Concentrations of neutrophil gelatinase-associated lipocalin are increased in serum and peritoneal fluid from horses with inflammatory abdominal disease and non-strangulating intestinal infarctions.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is produced in response to inflammation in horses, but it has not yet been investigated as a biomarker in horses with the abdominal disease. OBJECTIVES: To investigate NGAL in serum and peritoneal fluid in horses with the abdominal disease. STUDY DESIGN: Retrospective clinical study. METHODS: Data from medical records of 270 horses admitted with acute abdominal disease (simple obstructions [n = 43], strangulating obstructions [n = 104], inflammatory abdominal disease [n = 99], non-strangulating intestinal infarction [NSII, n = 24]) and 9 healthy control horses were reviewed, and serum and peritoneal fluid samples were retrieved from a biobank. NGAL was measured in serum and peritoneal fluid by ELISA. Differences in NGAL concentrations between groups were assessed. RESULTS: Healthy horses had low serum and peritoneal fluid concentrations of NGAL (median = 21.0 and 9.5 µg/L, respectively). Neither serum nor peritoneal fluid NGAL concentrations (median serum, peritoneal fluid) differed between healthy horses and horses with simple (28.1 µg/L, 13.0 µg/L) and strangulating intestinal obstructions (34.7 µg/L, 38.4 µg/L). Horses with NSII (308.0 µg/L, 2163.0 µg/L) and inflammatory abdominal disease (171.1 µg/L, 314.1 µg/L) had higher serum and peritoneal fluid concentrations of NGAL than the other groups (p < 0.001). Peritoneal fluid NGAL concentrations in horses with NSII were higher than in horses with inflammatory abdominal disease (p = 0.03). MAIN LIMITATIONS: Retrospective study design; small group of control horses. CONCLUSIONS: NGAL is a marker of inflammation in horses with the abdominal disease. The high peritoneal fluid concentration of NGAL in horses with NSII compared to all other groups may render NGAL useful for identifying this condition, which is otherwise difficult to differentiate from other types of peritonitis. Thereby, NGAL may potentially facilitate timely surgical intervention in this group of patients. The results of this study must be evaluated in larger, and preferably prospective, studies to obtain a more comprehensive evaluation of the diagnostic utility of NGAL.

CONTEXTO: Lipocalina associada à gelatinase de neutrófilos (NGAL) é produzida em resposta à inflamação em equinos, mas ainda não foi investigada como biomarcador de equinos com doenças abdominais. OBJETIVOS: Investigar a NGAL no soro e líquido peritoneal (LP) de equinos com doenças abdominais. DELINEAMENTO DO ESTUDO: Estudo retrospectivo. METODOLOGIA: Informação de prontuários médicos de 270 equinos com doença abdominal (obstrução simples [n = 43], obstrução estrangulante [n = 104], doença abdominal inflamatória [n = 99], lesão de infarto não estrangulante [NSII, n = 24]) e nove equinos saudáveis como controle foram revisados, e o soro e LP foram obtidos de um biobank. NGAL foi mensurado no soro e LP através de ELISA. Diferenças na concentração de NGAL entre os grupos foi avaliada. RESULTADOS: Cavalos saudáveis tiveram concentração de NGAL baixas no soro e no LP (mediana = 21.0 e 9.5 µg/L, respectivamente). A concentração de NGAL no soro e no LP não diferiram entre cavalos saudáveis e cavalos com obstrução simples (28.1,13.0) e obstrução estrangulante (34.7, 38.4). Cavalos com NSII (308.0, 2163.0) e doença abdominal inflamatória (171.1314.1) tiveram concentrações mais altas de NGAL no soro e LP quando comparados aos outros grupos (p < 0.001). A concentração de NGAL no LP em cavalos com NSII foi mais alta do que em cavalos com doença abdominal inflamatória (p = 0.03). PRINCIPAIS LIMITAÇÕES: Delineamento de estudo retrospectivo e pequeno grupo de equinos controle. CONCLUSÕES: NGAL é um marcador de inflamação em cavalos com doença abdominal. A concentração alta de NGAL no LP de cavalos com NSII comparado com os outros grupos indica que NGAL pode ser útil em identificar essa condição, o que é usualmente difícil de diferenciar de outros tipos de peritonite. Contudo, NGAL tem o potencial de facilitar a decisão rápida da necessidade de intervenção cirúrgica nesse grupo de pacientes. Os resultados deste estudo devem ser avaliados em uma população maior (e preferencialmente prospectiva) para obter uma melhor avaliação compreensiva da utilidade diagnóstica do NGAL.

Differentiating Strongylus vulgaris-associated, non-strangulating intestinal infarctions from idiopathic peritonitis and acute colitis in horses.

