Elucidating the role of ApxI in hemolysis and cellular damage by using a novel apxIA mutant of Actinobacillus pleuropneumoniae serotype 10

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin-resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.     

In vitro Inhibition of Canine Complement‐Mediated Hemolysis

Background

Immune‐mediated hemolytic anemia (IMHA) is the most common hematologic immune‐mediated disease in dogs. Complement fixation on erythrocytes causes hemolysis. Complement inhibition decreases hemolysis in people with the hemolytic disease and also may prove effective in treating IMHA in dogs.

Hypothesis/Objectives

Evaluate the in vitro efficacy of 2 complement inhibitors used in humans against canine complement.

Methods

The inhibitory activity of the C3‐inhibitor compstatin and recombinant human C1‐esterase inhibitor (C1‐INH) was evaluated using an in vitro hemolytic assay and spectrophotometric measurement of released hemoglobin. Dose‐response curves for each inhibitor were generated.

Results

Compstatin decreased approximately 50% of canine complement‐mediated hemolysis in initial experiments. This inhibition largely was lost when a new lot of drug was purchased. C1‐INH showed a dose‐dependent inhibition. The highest concentration of C1‐INH tested (500 μg/mL) decreased >80% of canine complement‐mediated hemolysis, and the lowest concentration tested (31.25 μg/mL) decreased hemolysis >60%.

Conclusions and Clinical Importance

Human C1‐INH is a robust inhibitor of canine complement‐mediated hemolysis, whereas compstatin was minimally and variably effective. Human C1‐INH may substantially decrease complement‐mediated hemolysis in dogs with IMHA and warrants further investigation.     

Protection of Clitoria ternatea flower petal extract against free radical-induced hemolysis and oxidative damage in canine erythrocytes

The present study assessed the antioxidant activity and protective ability of Clitoria ternatea flower petal extract (CTE) against in vitro 2,2′-azobis-2-methyl-propanimidamide dihydrochloride (AAPH)-induced hemolysis and oxidative damage of canine erythrocytes. From the phytochemical analysis, CTE contained phenolic compounds, flavonoids, and anthocyanins. In addition, CTE showed antioxidant activity as measured by oxygen radical absorbance capacity (ORAC) method and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. CTE (400 µg/ml) remarkably protected erythrocytes against AAPH-induced hemolysis at 4 h of incubation. Moreover, CTE (400 µg/ml) reduced membrane lipid peroxidation and protein carbonyl group formation and prevented the reduction of glutathione concentration in AAPH-induced oxidation of erythrocytes. The AAPH-induced morphological alteration of erythrocytes from a smooth discoid to an echinocytic form was effectively protected by CTE. The present results contribute important insights that CTE may have the potential to act as a natural antioxidant to prevent free radical-induced hemolysis, protein oxidation and lipid peroxidation in erythrocytes.     

Identification and characterization of serum complement activity in the American crocodile (Crocodylus acutus)

Incubation of unsensitized sheep red blood cells with serum from the American crocodile (Crocodylus acutus) resulted in a concentration-dependent hemolysis. The hemolytic activity was heat-sensitive, and inhibited by EDTA in a concentration-dependent manner. The EDTA-inhibited SRBC hemolysis could be restored by the addition of excess Ca²⁺ or Mg²⁺, but not Ba²⁺ or Cu²⁺, revealing the specificity of this activity for these two divalent cations. The hemolytic activity of crocodile serum was titer-dependent, with 329 μL producing 50% of maximal SRBC hemolysis. The complement activity was also temperature-dependent, with decreased activity at lower temperatures (5–15 °C) and maximal activity occurred at 30–40 °C. The hemolysis occurred relatively slowly, with near zero activity after 10 min, 40% of activity observed within 15 min of exposure to SRBCs, and maximal activity at 30 min.     

The hemolytic activity of bracken extracts in guinea pigs

This study was conducted to elucidate the hemolytic activity of a new toxic substance in bracken fern. A crude extract (CE) was prepared from the methanol extracts of bracken by the column chromatography. When the CE was injected subcutaneously in guinea pigs, the hemoglobinuria and hemolysis were observed within 6 hr, and 3 days later edema and hemorrhages in the urinary bladder were observed. The CE was then fractionated by high performance liquid chromatography (HPLC), and three (HF, BF and CF) of the fractions showed the toxic activities in guinea pigs. The HF caused the hemolysis, whereas both the BF and the CF caused the hemorrhagic cystitis without any hemolytic activities. The HF was further fractionated by the HPLC, resulting of the 3 fractions (HF-I, II and III). The hemolysis was caused only with the HF-II, and HF-II as well as HF did not cause the hemorrhagic cystitis. HPLC analysis revealed that both BF and CF contains braxin B and braxin C, respectively, and both HF and HF-II do not contain braxin A, B or C. These facts suggest that bracken fern contains a new toxic substance (hemolysin) which induces the acute hemolysis in guinea pigs.     

