Neutrophil function and plasma opsonic capacity in colostrum-fed and colostrum-deprived neonatal kittens

OBJECTIVE: To determine whether passive transfer of IgG in neonatal kittens affects plasma opsonic capacity and neutrophil phagocytic and oxidative burst responses to bacteria in vitro. ANIMALS: 22 kittens from 6 specific pathogen-free queens. PROCEDURE: Kittens were randomized at birth into the following treatment groups: colostrum-fed, colostrum-deprived, or colostrum-deprived supplemented with feline or equine IgG. Blood samples were collected at intervals from birth to 56 days of age. Plasma IgG concentrations were determined by radial immunodiffusion assay. Neutrophil function was assessed by a flow cytometry assay providing simultaneous measurement of bacteria-induced phagocytosis and oxidative burst. The opsonic capacity of kitten plasma was determined in an opsonophagocytosis assay with bacteria incubated in untreated or heat-inactivated plasma. RESULTS: Among treatment groups, there were no significant differences in neutrophil phagocytic and oxidative burst responses to bacteria or opsonic capacity of plasma. In all samples of plasma, inactivation of complement and other heat-labile opsonins significantly reduced the opsonic capacity. Plasma IgG concentrations in kittens did not correlate with neutrophil function or plasma opsonic capacity before or after inactivation of complement. CONCLUSIONS AND CLINICAL RELEVANCE: The plasma opsonic capacity and neutrophil phagocytic and oxidative burst responses in vitro of kittens receiving passive transfer of IgG via colostrum intake or IgG supplementation and those deprived of colostrum were similar. The alternate complement pathway or other heat-labile opsonins may be more important than IgG in bacterial opsonization and phagocytosis.     

Evaluation of intravenous administration of concentrated immunoglobulin G to colostrum-deprived foals.

Ten foals of various breeds were deprived of colostrum from birth to 36 hours of age, then were allotted to 2 groups. Foals of group 1 (n = 6) were given 20 g (200 ml) of purified equine IgG IV in a 10% solution, and foals of group 2 (n = 4) were given 30 g (300 ml) of the same preparation. Total administration time for each 10 g of IgG in 100 ml was approximately 10 minutes. Serum IgG concentration in foals was assessed prior to, between 24 and 48 hours, and at 7 and 14 days after IgG administration. Between 24 and 48 hours after IgG administration, mean serum IgG concentration in group-1 foals was 425 mg/dl (range, 350 to 480 mg/dl). Mean body weight for this group of foals was 50.3 kg (range, 43.3 to 54.7 kg). For group-2 foals, mean serum IgG concentration was 768 mg/dl (range, 640 to 920 mg/dl) between 24 and 48 hours after administration of IgG. Foals of this group had mean body weight of 43.2 kg (range, 36.5 to 47.5 kg). Serum IgG concentration in group-2 foals at 24 to 48 hours was significantly (P = 0.005) greater than that in group-1 foals. Mean total IgG recovery at 24 to 48 hours, calculated on the basis of 94.5 ml of plasma volume/kg of body weight, was approximately 100%. Values of IgG measured in all foals 1 and 2 weeks after administration of the IgG concentrate were equivalent to values expected after normal decay of passively acquired IgG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of partial arytenoidectomy as a treatment for equine laryngeal hemiplegia

