黄斑蓝子鱼皮肤黏液对刺激隐核虫及一些病原菌的抑杀作用

黄斑蓝子鱼(Siganus oramin)具有天然抗刺激隐核虫(Cryptocaryon irritans)能力, 并从其血清中分离纯化了一种抗虫蛋白。以黄斑蓝子鱼皮肤黏液为对象, 研究了其对刺激隐核虫(C. irritans)、多子小瓜虫(Ichthyophthirius multifiliis)、布氏锥虫(Trypanosoma brucei brucei)及一些病原菌的杀灭和抑制效果。结果显示: 黄斑蓝子鱼黏液对刺激隐核虫、多子小瓜虫和布氏锥虫均具有明显的杀虫效果, 黏液蛋白对3种寄生虫的最低杀寄生虫浓度(Minimum Parasiticidal Concentration, MPC)分别为4.0、5.0和3.0 mg/mL。显微镜观察发现, 刺激隐核虫和多子小瓜虫的幼虫在经过黄斑蓝子鱼黏液作用后, 均出现纤毛脱落、虫体肿胀、外膜破裂和内容物泄漏等现象。黏液抑菌活性实验结果表明, 除大肠杆菌(Escherichia coli)外, 黄斑蓝子鱼皮肤黏液对金黄色葡萄球菌(Staphylococcus aureus)、霍乱弧菌(Vibrio cholerae)、海豚链球菌(Streptococcus iniae)、温和气单胞菌(Aeromonas sobria)、溶藻弧菌(Vibrio alginolyticus)、副溶血弧菌(Vibrio Parahaemolyticus)、星形诺卡氏菌(Nocadia asteroides)和美人鱼发光杆菌杀鱼亚种(Photobacterium damselae subsp.)皆有明显的抑菌效果。在最低抑菌浓度(Minimum Inhibitory Concentration, MIC)测定中, 黄斑蓝子鱼黏液对金黄色葡萄球菌、霍乱弧菌、海豚链球菌、溶藻弧菌、温和气单胞菌和副溶血弧菌的MIC 分别为0.16、0.16、0.08、0.63、0.63和0.63 mg/mL。黄斑蓝子鱼皮肤黏液对小麦赤霉菌(Gibberella saubinetii)和黑曲霉菌(Aspergillus niger)没有明显抑菌作用。       

Chemoattraction of Ichthyophthirius multifiliis (Ciliophora) theronts to host molecules

Mechanisms in the host-finding process of Ichthyophthirius multifiliis were studied in vitro by a novel bioassay using 24-well multidishes supplied with bottom layers of agar with chemoattractants. It was shown that low molecular weight molecules (carbohydrates, amino acids, fatty acids, urea) did not attract theronts. In contrast, sera and mucus from a range of teleosts (including marine fish) were effective attractants. Fractionation by gel filtration of fish serum allowed determination of the molecular size of the attracting proteins. Further biochemical studies suggested the chemoattractants to be present in fractions with host immunoglobulin and some still undetermined proteins. No clear association between enzyme activity and chemotactic potential was seen. The high chemoattractive effect of serum from various unrelated teleosts corresponds to the low host specificity of I. multifiliis and suggests that serum factors in mucus could be involved in host finding of the parasite. Society for Parasitology Inc.     

Antiprotozoan activity of nonspecific cytotoxic cells (NCC) from the channel catfish (Ictalurus punctatus)

Nonspecific cytotoxic cells (NCC) obtained from channel catfish (Ictalurus punctatus) kill Tetrahymena pyriformis, an opportunistic parasite in fish. Based upon this fact, a new mechanism for nonspecific cellular anti-parasitic immunity in fish is proposed. Optimum in vitro conditions for NCC killing of deciliated T. pyriformis were first obtained. Lysis of T. pyriformis by NCC occurred by 10 hr of cocultivation of effector and target cells. During this time period, 50 to 60% cytotoxicity occurred. Fish anti-T. pyriformis serum enhanced NCC killing of T. pyriformis either by prolonging immobilization (after the cilia regeneration period) or by delaying cilia regeneration. Shared antigenic determinants between T. pyriformis, Ichthyophthirius multifiliis, and NC-37 target cells were demonstrated by binding-depletion experiments. For these studies, NCC were depleted from anterior kidney cells (the hemopoetic organ in fish) by preincubating formalin-treated T. pyriformis, I. multifiliis, or viable NC-37 target cells with NCC for 3 hr. Conjugates of effector and target cells were removed by overlaying on fetal bovine serum. Unconjugated fish anterior kidney cells were tested for cytotoxic activity against NC-37 or T. pyriformis target cells. Cold target inhibition experiments by using a 4-hr 51chromium cytotoxicity assay also demonstrated these shared antigenic determinants. Target-specific antisera, used to mediate the killing of T. pyriformis by NCC, were required only for immobilizing the targets, and did not function in an antibody-dependent cell-mediated (ADCC)-like mechanism. Scanning electron micrographs of NCC-T. pyriformis conjugates additionally demonstrated NCC binding to both cilia and cell surface determinants.     

