins in Serum and Peritoneal Fluid of Horses with Colic

Background The acute phase proteins (APP) serum amyloid A (SAA), haptoglobin, and fibrinogen are valuable blood biomarkers in equine inflammatory diseases, but knowledge of factors influencing their concentrations in blood and peritoneal fluid (PF) of horses with colic is needed. Objectives The objective of this study was to investigate the influence of demographics (age, sex, breed), disease process (simple obstruction, strangulating obstruction, inflammatory), disease location, disease duration, hypovolemia, and admission hospital on concentrations of APP, lactate and white blood cell counts (WBC) in horses with colic admitted to 2 referral hospitals. Animals The study included 367 horses with colic admitted at 2 referral hospitals. Methods Prospective multicenter observational study of clinical data, as well as blood and PF biomarkers. Associations between biomarker concentrations and clinical variables were analyzed using multivariate linear regression analysis. Results Increasing pre‐admission duration of colic was associated with increased concentrations of APP in blood and PF. Blood concentrations of SAA and fibrinogen were associated with disease process (inflammatory, strangulations, simple obstructions) in more colic duration groups (5–12 and >24 hours) than any of the other biomarkers. No relevant associations between demographic factors, hospital, or hydration status and the measured biomarkers were found. Conclusions and Clinical Importance In horses with colic, concentrations of APP are associated mainly with disease process and duration of colic and may thus be used for assessment of disease independently of demographic or geographic factors. Serum amyloid A may be a diagnostic marker for use in colic differential diagnosis, but further evaluation is needed.

Nonstrangulating intestinal infarction associated with Strongylus vulgaris in referred Danish equine cases.

REASONS FOR PERFORMING STUDY Strongylus vulgaris is a pathogenic helminth parasite infecting horses and was once considered to be the primary cause of colic. Migrating larvae cause ischaemia and infarction of intestinal segments. This knowledge is derived from case reports and experimental inoculations of parasite-naïve foals, and it remains unknown to what extent the parasite is associated with different types of colic. OBJECTIVES To evaluate the role of S. vulgaris as a risk factor for different types of colic in horses. STUDY DESIGN A retrospective case-control study among horses referred with abdominal pain to the University of Copenhagen Large Animal Teaching Hospital during 2009-2011. METHODS Each colic case was matched with an equid of the same type (pony, Warmblooded or Coldblooded), age, sex and admitted in the same month and year but for problems unrelated to the gastrointestinal tract. Serum samples were analysed for antibodies to migrating S. vulgaris larvae using a recently developed enzyme-linked immunosorbent assay (ELISA). The following 4 case definitions were used: colic sensu lato, i.e. all horses presenting with colic (n = 274), with further subgroups, i.e. undiagnosed colics (n = 48), strangulating obstructions (n = 76) and nonstrangulating infarctions (n = 20). RESULTS Strongylus vulgaris antibody levels were similar to control values in colics sensu lato and horses with undiagnosed colic. In contrast, nonstrangulating intestinal infarctions were significantly associated with positive S. vulgaris ELISAs (odds ratio 5.33, 95% confidence interval 1.03-27.76, P = 0.05). Also, horses with nonstrangulating infarctions had a significantly higher occurrence of positive ELISAs than horses with strangulating obstructions (odds ratio 3.79, 95% confidence interval 1.34-10.68, P = 0.01) and the colic sensu lato group (odds ratio 3.09, 95% confidence interval 1.20-8.01, P = 0.02). CONCLUSIONS Nonstrangulating intestinal infarction was strongly associated with S. vulgaris-specific antibodies, whereas the more broadly defined colic categories were not associated with positive ELISA results. Thus, the ELISA holds potential to become a helpful adjunct in diagnosis and management of horses with colic.

Short-term survival and mortality rates in a retrospective study of colic in 1588 Danish horses

BackgroundOutcomes of colic treatment are of great interest to clinicians, horse owners and insurers. One commonly used criterion of success is the overall short-term survival rate. This is used as to compare treatments and to measure quality of veterinary care, but may be biased by demographic or social factors such as attitudes towards animal suffering and euthanasia. The aims of this study were to 1) describe and analyse characteristics in horses with signs of colic referred to the University Hospital for Large Animals (UHLA), University of Copenhagen, Denmark over a 10-year period and 2) to compare these rates with those published in other comparable studies.ResultsThe overall survival rate for colic horses over the 10-year study period was 68% (confidence intervals (CI): 66–71%; 1087/1588). In the medical group, 1093 horses, short-term survival was 87% (CI: 85–89%). Thirty one % of referred horses were given diagnoses requiring surgical intervention (CI: 29–33%). In this group 32% of the horses were euthanized before surgery (CI: 28–36%; 159/495). Of the surgical cases 27% (CI: 23-31%) were euthanized or died during surgery. Of the horses that recovered from surgery 25% died or were euthanized (CI: 19–32%; 48/189), while 75% survived to discharge (CI: 68–81%).ConclusionsThe short term survival rates of Danish horses with colic were similar or lower to those reported from other countries. Apart from variability of veterinary care, attitudes towards euthanasia vary among the countries, which may bias the outcomes. This study indicates that qualitative interview studies on owners’ attitudes towards animal suffering and euthanasia need to be conducted. Our opinion is that survival rates are not valid as sole indicators of quality of care in colic treatment due to selection bias. If the survival rates are to be compared between hospitals, techniques or surgeons, prospective studies including mutually agreed-on disease severity scores and a predefined set of reasons for euthanasia are needed.