BACKGROUND: Horses with non-strangulating intestinal infarction (NSII) are often misdiagnosed with idiopathic peritonitis or acute colitis. Early diagnosis is essential to ensure early surgical intervention and improve survival. METHODS: Clinical and laboratory data from horses admitted to the University of Copenhagen Large Animal Teaching Hospital with NSII, idiopathic peritonitis or acute colitis between 2009 and 2018 were used for univariate comparisons and a multivariable logistic regression model for prediction of NSII. RESULTS: Two hundred and thirty-one horses were included. A multivariable model for the prediction of NSII included gastric reflux (more than 5 L) (odds ratio [OR] 8.7; 95% confidence interval [CI] 2.1-36.2), abnormal findings palpated per rectum (intestinal dilatations/impactions [OR 4.43; 95% CI 1.43-13.38], colon displacements [OR 23.16; 95% CI 5.26-101.97] or intestinal mass [OR 179.7; 95% CI 23.5-1375.5]), white blood cell count (OR 1.2; 95% CI 1.1-1.4), packed cell volume (OR 0.9; 95% CI 0.8-0.9), age (OR 0.9; 95% CI 0.8-1.0) and heart rate (OR 1.1; 95% CI 1.0-1.1). The model had a low false positive rate (5%), but a high false negative rate (50%). LIMITATIONS: Due to the retrospective nature of the study, sample collection was inconsistent, resulting in missing values. CONCLUSION: The model had some capability in predicting NSII. However, the high risk of false negatives means that exploratory laparotomy should be considered in horses with peritonitis of unknown aetiology in areas where Strongylus vulgaris is prevalent and occurrence of idiopathic peritonitis is low.

Investigation of two different human d-dimer assays in the horse.

BACKGROUND: D-dimer has value as a marker of thrombosis in critically ill horses and can provide additional information about prognosis. However, there are currently no equine species-specific d-dimer assays available, nor has there been any formal investigation of the applicability of human d-dimer assays in horses, so it is unknown, which assay performs best in this species. The aim of this study was therefore to evaluate and compare two human d-dimer assays for their applicability in horses. The study included four groups of horses: clinically healthy horses, horses with gastrointestinal (GI) disease and mild systemic inflammation based on low serum amyloid A (SAA) (low SAA group), horses with GI disease and strong systemic inflammation based on high SAA (high SAA group) and, horses with thrombotic GI disease caused by Strongylus vulgaris (also called non-strangulating intestinal infarction (NSII)) (NSII group). The assays evaluated were the STAGO STA-Liatest D-di + (Stago) and NycoCard™ D-dimer (NycoCard). Intra- and inter-coefficients of variation (CV) were assessed on two d-dimer concentrations, and linearity under dilution was evaluated. A group comparison was performed for both assays across the four groups of horses. A Spaghetti plot, Spearman Correlation, Passing Bablok regression and Bland-Altman plot were used to compare methods in terms of agreement. RESULTS: Ten horses were included in the clinically healthy group, eight in the low SAA group, eight in the high SAA group, and seven in the NSII group. For the Stago assay, intra- and inter-CVs were below the accepted level except for one inter-CV. The NycoCard assay did not meet the accepted level for any of the CVs. The linearity under dilution was acceptable for both the Stago and NycoCard. In the group comparison, both methods detected a significantly higher d-dimer concentration in the high SAA and NSII groups compared to the clinically healthy group. Method agreement showed slightly higher d-dimer concentrations with NycoCard compared to Stago. The overall agreement was stronger for the lower d-dimer concentrations. CONCLUSION: Both the Stago and the NycoCard were found to be applicable for use in horses but were not directly comparable.

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.

Acute-phase proteins as diagnostic markers in horses with colic.

OBJECTIVE: To investigate the diagnostic potential of the concentrations of acute-phase proteins serum amyloid A (SAA), haptoglobin (Hp), and fibrinogen in blood and peritoneal fluid (PF) for differentiating horses with inflammatory colic (entero-colitis and peritonitis) from those with surgical colic. DESIGN: Prospective observational multicenter study. SETTING: Two university referral hospitals. ANIMALS: Horses referred for severe acute abdominal pain to Hospital 1 (n = 148) or Hospital 2 (n = 78). INTERVENTION: Blood and PF samples collected at admission were used for acute-phase protein concentration measurement. MEASUREMENTS AND MAIN RESULTS: A multivariable logistic model including clinical parameters (lethargy, rectal temperature >38°C [100.4°F], normal rectal examination findings, and gastric reflux of 5-10 L) recorded at admission was constructed from Hospital 1 data. The ability of the model to correctly differentiate inflammatory from surgical colic was 86% determined as area under the receiver operating characteristic curve. Adding blood parameters (WBC, PCV, total plasma protein, lactate, SAA, Hp, and fibrinogen concentrations) to the logistic model based on clinical parameters revealed that only WBC and SAA and fibrinogen concentrations improved the model. With SAA included in the model no additional blood parameters improved the model, and the final model had an area under the curve of 90%. Addition of PF parameters (hemolysis, total protein concentration, WBC, SAA, or Hp concentrations) did not improve the model. When validated in Hospital 2 data, the models had good integrity and diagnostic performance. CONCLUSIONS: Evaluation of SAA in serum improved the ability to differentiate horses with acute inflammatory colic requiring medical treatment from horses with colic requiring surgery, as it allowed an additional 4% of horses to be correctly classified into medical and surgical cases. Improved differentiation of these 2 groups of horses with colic may minimize the risk of unnecessary or delayed surgery.

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.

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

BACKGROUND: Outcomes 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. RESULTS: The 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%). CONCLUSIONS: The 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.