Partial cross protection against Ichthyophthirius multifiliis in Gyrodactylus derjavini immunized rainbow trout.

Partial cross protection against a skin-parasitic ciliate has been recorded in rainbow trout previously immunized with an ectoparasitic platyhelminth. The susceptibility to infection by Ichthyophthirius multifiliis differed significantly between naive and Gyrodactylus derjavini immunized rainbow trout. Fish partly immune to the ectoparasitic monogenean G. derjavini became less infected and experienced lower mortality than naive fish when exposed to I. multifiliis infections. In vitro studies on immobilization of theronts using decomplemented (heat-inactivated) serum from G. derjavini immune or non-immune hosts showed no immobilization. However, untreated serum from both immune and non-immune fish containing intact complement immobilized theronts (titre 128-256). In addition, non-specific priming of the host response with interleukin (IL-1), bacterial lipopolysaccharide (LPS), concanavalin A (Con A) or mannan did confer a partial resistance to I. multifiliis infection. This will suggest that non-specific factors including complement could be partly responsible for the host response against infections with this ciliate.     

Tetrahymena pyriformis as a protective antigen against Ichthyophthirius multifiliis infection: comparisons between isolates and ciliary preparations

Channel catfish, Ictalurus punctatus , were immunized with cilia from three isolates of Tetrahymena pyriformis and challenged with Ichthyophthirius multifiliis using a reproducible quantitative procedure. Two different methods of deciliation were used in antigen preparation. Results indicate that T. pyriformis cilia elicit an immune response in channel catfish against I. multifiliis , and that the protective ability of the cilia varies between T. pyriformis strains.     

The I-antigens of Ichthyophthirius multifiliis are GPI-Anchored Proteins

The parasitic ciliate Ichthyophthirius multifiliis has abundant surface membrane proteins (i-antigens) that when clustered, trigger rapid, premature exit from the host. Similar antigens are present in free-living ciliates and are GPI-anchored in both Paramecium and Tetrahymena. Although transmembrane signalling through GPI-anchored proteins has been well-documented in metazoan cells, comparable phenomena have yet to be described in protists. Since premature exit of Ichthyophthirius is likely to involve a transmembrane signalling event, we sought to determine whether i-antigens are GPI-anchored in these cells as well. Based on their solubility properties in Triton X-114, the i-antigens of Ichthyophthirius are amphiphilic in nature and partition with the detergent phase. Nevertheless, following treatment of detergent lysates with phospholipase C, the same proteins become hydrophilic. Concomitantly, they are recognized by antibodies against a cross-reacting determinant exposed on virtually all GPI-anchored proteins following cleavage with phospholipase C. Finally, when expressed in recombinant form in Tetrahymena thermophila, full-length i-antigens are restricted to the membrane, while those lacking hydrophobic C-termini are secreted from the cell. Taken together, these observations argue strongly that the i-antigens of Ichthyophthirius multifiliis are, in fact, GPI-anchored proteins.     

Differences in initial and acquired resistance to Ichthyophthirius multifiliis between populations of rainbowfish

Wild-caught rainbowfish Melanotaenia spp. originating from three isolated populations were infected with a quantified dosage of parasites Ichthyophthirius multifiliis in a controlled environment. The Melanotaenia eachamensis from Dirran Creek were much more susceptible to ichthyophthiriasis than were M. splendida from the Lake Tinaroo or Bluewater Creek populations. When the highly susceptible Dirran Creek rainbowfish were crossed with rainbowfish from a fourth population, Lake Eacham M. eachamensis , they produced hybrids with significantly higher resistance than pure-bred Dirran Creek, but not higher than pure-bred Lake Eacham fish. Hence, intraspecific hybridization increased resistance to I. multifiliis infection in M. eachamensis. Hosts from all three populations were much less susceptible to infection on their second exposure to the parasite. However, the Bluewater Creek population was better able to acquire immunity to I. multifiliis than either the Dirran Creek or Lake Tinaroo populations. It is tentatively suggested that there may be a link between the heterozygosity of populations of rainbowfish and their initial ability to resist infection by Ichthyophthirius multifiliis.     