Safety Pharmacology, Toxicology and Pharmacokinetic Assessment of Human Gc Globulin (Vitamin D Binding Protein)

Gc globulin is an important protein of the plasma actin-scavenger system. As such, it has been shown to bind free actin and prevent hypercoagulation and shock in patients with massive actin release resulting from severe tissue injuries. Treatment of such patients with Gc globulin could therefore potentially be life-saving. This article presents pre-clinical toxicology experiments conducted on purified plasma-derived human Gc globulin. The Gc globulin formulation was shown to be stable for at least 4 years with full retention of actin-binding capacity. In vitro studies did not reveal activation of the kallikrein system or the complement system and cellular studies showed no toxic effects on a variety of human cell lines. In vivo studies showed no acute toxic effects in mice, rats or guinea pigs upon intravenous infusion. A 14-day local tolerance study in rabbits showed no adverse effects, and 14-day toxicity studies in rats and horses did not show any unwanted reactions. In a 14-day toxicology study in beagle dogs, formation of antibodies was seen and in the end of the study period, three out of four dogs showed clinical immunological reactions, which could be ascribed to the formation of antibodies. The half-life, T, for human Gc globulin was 12 hr in rats, 16 hr in horses and 30 hr in dogs. The safety profile of plasma-derived Gc globulin is concluded to be consistent to that required for use in man.

Polyclonal Peptide Antisera.

Polyclonal antibodies are relatively easy to produce and may supplement monoclonal antibodies for some applications or even have some advantages. The choice of species for production of (peptide) antisera is based on practical considerations, including availability of immunogen (vaccine) and animals. Two major factors govern the production of antisera: the nature of adaptive immune responses, which take place over days/weeks and ethical guidelines for animal welfare. Here, simple procedures for immunization of mice, rabbits, sheep, goats, pigs, horses, and chickens are presented.

Changes in Hemostatic Indices in Foals Naturally Infected With Strongylus vulgaris

Abstract Strongylus vulgaris has been found endemic in equine populations subject to parasite control by targeted selective anthelmintic therapy. This study investigated hemostasis in foals naturally infected with S. vulgaris and monitored this response over the course of progressing infection stages. The hemostatic indices D‐dimer, antithrombin III (ATIII), fibrinogen, prothrombin time (PT), and activated partial thromboplastin time were evaluated in weekly blood samples for up to 50 weeks in 12 foals born into a herd with high prevalence of S. vulgaris. Results were compared with weekly S. vulgaris antibody enzyme‐linked immunosorbent assay values in all foals using a linear mixed effects model with repeated measures and to total numbers of S. vulgaris larvae in nine foals at necropsy with Pearson linear correlation. In the first week of life, all evaluated indices of hemostasis were significantly different from those observed in the rest of the study weeks, corresponding to previously demonstrated aberrancies in neonates. Significant changes were seen for D‐dimer in weeks 11–24, 26–27, 30, and 39 compared with week 2, for PT in weeks 12–13 compared with week 6, and for ATIII in week 15 compared with week 4. Strongylus vulgaris antibody levels were statistically associated with D‐dimer (P = .0076) and fibrinogen (P = .0004) concentrations. Naturally acquired infection with S. vulgaris was associated with changes suggestive of mild activation of coagulation, fibrinolysis, and inflammation. The results of this study may help elucidate the pathogenesis of the endarteritis, thromboembolism, and nonstrangulating intestinal ischemia that is observed in horses with S. vulgaris infection. HighlightsIncreased levels of D‐dimer and fibrinogen are associated with arterial Strongylus vulgaris infection.Findings are supportive of on‐going hemostasis, fibrinolysis, and mild inflammation.Results may help elucidate the pathogenesis of verminous arteritis.

Characterization of equine vitamin D-binding protein, development of an assay, and assessment of plasma concentrations of the protein in healthy horses and horses with gastrointestinal disease

OBJECTIVE To purify and characterize equine vitamin D-binding protein (VDBP) from equine serum and to evaluate plasma concentrations of VDBP in healthy horses and horses with gastrointestinal injury or disease. ANIMALS 13 healthy laboratory animals (8 mice and 5 rabbits), 61 healthy horses, 12 horses with experimentally induced intestinal ischemia and reperfusion (IR), and 59 horses with acute gastrointestinal diseases. PROCEDURES VDBP was purified from serum of 2 healthy horses, and recombinant equine VDBP was obtained through a commercial service. Equine VDBP was characterized by mass spectrometry. Monoclonal and polyclonal antibodies were raised against equine VDBP, and a rocket immunoelectrophoresis assay for equine VDBP was established. Plasma samples from 61 healthy horses were used to establish working VDBP reference values for study purposes. Plasma VDBP concentrations were assessed at predetermined time points in horses with IR and in horses with naturally occurring gastrointestinal diseases. RESULTS The working reference range for plasma VDBP concentration in healthy horses was 531 to 1,382 mg/L. Plasma VDBP concentrations were significantly decreased after 1 hour of ischemia in horses with IR, compared with values prior to induction of ischemia, and were significantly lower in horses with naturally occurring gastrointestinal diseases with a colic duration of < 12 hours than in healthy horses. CONCLUSIONS AND CLINICAL RELEVANCE Plasma VDBP concentrations were significantly decreased in horses with acute gastrointestinal injury or disease. Further studies and the development of a clinically relevant assay are needed to establish the reliability of VDBP as a diagnostic and prognostic marker in horses.