The efficacy of partial arytenoidectomy was assessed in 6 Standardbred horses, with surgically induced laryngeal hemiplegia, at rest (Period A) and during exercise at speeds corresponding to maximum heart rate (Period C) and 75% of maximum heart rate (Period B). Peak expiratory and inspiratory airflow rate (PEF and PIF), and expiratory and inspiratory transupper airway pressure (P_UE and P_UI) were measured and expiratory and inspiratory impedance (Z_E and Z_I) were calculated. Simultaneously, tidal breathing flow-volume loops (TBFVL) were acquired using a respiratory function computer. Indices derived from TBFVL included airflow rates at 50 and 25% of tidal volume (EF₅₀, IF₅₀, EF₂₅. and IF₂₅) and the ratios of expiratory to inspiratory flows. Measurements were made before left recurrent laryngeal neurectomy (baseline), 2 weeks after left recurrent laryngeal neurectomy (LRLN) and 16 weeks after left partial arytenoidectomy coupled with bilateral ventriculectomy (ARYT). After LRLN, during exercise Periods B and C, Z₁ and the ratio of EF₅₀/IF₅₀ significantly increased and PIF, IF₅₀ and IF₂₅ significantly decreased from baseline values. At 16 weeks after ARYT, Z₁ returned to baseline values during Periods B and C. Although PIF, IF₅₀, IF₂₅, PEF/PIF, and EF₅₀/IF₅₀ returned to baseline values during Period B, these indices remained significantly different from baseline measurements during Period C. After ARYT, TBFVL shapes from horses during Period C approached that seen at the baseline evaluation. Partial arytenoidectomy improved upper airway function in exercising horses with surgically induced left laryngeal hemiplegia, although qualitative and quantitative evaluation of TBFVLs suggested that some flow limitation remains at near maximal airflow rates. These results indicate that, although the procedure does not completely restore the upper airway to normal, partial arytenoidectomy is a viable treatment option for failed laryngoplasty and arytenoid chondropathy in the horse.     

Investigation of the antigenic relationship between equine IgG and IgGT

The antigenic cross reactivity between equine IgG and IgGT was investigated. On the basis of immunodiffusion and immunoelectrophoresis reactions using an antiserum raised against the Fc fraction of IgGT, this equine immunoglobulin can be unequivocally classified as a subclass of IgG.     

Evaluation of colostral IgG1 absorption in newborn calves after treatment with alkalinizing agents.

The effects of alkalinizing agents, administered prior to feeding colostrum, on blood-gas and acid-base values and on absorption of IgG1 were determined in 40 newborn Holstein calves. Two treatments, sodium bicarbonate (3 mEq/kg of body weight, IV) and doxapram HCl (2 mg/kg, IV), were evaluated, using a randomized complete-block experimental design. These treatments resulted in significant (P less than 0.01) alteration of blood-gas and acid-base values, generally in the direction of normal values for adult cattle. Significant least squares mean effects were detected for sodium bicarbonate treatment on blood pH (+ 0.04 units, P less than 0.01), PCO2 (+ 4.1 mm of Hg, P less than 0.01), and HCO3 concentration (+ 4.4 mEq/L, P less than 0.01). Significant least squares mean effects were detected for doxapram HCl treatment on blood pH (+ 0.06 pH units, P less than 0.01) and PCO2 (-5.2 mm of Hg, P less than 0.01). Absorption of colostral IgG1 was not affected by the treatments given or by the altered blood-gas and/or acid-base status.     

Evaluation of progesterone treatment to create a model for equine endometritis.

To investigate a model for equine endometritis, 12 mares with normal reproductive tracts were divided into 2 groups. All mares received progesterone in oil, 250 mg im, daily. At 5 days after initiation of progesterone administration, the uteri were inoculated with 10(6) colony forming units of Pseudomonas aeruginosa. The day of inoculation was designated Day 0. On Day 6, endometrial swab samples yielded P. aeruginosa in 5 mares; samples from the other 7 mares yielded heavy growth of Escherichia coli, Streptococcus zooepidemicus, Klebsiella pneumoniae, Enterobacter spp., Citrobacter diversus, Staphylococcus epidermidis and Streptococcus morbillorum. On Days 6, 7 and 8, Group A mares received intrauterine infusions of 6 g ticarcillin disodium and 0.2 g clavulanate potassium in 100 ml sterile saline. Group B mares received infusions of saline only. The incidence of swab specimens yielding no bacterial growth was significantly higher in Group A than Group B mares on Days 8 and 13 (4/6 vs 0/6). Swab samples from 5 of the 6 mares in Group A yielded growth of fungi on Days 13 and 19. Mares in Group B were then similarly treated with ticarcillin/clavulanate infusions, on Days 19, 20 and 21. The incidence of swab specimens yielding no bacterial growth was 2/6 and 1/6 on Days 21 and 26, respectively; fungi were not recovered from these mares at any time. The incidence of no-growth swabs after antibiotic treatment tended to be higher in Group A and incidence of fungal recovery after antibiotic treatment was significantly higher in Group A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of surrogate markers for passive transfer of immunity in kittens