African trypanosomes: intracellular trafficking of host defense molecules

Trypanosoma brucei brucei is the causative agent of Nagana in cattle and can infect a wide range of mammals but is unable to infect humans because it is susceptible to the innate cytotoxic activity of normal human serum. A minor subfraction of human high-density lipoprotein (HDL), containing apolipoprotein A-I (APOA1), apolipoprotein L-I (APOL1) and haptoglobin-related protein (HPR) provides this innate protection against T. b. brucei infection. Both HPR and APOL1 are cytotoxic to T. b. brucei but their specific activities for killing increase several hundred-fold when assembled in the same HDL. This HDL is called trypanosome lytic factor (TLF) and kills T. b. brucei following receptor binding, endocytosis, and lysosomal localization. Trypanosome lytic factor is activated in the acidic lysosome and facilitates lysosomal membrane disruption. Lysosomal localization is necessary for T. b. brucei killing by TLF. Trypanosoma brucei rhodesiense, which is indistinguishable from T. b. brucei, is resistant to TLF killing and causes human African sleeping sickness. Human infectivity by T. b. rhodesiense correlates with the evolution of a human serum resistance associated protein (SRA) that is able to ablate TLF killing. When T. b. brucei is transfected with the SRA gene it becomes highly resistant to TLF and human serum. In the SRA transfected cells, intracellular trafficking of TLF is altered and TLF mainly localizes to a subset of SRA containing cytoplasmic vesicles but not to the lysosome. These findings indicate that the cellular distribution of TLF is influenced by SRA expression and may directly determine susceptibility.     

Experimental infection of striped marshfrog tadpoles (Limnodynastes peronii) by Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis, or white spot, is a well known and widely distributed parasite of freshwater fish. However, it is not know whether it can infect other aquatic vertebrates such as amphibians. This study uses a series of laboratory-based experiments to demonstrate that I. multifiliis can infect the tadpole stage of an amphibian, the striped marshfrog (Limnodynastes peronii) of Eastern Australia. The tadpoles did not appear to develop ichthyophthiriasis at low parasite levels (200 parasites per tadpole), but at high parasite levels (2,000 parasites per tadpole) 100% of the tadpoles developed ichthyophthiriasis. This is the first time that it has been demonstrated that I. multifiliis can infect a nonpiscine vertebrate host.     

Changes in the electrolyte composition of the blood and muscles in carp infection with Ichthyophthirius multifiliis (Ciliata, Ophryoglenidae)

A study of the electrolytic contents of blood serum, erythrocytes and muscles of carp infected with Ichthyophthirius multifiliis was carried out. Whatever the infection intensity, a characteristic increase in the cations K+ and Na+ ratio in the plasma and muscles of infected fishes takes place. The above dependence, which is expressed in the intensification of active ions transport, is associated with mobilization of organism's protective functions.     

Comparing tolerance of Ichthyophthirius multifiliis and Tetrahymena thermophila for new cryopreservation methods

Ichthyophthirius multifiliis is an obligate protozoan parasite of freshwater fishes that has a complex developmental cycle. It has not been successfully cryopreserved, so management studies are restricted to parasites obtained during outbreaks or perpetuated by passage in live fishes. To overcome this serious limitation, free-swimming I. multifiliis parasites were tested in a cryopreservation protocol routinely used for a related ciliate, Tetrahymena. In this protocol, I. multifiliis theronts retained infectivity for 3 days, although the protocol itself was ultimately lethal. Exposure of I. multifiliis and Tetrahymena thermophila to a battery of media and cryopreservative reagents showed that I. multifiliis was less hardy than T. thermophila and likely had significant biological and cytoskeletal differences. No combination of reagents, media, freezing rates, or dilution media permitted cryopreservation of I. multifiliis parasites that could then undergo development or infect fish. However, a vitrification protocol was formulated using Ficoll, 1,2-propanediol, and N,N-dimethylacetamide from which intact cryopreserved theronts with some motility were recovered. Understanding the effects of these reagents may lead to both a cryopreservation method for I. multifiliis and to improved understanding of the biology of ciliates.     

