Academic literature on the topic 'Fishes – Parasitic diseases – Chemotherapy'

SUMMARYMariculture is a rapidly developing industrial sector. Generally, fish are maintained in net cages with high density. Cage culture systems allow uncontrolled flow of sea water containing potentially infectious stages of fish parasites. In such culture conditions, prevention of such parasitic infections is difficult for parasites with life cycles that complete within culture sites, among which monogeneans and blood flukes are the most important platyhelminthes. Intense monogenean infections induce respiratory and osmo-regulatory dysfunctions. A variety of control measures have been developed, including freshwater bath treatment and chemotherapy. The potential to control monogenean infections through selective breeding, modified culture techniques to avoid infection, and general fish health management are discussed. It should be noted that mariculture conditions have provided some host-specific monogeneans with a chance to expand their host ranges. Blood flukes sometimes induce mass mortality among farmed fish. In-feed administration of praziquantel is the best solution to treat infected fish. Some cases are described that show how international trade in marine fish has resulted in the spread of hitherto unknown parasites into indigenous farmed and wild fish.

Pathological disorders caused due to metazoan parasitic infestation were studied in the hill stream fishes of northwest Himalayan region. Host specificity was found to be one of the fundamental features of metazoan parasites which belonged to the class Cestoda, Nematoda, Trematoda and phylum Acanthocephala. The study indicates that a successful co-evolution of the host and its parasite has caused the adaptation of the later by developing evading mechanisms in order to avoid extinction. Besides this it was also observed that some parasites have even understood to benefit from the well developed antiparasitic armament in fish intestinal epithelia. Thus, parasites are exploiting the antiparasitic response mechanism of the host to optimize, host finding, invasion and survival in the host. Such interaction between host and parasites are considered phylogenetically old. Some monogeneans, cestodes, digeneans and acanthocephalans were found to resist pronounced cellular and host reactions which even improved the attachment of parasite into the host predilection site. Scanning Electron Microscopy and hitstopathological examination was conducted on parasites recovered from fishes in order to understand the host parasite interaction and the damage inflicted by parasite on hill stream fish species.

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP

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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Parasitic diseases remain a health burden affecting more than 500 million people worldwide with malaria having the highest mortality rate. The parasites can be transferred to the human bodies either through the mouth by ingestion of contaminated food and water or through the skin by bug bites or direct contact to environments harbouring them. Epidemiological control seems to be impossible since there is failure to control the insect vectors as well as practice of hygiene. Therefore, this has led to the development of a number of vaccines, chemotherapy and disease control programs. However, parasites have increasingly developed resistance to traditionally used anti-parasitic drugs and due to that fact there is need for alternative medication for parasitic diseases. Heat shock protein 90 (Hsp90) facilitates the folding of proteins in all living cells and their role is more important to parasites because of their environmental changes, from vector to host. Hsp90s play a major role; therefore this justifies the need for a deeper analysis of the parasitic Hsp90s. Recent studies have revealed that, the Plasmodium sp. Hsp90 has an extended linker region which increases the protein’s affinity for ATP and its inhibitors. Therefore we hypothesize that there are also significant features in other parasitic Hsp90s which would lead to Hsp90 being defined as potential drug targets. In the present study an attempt was made to gain more insight into the differences in primary structure of human and parasitic Hsp90s. The sequences were retrieved from the NCBI database and analysis was done in three groups basing on the localization of the Hsp90. The physicochemical properties were calculated and in every group, the protozoan Hsp90s showed significant differences when compared to the human orthologs. Multiple sequence alignments (MSA) showed that endoplasmic reticulum Hsp90s have an extended region in the middle domain indicating their ability to bind to a unique subset of client proteins. Sequence identities between the human and parasites showed that the protozoan Hsp90s are less related to the human Hsp90s as compared to the other parasites. Likewise, motif analysis showed the trypanosomatids and apicomplexan groups have their own unique set of motifs and they were grouped together in the phylogenetic analysis. Phylogenetic analysis also showed that, the protozoan Hsp90s forms their own clades in each group while the helminths did not form in endoplasmic reticulum group. In this study, we concluded that, Hsp90 can be a potential drug target for the protozoan species and more specifically those from the apicomplexan and trypanosomatids groups.