Hemolytic anemia,spherocytosis, and thrombocytopenia associated with honey bee envenomation in a dog

This case report describes a massive honey bee envenomation in a 14‐month‐old male Belgian Malinois dog from St. Kitts, West Indies. Acute and delayed onsets of hemolytic anemia, echinocytosis, spherocytosis, thrombocytopenia, hemoglobinemia, and hemoglobinuria developed following envenomation. The dog recovered after treatment with glucocorticoids and supportive therapy. Spherocytosis, hemolysis, and thrombocytopenia in patients with massive bee envenomation are likely due to the direct toxic effects of the primary components of bee venom, melittin and phospholipase A₂ (PLA₂). Mellitin causes hemolysis by forming large pores in erythrocytes resulting in leakage of hemoglobin and also causes spectrin stiffening and resultant echinocyte and spherocyte formation. Melittin also stimulates PLA₂, a hydrolase that causes echinocytosis and spherocytosis, in vivo and in vitro, and mitochondrial breakdown in platelets. However, delayed manifestations could be attributed to immune‐mediated mechanisms from the generation of antibodies against damaged erythrocytes and platelet membrane proteins.     

Hemolytic activity of Trichomonas gallinae isolates does not correspond with clinical virulence

The hemolytic activity of 22 Trichomonas gallinae isolates was investigated using an 18 h erythrocyte hemolysis assay which has been shown to correlate with the clinical virulence of T. vaginalis. Absorbance of the assay supernatants was measured at 540 nm and expressed as percentage of complete hemolysis. Mean hemolytic activity of the T. gallinae isolates ranged from 3.5% to 53.4% and did not correspond with clinical virulence. The results of this investigation suggest hemolytic activity is not a useful in vitro virulence assay for T. gallinae.     

Hemolytic activity of Fusobacterium necrophorum culture supernatants due to presence of phospholipase A and lysophospholipase.

Culture supernatants of Fusobacterium necrophorum demonstrated hemolytic activity. The hemolysin(s), which was partially purified by ammonium sulfate precipitation, was temperature-dependent and heat labile. The spectrum of hemolytic activity against various erythrocytes included rabbit, human, and dog erythrocytes. Goats, sheep, and bovine erythrocytes showed only trace hemolysis. According to results of thin-layer chromatography, the hemolysin hydrolyzed rabbit erythrocyte phosphatidyl choline, phosphatidyl ethanolamine, lysophosphatidyl choline, and bovine phosphatidyl choline. Hydrolysis of egg yolk phosphatidyl choline, bovine phosphatidyl ethanolamine, cholesterol, 1,2-dipalmitin, 1,3-dipalmitin, sphingomyelin, or triolein was not detected by thin layer chromatography. A more sensitive procedure utilizing gas-liquid chromatography revealed that, of the substrates tested, the following were bein hydrolyzed: bovine and egg yolk phosphatidyl choline, lysophosphatidyl choline, alpha-palmito-beta-eleoyl-L-alpha lecithin and alpha-oleoyl-betal-palmitoyl-L-alpha lecithin. Substrates which were weakly hydrolyzed were bovine phosphatidyl ethanolamine, DL-alpha-hosphatidyl ethanolamine dipalmitoyl, 1,2-dipalmitin, 1,3-dipalmitin, and triolein.     

The use of a passive hemolysis system to evaluate the complement activities of six mammalian species

A passive hemolysis assay system was developed which permitted comparisons of the hemolytic activities of complement (C) from six species. This system employs a single antigen and an antiserum raised in one species. Thus, variations resulting from different target antigens and those inherent in using antibodies (of different affinities and isotypes) raised in a variety of species were minimized. Of the erythrocytes (E) examined, those from horses and guinea pigs were most susceptible to lysis, and either would be suitable, as a tentative choice, for measuring C activity of a previously unstudied species. Horse serum had the lowest C activity of any of the sera tested. It lysed certain cells only at high concentrations, and the hemolytic activity dropped off sharply with minimal dilution. The data presented in this paper could also be used for selecting target E for C studies using direct lysis with antibodies raised against the cells.     