Effect of immunization of channel catfish with inactivated trophonts on serum and cutaneous antibody titers and survival against Ichthyophthirius multifiliis

Two trials were conducted to determine the effect of immunization of channel catfish with inactivated trophonts on serum and cutaneous antibody titers and survival against Ichthyophthirius multifiliis Fouquet (Ich). In trial I, catfish were immunized intraperitoneally (IP) with: 1) 1% formalin-inactivated trophonts, 2) 3% formalin-inactivated trophonts and 3) freeze-thawed trophonts. Positive and negative control catfish were immunized with live theronts and 5% bovine serum albumin (BSA), respectively. At day 14, 28 and 50 post-immunizations, no statistical difference was noted in serum or cutaneous anti-Ich antibody titers to formalin-inactivated trophonts or freeze-thawed trophonts. The survival of catfish challenged with live theronts ranged from 33.3% to 43.3% for the formalin-inactivated or freeze-thawed trophonts at 50 d post-immunization. The survival of catfish immunized with live theront and BSA was 93.3 and 0%, respectively. In trial II, catfish were IP immunized with sonicated trophonts at doses of 1) 5 trophonts or 10.2 microg protein g(-1) fish, 2) 10 trophonts or 20.4 microg protein g(-1) fish, 3) 20 trophonts or 40.8 microg protein g(-1) fish, and 4) 5% BSA as the control. Fish immunized with 10 or 20 trophonts g(-1) fish showed highest serum (1/210 to 1/480) and cutaneous antibody titers (1/48 to 1/52), respectively, at 22 d post-immunization and survival (63.3-60.0%). The fish immunized with 5 trophonts g(-1) fish had titers of 1/52 and 1/12 for serum and cutaneous antibody and survival of 23.3%. BSA immunized catfish had background titers and a survival of 6.7%. There was a significant correlation between doses of sonicated trophonts used to immunize and catfish survival (correlation coefficient = 0.859, p < 0.01). These results indicate that doses of sonicated trophonts, but not formalin-inactivated or freeze-thawed trophonts provided both serum and cutaneous antibody responses and survival to live trophont challenge.     

Role of serum in the intracellular killing of staphylococci in rabbit monocytes

Shayegani, Mehdi G. (U.S. Veterans Administration Hospital, Philadelphia, Pa.), and Stuart Mudd. Role of serum in the intracellular killing of staphylococci in rabbit monocytes. J. Bacteriol. 91:1393-1398. 1966.-Although some intracellular killing occurs in rabbit monocytes with heated normal serum or even in monocytes washed three times with Hanks' solution and with staphylococci not exposed to serum, efficient killing of coagulase-positive Staphylococcus aureus cells in the mononuclear phagocytes of rabbits is shown to require heat-labile components of serum. The effect of serum in promoting phagocytosis and intracellular killing may be exhibited either by presensitization of the staphylococcal cells before contact with leukocytes or by the presence of serum in the phagocytic system. Under any conditions studied the rate of intracellular killing of S. aureus is very slow.     

Protection of channel catfish, Ictalurus punctatus Rafmesque, against Ichthyophthirius multifiliis Fouquet by immunization

Channel catfish, Ictalurus punctatus Rafmesque, were immunized with ciliary and whole cell preparations of Ichthyophthirius multifiliis and subsequently challenged with I. multi-filiis . Similar Telrahymena pyriformis preparations were used to determine if this heterologous immunization would elicit a protective immune response against I. mullifiliis infestation. Results indicate that T. pyriformis ciliary antigens provided the greatest degree of protection.     

Serum resistance-associated protein blocks lysosomal targeting of trypanosome lytic factor in Trypanosoma brucei

Trypanosoma brucei brucei is the causative agent of nagana in cattle and can infect a wide range of mammals but is unable to infect humans because it is susceptible to the innate cytotoxic activity of normal human serum. A minor subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I (apoA-I), apolipoprotein L-I (apoL-I), and haptoglobin-related protein (Hpr) provides this innate protection against T. b. brucei infection. This HDL subfraction, called trypanosome lytic factor (TLF), kills T. b. brucei following receptor binding, endocytosis, and lysosomal localization. Trypanosoma brucei rhodesiense, which is morphologically and physiologically indistinguishable from T. b. brucei, is resistant to TLF-mediated killing and causes human African sleeping sickness. Human infectivity by T. b. rhodesiense correlates with the evolution of a resistance-associated protein (SRA) that is able to ablate TLF killing. To examine the mechanism of TLF resistance, we transfected T. b. brucei with an epitope-tagged SRA gene. Transfected T. b. brucei expressed SRA mRNA at levels comparable to those in T. b. rhodesiense and was highly resistant to TLF. In the SRA-transfected cells, intracellular trafficking of TLF was altered, with TLF being mainly localized to a subset of SRA-containing cytoplasmic vesicles but not to the lysosome. These results indicate that the cellular distribution of TLF is influenced by SRA expression and may directly determine the organism's susceptibility to TLF.     

