Ichthyophthirius multifiliis as a potential vector of Edwardsiella ictaluri in channel catfish

There is limited information on whether parasites act as vectors to transmit bacteria in fish. In this trial, we used Ichthyophthirius multifiliis and fluorescent Edwardsiella ictaluri as a model to study the interaction between parasite, bacterium, and fish. The percentage (23-39%) of theronts fluorescing after exposure to E.?ictaluri was significantly higher than control theronts (~?6%) using flow cytometry. Theronts exposed to E.?ictaluri at 4?×?10(7) CFU?mL(-1) showed a higher percentage (~?60%) of fluorescent theronts compared to those (42%) exposed to 4?×?10(3) CFU?mL(-1) at 4?h. All tomonts (100%) carried the bacterium after exposure to E.?ictaluri. Edwardsiella ictaluri survived and replicated during tomont division. Confocal microscopy demonstrated that E.?ictaluri was associated with the tomont surface. Among theronts released from tomonts exposed to E.?ictaluri, 31-66% were observed with attached E.?ictaluri. Sixty percent of fish exposed to theronts treated with 5?×?10(7) E.?ictaluri?mL(-1) were positive for E.?ictaluri at 4?h as determined by qPCR or fluorescent microscopy. Fluorescent E.?ictaluri were observed on trophonts in skin and gill wet mounts of dead fish. This study demonstrated that Ich could vector E.?ictaluri to channel catfish.     

Fate of fluorescent microspheres in developing Ictalurus punctatus following prolonged immersion

Particulate antigen uptake by the mucosa of developing channel catfish was determined by immersing larvae and fry [2-day post-hatch (dph), 1-, 2-, 3-, 4-, and 8-week post-hatch (wph)] to two forms of fluorescent microspheres (FMS): blue FMS were carboxylated, and green FMS were coated via conjugation with a crude extract of Edwardsiella ictaluri outer membrane protein (OMP). Phagocytosis, destination, and clearance appeared similar for the two types of FMS used. In the older age classes, primary uptake was observed in epithelial cells of the torso, fins, nares and to a lesser extent the gills. Fluorescent microspheres were less frequently observed within mononuclear phagocytes in the epidermis, dermis and underlying connective tissue of the tissue mentioned above. Limited FMS trafficking was observed from 4- to 24-h post-immersion (hpi). Significantly higher numbers of FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of the 4- and 8-wph age classes and in the anterior kidney and spleen of the 8-wph age class when compared to younger age classes (p < 0.05). Significantly higher FMS (blue and green)/mm(3) of tissue were observed in the posterior kidney of 4- and 8-wph fish when compared to all other organs (p < 0.05). The present study indicates that FMS uptake increases with age in channel catfish. The younger age classes may possess an increased ability to exclude particulate antigen, or lack the specific mechanisms that needed to take up particulates in the form of FMS.     

Tissue and Organ Specificity of Eimeria tenella (Railliet and Lucet, 1891) Fantham, 1909 in Cecectomized Chickens*†

SYNOPSIS. The ceca of 2-week-old chicks were surgically removed. One week post-operation each cecectomized bird was given 2 × 10⁶ sporulated Eimeria tenella oocysts per os. Birds from the same hatch, with intact ceca, served as controls and were infected the same time as the cecectomized birds. However, in order to reduce mortality, control birds were each given 1 × 10⁴ sporulated E. tenella oocysts per os. Four cecectomized and 5 control birds were killed 12, 24, 48, 72, 96, 120, and 144 hours after inoculation. Tissues from the small and large intestine of each bird proximal to the cecal junction were removed, processed by the dioxan method, and studied microscopically for developmental stages of the parasite. Developmental stages of the parasite were observed in all sections from the large intestine of cecectomized chickens. Initial sporozoite penetration and subsequent development of the parasite in this location was similar to that observed in the cecal mucosa of non-cecectomized chickens. No parasites were observed in sections of the small intestine of cecectomized birds 12 or 24 hours after inoculation, and findings after 48 and 72 hours were inconsistent. However, numerous parasites were observed in sections 96, 120, and 144 hours post-inoculation. In contrast, endogenous stages of the parasite were not seen in tissue sections of the small and large intestine of birds with intact ceca until 120 hours after inoculation. Numerous young gametocytes were then observed in sections from all birds. Similarly, mature gametocytes were observed in all fixed sections 144 hours after inoculation. No evidence was found that would indicate whether or not infection in the small and large intestine of birds with intact ceca or the small intestine of cecectomized birds was initiated by sporozoites or merozoites, nor was evidence found to suggest that development of any stage of the parasite was suppressed in these organs.     