OBJECTIVE: To identify surrogate markers of passive transfer of immunity in kittens. DESIGN: Prospective clinical trial. ANIMALS: 55 kittens from 12 specific-pathogen-free queens. PROCEDURE: Kittens were allocated at birth into colostrum-fed (n = 27) and colostrum-deprived (28) groups. Blood was collected at birth and on days 1, 2, 4, 7, 14, 28, and 56. Serum samples were analyzed for activities of alkaline phosphatase (ALP), alanine aminotransferase, aspartate aminotransferase, creatine kinase, lactate dehydrogenase, gamma-glutamyltransferase, amylase, and lipase and for concentrations of albumin, total protein, bilirubin, urea nitrogen, creatinine, cholesterol, glucose, calcium, phosphorus, and triglycerides by use of an automated analyzer. Total serum solids concentrations were estimated by use of refractometry. Serum IgG concentrations were quantified by use of radial immunodiffusion. RESULTS: All kittens were agammaglobulinemic at birth. Colostrum-fed kittens had significantly higher IgG concentrations than did colostrum-deprived kittens from 1 though 28 days of age. Transient significant differences in serum biochemical variables between the colostrum-deprived and colostrum-fed groups were substantially resolved by day 4. Passive transfer of immunity could be reliably determined at 1 day of age and to a lesser extent at 2 days of age only by measurement of serum activity of ALP. CONCLUSIONS AND CLINICAL RELEVANCE: Adequacy of passive transfer in kittens initially correlated with serum activity of ALP, but quantification of serum IgG concentration was necessary after 2 days of age.     

Evaluation of the equine cardiovascular system

A thorough examination of the cardiovascular system is an integral part of a physical examination in the horse. The normal equine cardiovascular parameters are discussed, with an emphasis on auscultatory findings. The availability and application of other diagnostic techniques are discussed based upon findings of the physical examination.     

Comparison of methods for depletion of albumin and IgG from equine serum

Background: Disease‐specific biomarkers hold diagnostic promise in both human and veterinary medicine, but serum biomarkers in low concentrations may be masked by the presence of abundant proteins, mostly albumin and IgG. Methods to deplete albumin and IgG exist, but efficacy of these methods for depleting equine serum of these proteins has not been established. Objective: The aim of this study was to determine if albumin and IgG could be depleted from equine serum using several commercially available kits and procedures. Methods: One‐dimensional gel electrophoresis followed by densitometry was used to determine percent of albumin, IgG, and both in pooled serum from 3 horses before and after application of 7 depletion methods. Repeatability was determined by applying the 2 best methods to serum samples from 6 grade horses. Results: For pooled serum, depletion rates varied from 35–90% for albumin and 0–94% for IgG. In the repeatability study, the ProteoExtract method combined with protein G Sepharose beads to remove additional IgG provided the best overall performance with 66% albumin depletion and 100% IgG depletion. A protocol using protein G Sepharose beads to remove IgG followed by ethanol precipitation of nonalbumin proteins with albumin remaining in the supernatant was the second most effective, with 85% albumin depletion and 55% IgG depletion. Although a multiprotein immunodepletion column effectively removed 90% of the albumin, the method was ineffective at removing IgG. Conclusion: Albumin and IgG removal kits optimized for human use have variable efficacy for equine serum. Combined use of the ProteoExtract kit and manual incubation with protein G Sepharose beads provided the most effective depletion.     

IgG antibody subclass response against equine herpesvirus type 4 in horses

In this study, IgG subclass responses against equine herpesvirus type 4 (EHV-4) were examined by enzyme-linked immunosorbent assay (ELISA) using a type-specific region of EHV-4 glycoprotein G (gG). ELISA using sera collected from horses experimentally infected with EHV-4 revealed that IgGa and IgGb antibodies were detected at high level, but IgGc and IgG(T) antibody responses were detected at low level or were undetectable. The IgGa antibody response reached its peak on day 10 post-infection, and then dropped. The IgGb antibody response reached its maximum level on day 12 post-infection, and then the level was sustained during at least 28 days after infection. Forty healthy racehorses that had already been infected with EHV-4 possessed antibody against EHV-4. Although IgGa antibodies specific for EHV-4 were not detected in any horses, IgGb antibodies were detected and the levels correlated with total IgG antibodies against EHV-4 gG. The results suggest that EHV-4-specific IgGa and IgGb antibodies are induced in EHV-4-infected horses, and that IgGb antibody, but not IgGa, is long lasting.     