Reproduction of Ichthyophthirius multifiliis (Ciliata, Ophryoglenidae)

A case of reproduction of Ichthyophthirius multifiliis in the superficial tissues of larvae and fry of Amur wild carp is described. At a temperature of water from 28 to 29 degrees trophonts of Ichthyophthirius encysted on fishes. Inside cysts repeated cell division occurred but this process did not result in swarm spores formation. Later on with the increase of temperature to 29.5--31.5 degrees cysts degenerated.     

Heterosis in resistance to Ichthyophthirius multifiliis infections in poeciliid fish

Resistance to experimental infections of Ichthyophthirius multifiliis was investigated in the poeciliid fish Xiphophorus maculatus, X. variatus and their two reciprocal hybrids. A third species, the goodeid Ameca splendens , was infected for comparative purposes. Infection data were analysed using a generalized linear interactive modelling package (GLIM). Infection levels were not significantly influenced by the sex of the fish, temperature, or speciestemperature interactions, but body area and time of infection did affect susceptibility. After accounting for variation due to these factors, it was shown that the two reciprocal hybrids of X. maculatus and X. variatus had significantly lower infection levels than either parental stock. Heterosis, H , was calculated from both raw data (16.2%) and from modelled values (31.3%) after removal of environmental factors. This confirms the existence of genetic factors determining resistance to /. multifiliis infection and has shown that gains in resistance can be made through hybridization.     

Hemoglobin is a co-factor of human trypanosome lytic factor

Trypanosome lytic factor (TLF) is a high-density lipoprotein (HDL) subclass providing innate protection to humans against infection by the protozoan parasite Trypanosoma brucei brucei. Two primate-specific plasma proteins, haptoglobin-related protein (Hpr) and apolipoprotein L-1 (ApoL-1), have been proposed to kill T. b. brucei both singularly or when co-assembled into the same HDL. To better understand the mechanism of T. b. brucei killing by TLF, the protein composition of TLF was investigated using a gentle immunoaffinity purification technique that avoids the loss of weakly associated proteins. HDL particles recovered by immunoaffinity absorption, with either anti-Hpr or anti-ApoL-1, were identical in protein composition and specific activity for T. b. brucei killing. Here, we show that TLF-bound Hpr strongly binds Hb and that addition of Hb stimulates TLF killing of T. b. brucei by increasing the affinity of TLF for its receptor, and by inducing Fenton chemistry within the trypanosome lysosome. These findings suggest that TLF in uninfected humans may be inactive against T. b. brucei prior to initiation of infection. We propose that infection of humans by T. b. brucei causes hemolysis that triggers the activation of TLF by the formation of Hpr-Hb complexes, leading to enhanced binding, trypanolytic activity, and clearance of parasites.     

Chloramine-T for the control of Ichthyophthirim multifiliis (Fouquet)

Tests were conducted to determine the toxicity of chloramine-T to the free-living stages of the protozoan parasite Ichthyophthirius multifiliis (Fouquet) and the fish Rasbora heteromorpha (Duncker) and Rutilus rutilus (L.) with the view to using the reagent to control ichthyophthiriasis. The toxicity was dependent on the p H value of the water and, to a lesser extent, its hardness. A method of treating the disease is proposed and discussed.     

Treatment of ichthyophthiriasis after malachite green. II. Earth ponds at salmonid farms

We tested formalin, chloramine-T-formalin and Desirox-formalin, for use against white spot disease (ichthyophthiriasis) caused by Ichthyophthirius multifiliis at 3 salmonid farms (Salmo salar and S. trutta smolt reared in earth ponds). I. multifiliis disappeared from most individuals 4 to 5 wk after the first treatment (and after the first I. multifiliis were found) with all chemicals, indicating that combinations of these chemicals, and even formalin alone, can be used to lower the parasite burden in earth ponds to such a level that no mortality occurs. This was the case when the fish were treated frequently at the beginning of the infection. Treatment can be stopped once the fish have achieved immunity to ichthyophthiriasis. The developing immunity was also revealed by the distribution of ciliates in the course of the disease. At the beginning of the infection I. multifiliis individuals were randomly distributed among the fish, but after 2 to 3 wk, when all the fish were infected, ciliates had increased in numbers and were aggregated in such a way that some fish carried quite heavy burdens. However, over 60% of the fish were free of the parasites after 4 to 5 wk, and had few or no ciliates, meaning that the distribution was even more aggregated. Sea trout had fewer parasites than salmon, and they also recovered from infection earlier even though the treatments and ponds were similar, indicating variation in resistance to I. multifiliis between fish species. It was also evident that the chemicals and their concentrations must be planned carefully to suit the conditions at each farm.