Evaluation of heat-sensitive, neutrophil-toxic, and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae

Cytotoxic and hemolytic activity of Haemophilus (Actinobacillus) pleuropneumoniae serotype 1 strain CM5 was investigated because of the potential role as a virulence determinant. Viable bacteria were toxic for porcine and bovine neutrophils, whereas bacteria killed by heat treatment at 60 C for 1 hour were not. Similarly, bacteria-free culture supernatant was cytotoxic and hemolytic in assays that used porcine neutrophils and erythrocytes, whereas supernatant treated at 60 C for 1 hour had no activity. Erythrocytes from various species were susceptible to the hemolytic activity of bacteria-free culture supernatant, with ovine and bovine erythrocytes being most sensitive. The neutrophil-toxic and hemolytic activity of bacteria-free culture supernatant was inhibited by cholesterol and oxygen and abolished after trypsin digestion. The neutrophil-toxic and hemolytic activity was preserved during storage at or less than 4 C, but was lost rapidly at 56 C or 80 C. Neutralizing antibodies were demonstrated in serum of pigs and rabbits immunized with 10-fold concentrated culture supernatant of strain CM5 and in field pigs that had recovered from natural infection with H pleuropneumoniae serotype 1. Bacteria-free culture supernatants of 18 strains, including H pleuropneumoniae serotypes 1 through 10, Actinobacillus suis, and Haemophilus taxon minor group, were tested for heat-sensitive, neutrophil-toxic, and hemolytic activity. Fifteen strains were neutrophil toxic, but only 10 of these were hemolytic. Haemophilus pleuropneumoniae, serotype 1, strain VLS557; serotype 5, strain K17; and Haemophilus taxon minor group strain 33PN were neither cytotoxic nor hemolytic.     

Acute intravascular hemolytic anemia in the black rhinoceros: hematologic and immunohematologic observations

To investigate the syndrome of acute intravascular hemolytic anemia in the black rhinoceros (Diceros bicornis), laboratory techniques used in the differential diagnosis of hemolytic anemias were performed on blood samples from 6 black rhinoceroses: 3 nonrelated healthy rhinoceroses, 1 rhinoceros with iron deficiency anemia, and 2 rhinoceroses with intravascular hemolysis. Osmotic fragility, erythrocyte membrane protein composition, hemoglobin electrophoresis, and hemoglobin stability did not distinguish between healthy and affected (anemia or hemolysis) rhinoceroses. Polyclonal antiglobulin reagents were prepared in rabbits, using whole rhinoceros serum and purified rhinoceros immunoglobulin G. These reagents were nonreactive against erythrocytes of the healthy and iron-deficient rhinoceroses. Reactions with RBC from the rhinoceros with fatal hemolytic anemia indicated increased membrane coating by the third component of complement; this was not evident in a second rhinoceros that survived a hemolytic event.     

Purification and properties of cholesterol oxidase and choline phosphohydrolase from Rhodococcus equi.

Cholesterol oxidase (CO) and choline phosphohydrolase (CPH) exoenzymes were isolated from culture supernatants of Rhodococcus equi ATCC 33701 and their hemolytic and cytotoxic activities examined. The purifications involved differential ammonium sulphate precipitation, ion exchange and gel filtration chromatography. A purification of 32.8-fold and a yield of 0.3% of CO were determined by synergistic hemolysis of sheep red blood cells (SRBC) presensitized with Staphylococcus aureus beta toxin. The enzymatic activity of CO was also demonstrated by oxidation of aqueous cholesterol suspensions. The activity of CO was reversibly inhibited by concentration. A purification of 412.4-fold and a yield of 1.7% of CPH were determined by hydrolysis of p-nitrophenyphosphorylcholine. Purity of both exoenzymes was confirmed by immunoblotting. On sodium dodecyl sulphate polyacrylamide gel electrophoresis, the CO had a molecular mass (Mr) of 60 kd and the CPH a Mr of 65 kd. Choline phosphohydrolase did not hydrolyse sphingomyelin. Sphingomyelinase C (SMC) activity was however demonstrated in concentrated culture supernatants. This dissociation of SMC from CPH activity indicates that R. equi produces two distinct phospholipase C exoenzymes, a CPH and a SMC. Both CO and CPH combined, or individually, did not lyse native SRBC even with subsequent chilling of the cells at 4 degrees C ("hot-cold" treatment). Purified CO lysed beta toxin-sensitized SRBC. The CPH showed only minor hemolytic activity against such sensitized SRBC even at high concentrations. Combination of CO and CPH in lysis of beta toxin sensitized SRBC showed only minor additive rather than synergistic effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibody-dependent cell-mediated cytotoxicity in sheep