Production and characterisation of two monoclonal antibodies recognising equine IgG Fc receptors

Despite the importance of IgG Fc receptors in the regulation of various immunological mechanisms, these receptors have not been well characterised in the domesticated animals including equines. This paper describes the production of two monoclonal antibodies (CVS 59 and CVS 61) that recognise equine IgG Fc receptors. Fusions were conducted using BALB/c mice hyperimmunised with equine peripheral blood mononuclear cells. Hybridoma supernatants were screened on the basis of their ability to inhibit the rosetting of equine antibody coated sheep erythrocytes with equine peripheral blood mononuclear cells. The mAbs were then characterised for cellular distribution of the antigen by flow cytometry. Using immunoprecipitation, it was shown that both under reducing and non-reducing conditions, CVS 59 and CVS 61 precipitated a 80 and 40 kD protein, respectively. From the functional study coupled with cellular distribution and molecular weight of the antigen recognised by these mAbs, it would appear that CVS 59 and CVS 61 recognise an epitope on equine equivalent of human and murine FcgammaRIII and FcgammaRII, respectively.     

Opsonization of yeast cells with equine iC3b, C3b, and IgG

The main opsonins in serum are antibodies and complement factor C3. The opsonization mechanisms including complement activation and deposition are important in studies of phagocytosis and of mechanisms of microbial immune evasion. The objective of the present study was to monitor the deposition of complement C3 and IgG from equine serum on yeast cells (Saccharomyces cerevisiae) using a flow cytometric immunoassay. Correlations were made between the opsonic coating and phagocytic capacity using equine blood neutrophils. In addition, the bound C3 fragments were characterized by SDS–PAGE and Western blot analyses.

Opsonic coating of yeast with equine C3 and IgG occurred rapidly with detectable levels with as little as 0.75% serum. C3 deposition was a result of complement activation and no passive adsorption was observed. When complement was inactivated, the fluorescence indicating IgG deposition increased 3–6-fold, indicating spatial competition between C3 and IgG at binding.

Opsonization with 1.5% serum led to suboptimal equine neutrophil phagocytosis of yeast cells which was dependent on complement activation by the classical pathway. With ≥6.25% serum, IgG contributed to opsonization and phagocytosis. With 50% serum and more, C3 was deposited also by the alternative pathway. Phagocytosis rates became optimal with 3% serum, and did not increase further with higher serum concentrations. The main form of C3 on the yeast cells was iC3b and the rest was C3b without any detectable breakdown products (C3c or C3dg). The equine complement components are similar in size to the human equivalents.

It may be concluded that opsonization of yeast particles leading to phagocytosis, occurs at very low serum concentrations (1.5%) and that it is dependent on activation of the classical complement pathway at this low opsonic level. This is an important finding for efficient host defense, e.g. extravascular phagocytosis at infection sites.     

Evaluation of lipopolysaccharide-induced activation of equine neutrophils

OBJECTIVE: To evaluate lipopolysaccharide (LPS)-induced activation of equine neutrophils in blood. SAMPLE POPULATION: Blood samples from 5 healthy adult Thoroughbreds. PROCEDURES: Neutrophil integrin (CD11/CD18) expression, size variation, degranulation, and deformability were measured with and without incubation with LPS. Time and concentration studies were done. The mechanism of endotoxin-induced neutrophil activation was investigated by inactivating complement or preincubating neutrophils with inhibitors of tumor necrosis factor-alpha (TNF-alpha) synthesis, prostaglandin-leukotriene synthesis, or platelet-activating factor. RESULTS: Incubation of equine neutrophils with LPS increased cell surface expression of CD11/CD18, decreased neutrophil deformability, increased and decreased neutrophil size, and induced neutrophil degranulation. The LPS-induced neutrophil activation was attenuated by addition of inhibitors of TNF-alpha and prostaglandin-leukotriene synthesis. CONCLUSIONS AND CLINICAL RELEVANCE: Equine neutrophils are readily activated in vitro by LPS, resulting in increased expression of integrin adhesion molecules, decreased deformability, variation in neutrophil size, and degranulation. The tests used to detect activated neutrophils in this study may be useful in detecting in vivo neutrophil activation in horses with sepsis and endotoxemia.     