Early Development and Tissue Distribution of Pseudoloma neurophilia in the Zebrafish,Danio rerio

The early proliferative stages of the microsporidian parasite, Pseudoloma neurophilia were visualized in larval zebrafish, Danio rerio, using histological sections with a combination of an in situ hybridization probe specific to the P. neurophilia small‐subunit ribosomal RNA gene, standard hematoxylin‐eosin stain, and the Luna stain to visualize spores. Beginning at 5 d post fertilization, fish were exposed to P. neurophilia and examined at 12, 24, 36, 48, 72, 96, and 120 h post exposure (hpe). At 12 hpe, intact spores in the intestinal lumen and proliferative stages developing in the epithelial cells of the anterior intestine and the pharynx and within hepatocytes were observed. Proliferative stages were visualized in the pancreas and kidney at 36–48 hpe and in the spinal cord, eye, and skeletal muscle beginning at 72 hpe. The first spore stages of P. neurophilia were observed at 96 hpe in the pharyngeal epithelium, liver, spinal cord, and skeletal muscle. The parasite was only observed in the brain of larval fish at 120 hpe. The distribution of the early stages of P. neurophilia and the lack of mature spores until 96 hpe indicates that the parasite gains access to organs distant from the initial site of entry, likely by penetrating the intestinal wall with the polar tube.     

Relations between histopathology and parasitaemias in Oncorhynchus mykiss infected with Cryptobia salmositica, a pathogenic haemoflagellate

Ichthyophthirius multifiliis is a ciliated protozoan parasite that infects the skin and gills of freshwater fish. This report describes the unusual finding of I. multifiliis within the peritoneal cavities of experimentally infected channel catfish Ictalurus punctatus. Twenty catfish fingerlings were exposed to I. multifiliis theronts using a standardized protocol. Five infected fish and 2 control fish were killed at various time points after infection and their tissues examined. Formalin-fixed, paraffin embedded sections were processed for light microscopy and immunohistochemical detection of I. multifiliis immobilization antigen. Trophonts were observed in skin and gill sections of all exposed fish. Parasites were associated with epithelial hyperplasia, focal areas of cellular disruption and necrosis. In addition to these usual sites of infection, individual trophonts were unexpectedly found within the peritoneal cavities of 4 fish. Staining for parasite antigen facilitated their detection within abdominal adipose tissue or adjacent to intestines. This discovery is interesting as it suggests I. multifiliis may be found in tissues other than the skin and gills during the course of a normal infection.     

The effects of elevated temperatures and various time-temperature combinations on the development of Brachiola (Nosema) algerae N. Comb. in mammalian cell culture

Nosema algerae Vávra and Undeen 1970, a microsporidian known to cause infection in mosquitoes, develops in mammalian cell cultures at 24-35 degrees C and in the tails and footpads of athymic mice. More recently it has been reported to grow at 38 degrees C in human cell culture. The present study is a two-part temperature/development examination. The first part examines the development of N. algerae in rabbit kidney cell culture at 29 degrees C, which permits the formation of functional spores within 72 h, and compares the effect of elevated temperatures (36.0, 36.5, 37 degrees C) on parasite development. At these elevated temperatures, N. algerae infects but undergoes only one or two proliferative divisions, with no evidence of sporogony by 72 h post-inoculation. During this time, however, the host cells continue to divide resulting in fewer infected cells over time and giving the appearance of a diminished parasitemia. Additionally, at 37 degrees C some organisms degenerate/hibernate by 72 h while others remain viable/active. It is not until 96 h that the parasites appear in large clusters of proliferative stages in the few host cells that are infected. By 120 h post-inoculation, proliferative cells, sporoblasts, and early spores are observed. These results suggest that elevated temperatures impede proliferation rates and the onset of sporogony. The second part of this study evaluates developmental changes in N. algerae when incubation temperatures and times are varied during parasite growth, resulting in abnormal parasite morphology. These abnormalities were not present when parasites were grown at constant temperature (29-37 degrees C). This report demonstrates that N. algerae can successfully develop at high temperatures (37 degrees C), justifying its taxonomic relocation to the genus Brachiola.     