The ability of sheep leukocytes to mediate antibody — dependent cell-mediated cytotoxicity (ADCC) and that of sheep serum IgG₁ and IgG₂ to induce ADCC were investigated. Partial characterization of effector cells was attempted. These investigations revealed that ADCC occurs in sneep. With chicken erythrocytes (CRBC) as the target cells, polymorphonu-cleated cells (PMN), and monocytes, were the most effective leukocytes. Ovine peripheral blood lymphocytes (PBL) also mediated ADCC, and within the PBL population, T-cells were capable of mediating ADCC. The T-cells were obtained by nylon wool fractionation and selective agglutination by peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA). Both nylon wool adherent and non-adherent fractions were active in ADCC, although the former were more active, implying heterogeneity in nylon wool adherence among ovine K-cells. Depletion of B (SIg⁺) cells did not affect ADCC activity of the remaining cells. Depletion of Fc⁺ cells markedly reduced cytotoxic activity of PBL. Both sheep IgG₁ and IgG₂ anti-CRBC immunoglobulins were able to induce ADCC.     

Effects of Moraxella bovis and culture filtrates on 51Cr-labeled bovine neutrophils

The cytotoxicity of Moraxella bovis whole cells and culture filtrates was studied, using 51Cr-labeled bovine and human blood neutrophils. The cytotoxicity of living M bovis was directly related to the concentration of bacteria in the neutrophil cultures, and was maximal at an approximate neutrophil to bacteria ratio of 1:10. Cytotoxicity was maximal by 30 minutes after living bacteria were added to the suspension of the 51Cr-labeled neutrophils. Expression of the cytotoxicity was dependent on the presence of Ca2+ in the media, and was independent of the presence of Mg2+. Cytotoxic activity was eliminated by inactivating M bovis in buffers containing formalin or sodium azide. Hemolytic and nonhemolytic isolates of M bovis were examined for cytotoxic activity. All 7 of the hemolytic isolates were cytotoxic for bovine neutrophils, but all 4 of the nonhemolytic isolates were devoid of cytotoxic activity. None of the 11 isolates were cytotoxic for human neutrophils. Sterile filtrates from 6-hour shaker cultures of a hemolytic M bovis isolate were cytotoxic for bovine neutrophils. Cytotoxicity of the filtrate was eliminated by heating, incubation with trypsin, or addition of EDTA to the media. Bacterial homogenates or sterile filtrates prepared from statistically incubated cultures of M bovis were not toxic for bovine neutrophils.     

Heinz body hemolytic anemia with eccentrocytosis from ingestion of Chinese chive (Allium tuberosum) and garlic (Allium sativum) in a dog

A 4-year-old, intact male miniature schnauzer was presented with anorexia. The dog had ingested some Chinese steamed dumplings 2 days before, which contained Chinese chive (Allium tuberosum) and garlic (Allium sativum). Hematological examinations revealed severe Heinz body hemolytic anemia with eccentrocytosis and an increased concentration of methemoglobin, which was thought to result from oxidative damage to erythrocytes by constituents in these Allium plants. In this case, eccentrocytosis was a hallmark finding and could be detected easily, suggesting that this hematological abnormality is useful in diagnosing Allium plant-induced hemolysis.     

Cholinesterase and Phosphatase Activities in Adults and Infective-stage Larvae of Levamisole-resistant and Levamisole-susceptible Isolates of Haemonchus contortus

Cholinesterase (ChE) and acid phosphatase (AP) activities, but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of adult and infective-stage larvae of levamisole-resistant and levamisole-susceptible Haemonchus contortus. In contrast to other gastrointestinal nematodes, the ChE activity was higher in L₃ than in adults and, in both cases, was mainly associated with membranes. ChE activity was inhibited by Triton X-100 and was only detected in membrane-bound fractions when the detergent was removed. Differences between resistant and susceptible L₃ were observed in the response to inhibitors (cytosolic fraction) and in the enzymatic content (membrane-bound fraction). Phosphatase activity was detected at acidic pH in all fractions, being higher in the adult than in the L₃ stage. In the former, most of the enzyme was localized in the membrane-bound fractions, whereas in the latter it was mainly in cytosolic fractions. This difference could be correlated with the activity in the gut. In inhibition assays, a difference between cytosolic fractions from resistant and susceptible adults was observed in their response to 1 mmol/L tartaric acid.     