Evaluation of Carolina Rinse solution as a treatment for ischaemia reperfusion of the equine jejunum

REASONS FOR PERFORMING STUDY: Ileus and peritoneal adhesions are the most common complications following surgery for small intestinal obstruction. Carolina Rinse (CR) has been shown to decrease reperfusion injury in intestine and other organs. HYPOTHESIS: CR decreases intestinal inflammation and subsequent scarring associated with reperfusion injury. METHODS: CR was infused intra-arterially and applied topically just prior to reperfusion in jejunum exposed to experimental ischemia. Vascular permeability, neutrophil accumulation and serosal scarring were compared in treated and untreated intestine. RESULTS: CR maintained a normal osmotic reflection coefficient and decreased migration of neutrophils into the serosa during reperfusion. After 10 days, treated intestine was normal in appearance with a trend toward less serosal scarring and fibroblast proliferation. There was a significant decrease in fibroplasia at biopsy sites in treated intestine. CONCLUSIONS: Arterial perfusion combined with topical application of CR during jejunal ischaemia decreases immediate reperfusion injury and limits post operative scarring. POTENTIAL RELEVANCE: CR should be used as a local perfusate rather than a systemic treatment; it may best be applied topically and intraluminally to avoid damaging mesenteric arteries. CR should be considered an adjunct treatment as part of overall surgical management and post operative care.     

马属动物附红虫病的诊断与防治

马属动物附红虫是寄生于马类家畜血液里 ,附着在红细胞表面的一种多型性单细胞微生物。该病在临床上主要表现为黄疸、贫血和发热 ,并继发其它疾病。1　流行病学本病主要是由节肢动物传播 ,在自然界里主要由虱、蚤、螨蜱等传播 ,也可通过病畜和带虫者的血传播 ,还可通过被污染的针头、器械感染。同群马类畜感染率为 1 0 0 %。据有关报道 ,本病病原体十分脆弱 ,对干燥与化学药品的抵抗力很低 ,一般的消毒药几分钟即可杀死。2　临床症状一般病畜精神不振 ,食欲减退 ,逐渐乏瘦 ,重症病畜鼻流浆液性粘液 ,口吐白沫 ,可视粘膜苍白并黄染 ,有的病畜…     

Systemic antibody response in colostrum-deprived pigs experimentally infected with Haemophilus parasuis

The serum antibody response to an experimental infection by Haemophilus parasuis, the etiological agent of Glässer’s disease in pigs, was characterized by ELISA measuring IgM and IgGt levels against whole-cells and outer-membrane-proteins (OMPs) as antigens. Five groups of pigs were studied, four of those were previously immunized with different formulations, and the fifth was maintained as non-immunized control. All groups were challenged with 5 × 10⁹ CFU of H. parasuis. The non-commercial bacterin induced a full protection against disease, the OMP-vaccine and the exposure to a sublethal dose of 10⁵ CFU protected only partially, and the recombinant TbpB-vaccine conferred no protection. The humoral response in the pigs that died after infection (all controls, all those vaccinated with the recombinant TbpB, and two of both those inoculated with OMPs and those exposed to the sublethal dose) could be only measured before it, but it was irrelevant in all cases. However, a specific IgM and IgGt production was observed before challenge in all the surviving pigs, irrespective of the type of immunization received. This antibody response was even greater after H. parasuis infection, especially in those survivors receiving the sublethal dose. These results suggest a role of the antibodies developed after the different immunization protocols in preventing infection and death; therefore, the humoral immunity is protective against experimental Glässer’s disease.