Haemocyte reactions in WSSV immersion infected Penaeus monodon

White spot syndrome virus (WSSV) has been a major cause of shrimp mortality in aquaculture worldwide in the past decades. In this study, WSSV infection (by immersion) and behaviour recruitment of haemocytes is investigated in gills and midgut, using an antiserum against the viral protein VP28 and a monoclonal antibody recognising haemocytes (WSH8) in a double immunohistochemical staining and in addition transmission electron microscopy was applied. More WSH 8(+) haemocytes were detected at 48 and 72 h post-infection in the gills of infected shrimp compared to uninfected animals. Haemocytes in the gills and midgut were not associated with VP28-immunoreactivity. In the gills many other cells showed virus replication in their nuclei, while infected nuclei in the gut cells were rare. Nevertheless, the epithelial cells in the midgut showed a clear uptake of VP28 and accumulation in supranuclear vacuoles (SNV) at 8h post-infection. However, epithelial nuclei were never VP28-immunoreactive and electron microscopy study suggests degradation of viral-like particles in the SNV. In contrast to the gills, the midgut connective tissue shows a clear increase in degranulation of haemocytes, resulting in the appearance of WSH8-immunoreactive thread-like material at 48 and 72 h post-infection. These results indicate recruitment of haemocytes upon immersion infection in the gills and degranulation of haemocytes in less infected organs, like the midgut.     

Kinetic study of the replication of a cell-culture-adapted hepatitis A virus

The kinetics of replication of hepatitis A virus (LCDC-01) was studied in foetal rhesus monkey kidney cells (FRhK-4). Cells infected at a multiplicity of infection (MOl) of 2.0 showed no viral antigen production until 12 h post-infection using radioimmuno assay (RIA); however, at 48 h post-infection a logarithmic increase in antigen concentration began, which peaked by day 7. Similar patterns were observed with cultures infected with lower MOI (0.20 and 0.02) but events were delayed by about 24 h. In contrast, detection of antigen by fluorescence antibody methods occured at only 72 h after inoculation, with either 2.0 or 0.02 MOI, and peaked by day 9. The production of infectious virus did not begin until 24 h post-infection as measured by RIA and gradually peaked by day 6. Viral RNA was first detected 24 h post-infection by hybridization assay. The amount of viral RNA in the infected cells increased significantly between days 4 to 7. Restriction in the synthesis of RNA or infectious virus was not observed.     

Susceptibility of European catfish (Silurus gianis) to Edwardsiella ictaluri

European catfish (Silurus glanis) were tested for their susceptibility to the bacterium Edwardsiella ictaluri. The LD₅₀ of E. ictaluri when injected into European catfish was 5.4 × 10⁶ compared to 7.1 times; 10⁴ for channel catfish (Ictalurus punctatus). E. ictaluri was isolated from dead and moribund European catfish and the bacterium was also detected in kidney smears by an indirect fluorescent antibody (FA) technique. The bacterium was not isolated or detected by FA from surviving fish 15 days after injection. No clinical signs of E. ictaluri infection were noted in European catfish, but these were prevalent in the channel catfish. These experiments indicate that under experimental conditions European catfish are not as susceptible to E. ictaluri as channel catfish.     

饲料中维生素D3的添加水平对黄颡鱼幼鱼生长和Toll样受体TLR18、TLR19和TLR21的影响

为了探讨饲料中维生素D3添加水平对黄颡鱼(Pelteobagrus fulvidraco)生长和Toll样受体的影响,研究设计了5个不同浓度梯度的维生素D3饲料(1120、2260、3950、8030和16600 IU/kg),对体重为(5.0±0.2) g的黄颡鱼进行了为期12周的生长实验,并在生长实验结束后进行鮰爱德华氏菌(Edwardsiella ictaluri)攻毒72h。于攻毒前(0)和攻毒后(72h)采样,每个饲料组分别取6尾鱼的脾脏、头肾、肝脏和前肠四个组织,检测不同浓度维生素D3处理对攻毒前和攻毒后TLR18、TLR19和TLR21基因表达量的影响。同时另取6条新鲜黄颡鱼的肌肉、头肾、肾脏、皮肤、脑、鳃、脾脏、胃上皮、小肠和肝脏,检测TLR18、TLR19和TLR21基因在黄颡鱼中的组织分布。结果表明:不同的维生素D3添加水平会显著影响黄颡鱼幼鱼的生长性能; TLR18、TLR19和TLR21基因在所检测的组织中均有表达,但在脾脏中表达量最高;饲料中不同维生素D3含量在攻毒前后均会显著影响TLR18、TLR19和TLR21在头肾、脾脏、肝脏和前肠中的表达,攻毒后基因的...     