Enterotoxigenicity,hemagglutination and cell-surface hydrophobicity in Aeromonas hydrophila,A. Sobria and A. Salmonicida

Thirty-one Aeromonas hydrophila, 13 A. sobria and two A. salmonicida strains of diverse sources were tested for enterotoxigenicity, hemagglutination and cell surface hydrophobicity. Although 93% of the culture supernatant fluids of the Aeromonas strains exhibited cytotoxic effects on Y1 adrenal and Chinese hamster ovary (CHO) cells, typical rounding of Y1 adrenal cells was reproducibly observed before cytotoxicity for 80% of the isolates within 1 h of exposure.Twenty-eight strains were positive for delayed permeability factor (DPF) activity in rabbit skin. Culture filtrates of 16 of 20 strains that were positive both in the Y1 adrenal cell test and for DPF activity elicited fluid accumulation in rabbit ileal loops. The DPF and ileal loop activities were neutralizable by cholera antitoxin. All, except two strains each of A. sobria and A. hydrophila, produced a heat-stable, rapid permeability factor (RPF) detected in rabbit skin. Heat-treated culture supernatant fluids of two A. hydrophila and one A. sobria isolate gave positive responses in the infant mouse assay. Nine other strains gave borderline reactions.When A. hydrophila and A. sobria isolates were grown in broth, approximately 90% agglutinated bovine, chicken, human group A and guinea-pig erythrocytes in the presence of mannose at 4°C and/or 20°C. The two A. salmonicida isolates produced mannose resistant hemagglutination (MRHA) of these four blood types.Hydrophobic interaction chromatography indicated adhesive potential in 61% A. hydrophila and 100% A. sobria strains expressing weak to strong hydrophobic cell surface properties. The results of these investigations strongly imply that the Aeromonas strains produce a cytotonic enterotoxin immunologically related to cholera toxin. Adhesive characteristics were commonly found in both clinical and routine isolates.     

Antiviral activity against transmissible gastroenteritis virus, and cytotoxicity, of natural porcine interferons alpha and beta.

Porcine interferon (POIFN)-alpha prepared in primed peripheral blood leukocyte cultures induced with Newcastle disease virus and POIFN-beta from PK-15 cell cultures induced with polyinosinic:polycytidylic acid were partially purified by precipitation with potassium thiocyanate and anion exchange chromatography. Mean purification factors in terms of units of POIFN per mg of protein, of 37 and 12 were obtained for POIFN-alpha and POIFN-beta respectively. In yield reduction assays in swine testis and pig kidney cell cultures, POIFN-alpha and POIFN-beta had greater antiviral activity against vesicular stomatitis virus than against transmissible gastroenteritis virus (TGEV). The antiviral effects were greater at higher concentrations of interferon (IFN), and when the IFN treatments were continued postinfection. Porcine interferon-beta showed greater antiviral activity against TGEV than POIFN-alpha, but this may have been partly due to cytotoxicity. There were no major differences in the antiviral activities of crude and partially purified IFN preparations. Both types of IFN showed antiviral activity against TGEV in yield reduction assays in porcine intestinal explant and intestinal epithelial cell cultures. Crude POIFN-beta was found to be rapidly cytotoxic, especially in porcine cells, and some fractions of partially purified POIFN-beta were also cytotoxic. The cytotoxicity of POIFN-beta was partially neutralized by antibodies against human IFN-beta, but human IFN-beta was not cytotoxic for porcine or bovine cells.     

Suppressive effect of culture supernatant of erythrocytes and serum from dogs infected with Babesia gibsoni on the morphological maturation of canine reticulocytes in vitro

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5''-nucleotidase subclass I and purine-specific 5''-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5''-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.     

Effects of various treatments of bovine complement on its lytic efficacy measured by two different tests

Bovine complement was treated with various agents known to activate or inactivate one or more of the cascade components. The treated complement was then assessed for remaining hemolytic activity by a tube titration test and a radial hemolysis method. Divalent cation chelators (EDTA and EGTA); immune complexes prepared with serum and IgM, IgG1, IgG2, and IgA isotypes; smooth and rough lipopolysaccharides and lipid A; hydrazine; zymosan; cobra venom factor and brown recluse spider venom caused depletion of complement as determined in the tube titration test. Immune complexes (prepared with serum); hydrazine; cobra venom factor; EDTA and smooth lipopolysaccharide caused loss of hemolytic activity in the radial hemolysis test. This evidence suggests that the radial hemolysis test assessed complement consumed by the alternate pathway, while the tube titration method measured classical pathway consumption.