鸭呼肠孤病毒强毒株致鸭胚原代成纤维细胞凋亡的研究

通过光镜、电镜、DNA Ladder法、流式细胞术、荧光染色对鸭呼肠孤病毒(DRV)诱导鸭胚原代成纤维细胞(DEF)凋亡情况进行检测.结果显示,光镜可见细胞形态学上出现细胞皱缩,染色质浓染边移;电镜观察到细胞胞浆浓缩,细胞核染色质凝聚、部分形成凋亡小体;荧光染色结果显示,在感染后24h有激发绿色荧光的凋亡细胞出现,随着时间的推移,激发红色荧光的死亡细胞数量增多;DNA Ladder检测到感染后24～144h的DNA样品呈梯形条带;流式细胞术于感染后24h检测到凋亡细胞,其数量在72～96h达到高峰,144h开始下降.研究结果表明,DRV在DEF增殖的过程中具有诱导宿主细胞凋亡的作用.     

The aspartate-semialdehyde dehydrogenase of Edwardsiella ictaluri and its use as balanced-lethal system in fish vaccinology

asdA mutants of gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd(+) plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd(+) plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd(+) expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd(+) vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd(+) plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry.     

Rapid plasmid analysis for identification of Edwardsiella ictaluri from infected channel catfish (Ictalurus punctatus).

Eighteen different strains of Edwardsiella ictaluri isolated from infected channel catfish (Ictalurus punctatus) were screened to determine whether plasmid DNA was present. Two plasmids of 5,700 and 4,900 base pairs were identified. Restriction enzyme analysis showed that each of the strains harbored these same two plasmids. Restriction maps of the separated plasmids indicated that these plasmids were not closely related to each other. A rapid screening technique was developed that would allow the presence of these plasmids from either broth cultures or single colonies of E. ictaluri to be determined within 2 to 3 h by agarose gel electrophoresis. These results suggest that plasmid fingerprinting of E. ictaluri should become a useful tool in the presumptive identification of this bacterium from infected channel catfish.     

Development of bioluminescent Edwardsiella ictaluri for noninvasive disease monitoring

Edwardsiella ictaluri is a facultative intracellular bacterium that causes enteric septicemia of catfish (ESC). In this study, we aimed to develop bioluminescent E. ictaluri that can be monitored by noninvasive bioluminescence imaging (BLI). To accomplish this, the luxCDABE operon of Photorhabdus luminescens was cloned downstream of the lacZ promoter in the broad host range plasmid pBBR1MCS4. Edwardsiella ictaluri strain 93-146 transformed with the new plasmid, pAKlux1, was highly bioluminescent. pAKlux1 was stably maintained in E. ictaluri without any apparent effect on growth or native plasmid stability. To assess the usefulness of the bioluminescent strain in disease studies, catfish were infected with 93-146 pAKlux1 by intraperitoneal injection and by bath immersion, and in vivo bacterial dissemination was observed using BLI. This study demonstrated that bioluminescent E. ictaluri can be used for real-time monitoring of ESC in live fish, which should enable observation of pathogen attachment sites and tissue predilections.     

Inflammatory response of rainbow trout Oncorhynchus mykiss (Walbaum, 1792) larvae against Ichthyophthirius multifiliis

At hatching, the immune system of the rainbow trout larva is not fully developed. The larva emerges from the egg and is exposed to the aquatic freshwater environment containing pathogenic organisms. At this early stage, protection from disease causing organisms is thought to depend on innate immune mechanisms. Here, we studied the ability of young post-hatch rainbow trout larvae to respond immunologically to an infection with Ichthyophthirius multifiliis and also report on the localization of 5 different immune relevant molecules in the tissue of infected and uninfected larvae. Quantitative RT-PCR (qPCR) was used to analyze the genetic regulation of IL-1β, IL-8, IL-6, TNF-α, iNOS, SAA, cathelicidin-2, hepcidin, IL-10, IL-22, IgM and IgT. Also, a panel of 5 monoclonal antibodies was used to investigate the presence and localization of the proteins CD8, SAA, MHCII, IgM and IgT. At 10 days (84 degree days) post-hatching, larvae were infected with I. multifiliis and sampled for qPCR at 3, 6, 12, 24, 48 and 72 h post-infection (p.i.). At 72 h p.i. samples were taken for antibody staining. The first of the examined genes to be up-regulated was IL-1β. Subsequently, IL-8 and cathelicidin-2 were up-regulated and later TNF-α, hepcidin, IL-6, iNOS and SAA. Immunohistochemical staining showed presence of CD8 and MHCII in the thymus of both infected and non-infected larvae. Staining of MHCII and SAA was seen at sites of parasite localization and weak staining of SAA was seen in the liver of infected larvae. Staining of IgT was seen at site of infection in the gills which may be one of the earliest adaptive factors seen. No positive staining was seen for IgM. The study illustrates that rainbow trout larvae as young as 10 days (84 degree days) post-hatch are able to regulate important immune relevant cytokines, chemokines and acute phase proteins in response to infection with a skin parasitizing protozoan parasite.     

Rapid plasmid analysis for identification of Edwardsiella ictaluri from infected channel catfish (Ictalurus punctatus)

Eighteen different strains of Edwardsiella ictaluri isolated from infected channel catfish (Ictalurus punctatus) were screened to determine whether plasmid DNA was present. Two plasmids of 5,700 and 4,900 base pairs were identified. Restriction enzyme analysis showed that each of the strains harbored these same two plasmids. Restriction maps of the separated plasmids indicated that these plasmids were not closely related to each other. A rapid screening technique was developed that would allow the presence of these plasmids from either broth cultures or single colonies of E. ictaluri to be determined within 2 to 3 h by agarose gel electrophoresis. These results suggest that plasmid fingerprinting of E. ictaluri should become a useful tool in the presumptive identification of this bacterium from infected channel catfish.     

Synchronization of Plasmodium falciparum erythrocytic stages in culture.

Synchronous development of the erythrocytic stages of a human malaria parasite, Plasmodium falciparum, in culture was accomplished by suspending cultured parasites in 5% D-sorbitol and subsequent reintroduction into culture. Immediately after sorbitol treatment, cultures consisted mainly of single and multiple ring-form infections. At the same time, varying degrees of lysis of erythrocytes infected with the more mature stages of the parasite was evident. Approximately 95% of the parasites were in the ring stage of development at 48 and 96 hr after sorbitol treatment-likewise, a high percentage of trophozoite and schizont stages was observed at 24, 72, and 120 hr. D-Mannitol produced similar, selective, lytic effects.     

Babesia gibsoni: detection during experimental infections and after combined atovaquone and azithromycin therapy

Babesia gibsoni is a protozoan parasite of dogs worldwide yet both an effective treatment and a reliable method for detecting subclinical cases of this emerging infection remain elusive. Experimental B. gibsoni infections were established in vivo to investigate the efficacy of combined atovaquone and azithromycin drug therapy and to determine the detection limits of a nested-PCR, IFAT and microscopy during various stages of infection. While atovaquone and azithromycin produced a reduction in parasitaemia, it did not eliminate the parasite and drug resistance appeared to develop in one dog. Polymerase chain reaction was found to be most useful in detecting infection in the pre-acute and acute stages, while IFAT was most reliable during chronic infections. Microscopy is suggested to be only effective for detecting acute stage infections. This study also describes the detection of B. gibsoni in tissue samples during chronic infections for the first time, suggesting possible sequestration of this parasite.     

Invasion of Ceratomyxa shasta (Myxozoa) and comparison of migration to the intestine between susceptible and resistant fish hosts

The myxozoan parasite Ceratomyxa shasta infects salmonids causing ceratomyxosis, a disease elicited by proliferation of the parasite in the intestine. This parasite is endemic to the Pacific Northwest of North America and salmon and trout strains from endemic river basins show increased resistance to the parasite. It has been suggested that these resistant fish (i) exclude the parasite at the site of invasion and/or (ii) prevent establishment in the intestine. Using parasites pre-labeled with a fluorescent stain, carboxyfluorescein succinimidyl diacetate (CFSE), the gills were identified as the site of attachment of C. shasta in a susceptible fish strain. In situ hybridization (ISH) of histological sections was then used to describe the invasion of the parasites in the gill filaments. To investigate differences in the progress of infection between resistant and susceptible fish, a C. shasta-susceptible strain of rainbow trout (Oncorhynchus mykiss) and a C. shasta-resistant strain of Chinook salmon (Oncorhynchus tshawytscha) were sampled at consecutive time points following exposure at an endemic site. Using ISH in both species, the parasite was observed to migrate from the gill epithelium into the gill blood vessels where replication and release of parasite stages occurred. Quantitative PCR verified entry of the parasite into the blood. Parasite levels in blood increased 4 days p.i. and remained at a consistent level until the second week when parasite abundance increased further and coincided with host mortality. The timing of parasite replication and migration to the intestine were similar for both fish species. The field exposure dose was unexpectedly high and apparently overwhelmed the Chinook salmon’s defenses, as no evidence of resistance to parasite penetration into the gills or prevention of parasite establishment in the intestine was observed.