The efficacy of a range of anti-parasitic chemotherapeutic agents against the salmon louse Lepeophtheirus salmonis following topical application was studied in vitro and in vivo. In general, adult and preadult lice were susceptible to a wide range of compounds with dose rates, following 1 hour exposures, ranging several orders of magnitude (10.0 - 0.001 mg/L). Overall the pyrethroid compounds which were tested were found to have the widest therapeutic ratios, indicating the potential of this group of chemotherapeutants for sea lice control. Resistance to the organophosphorus (OP) compounds dichlorvos and azamethiphos was detected in isolated populations of lice. Field trials with azamethiphos indicated that the compound was highly efficacious against sensitive lice (@ 0.1 mg/L; however, where resistance was present, efficacy (@ 0.2 mg/L) was highly variable. When used at the above dose rates, azamethiphos was found to be well tolerated by fish as indicated by a lack of significant brain acetylcholinesterase inhibition. Results on cross resistance (between pyrethroids and OPs) were inconclusive which was believed to be, primarily, due to the overall high toxicity of the group; but also to the variable responses from exposed lice. In a series of preliminary trials, one of the pyrethroid compounds, PHRDL-D, was found to effectively remove lice when administered orally to infected salmon, indicating the potential of pyrethroids as oral chemotherapeutants. A comparison of the relative toxicity of azamethiphos (OP), resmethrin (pyrethroid), ivermectin (avermectin) and the structurally similar compound SKB7 (milbemycin), indicated that chalimus stages were only susceptible to ivermectin and SKB7 following topical and intra-peritoneal injection to lice infected fish. In contrast, azamethiphos and resmethrin were found to be non toxic to chalimus larvae at dose rates which were highly toxic to both adult lice and treated fish. Preliminary studies on the uptake of r4C]azamethiphos in adult lice indicated that uptake was both concentration and time dependant, reaching a plateau at the onset of toxicity. Uptake appeared to be primarily associated with frontal plates, 1st antennae and anus. The fmdings indicated that several compounds/compound classes are highly active against lice and, given the limited number of compounds available for sea lice control and the development of resistance to OPs, might be considered as alternatives. In light of these findings, the potential of chemotherapy for the future control of sea lice is discussed.

Leishmaniose e doença de Chagas são parasitoses endêmicas causadas, respectivamente, pelos protozoários Leishmania spp. e Trypanosoma cruzi. Ante à escassez de quimioterápicos, à elevada toxicidade dos fármacos disponíveis e à baixa eficácia destes no combate às formas intracelulares, replicantes, dos parasitos há necessidade de buscar novas alternativas quimioterápicas. A atividade tripanomicida do hidroximetilnitrofural, base de Mannich do nitrofural, já era conhecida. O presente trabalho mostra que este derivado também apresenta atividade leishmanicida, quando ensaiado em formas promastigotas de L. amazonensis, L. chagasi e L. baziliensis. Com o objetivo de obter pró-fármacos potencialmente ativos em doença de Chagas e leishmanioses visceral e mucocutânea, planejaram-se e foram sintetizados derivados hidrossolúveis de hidroximetilnitrofural e quitosana, polissacarídeo que apresenta, também, atividade imunomoduladora. Para a aplicação tópica em leishmaniose cutânea sintetizaram-se membranas de quitosana ligada ao hidroximetilnitrofural. Membranas de quitosana copolimerizadas com enxertos de ácido acrílico e metacrilato de hidroxietila foram sintetizadas e avaliadas quanto à biocompatibilidade - trombogenicidade, citotoxicidade e potencial hemolítico. Aquelas com maior teor em metacrilato de hidroxietila não se mostraram citotóxicas, tampouco hemolíticas; aquelas com maior proporção em ácido acrílico, por sua vez, apresentaram excelentes características de intumescimento, mas certo grau de citotoxicidade e hemólise, possivelmente devido à presença de monômeros que não reagiram no material. A ligação do hidroximetilnitrofural à quitosana, por meio de espaçante succínico, produziu prófármaco com propriedades filmogênicas para a aplicação tópica. Os derivados obtidos pró-fármacos e transportadores (quitosanas modificadas) - foram analisados no infravermelho, por ressonância magnética nuclear (RMN 1H e RMN 13C) e por análise térmica - OMTA, TG, OSC. Ensaios de atividade tripanomicida e leishmanicida dos pró-fármacos poliméricos e membranas serão posteriormente efetuados.
Leishmaniasis and Chagas\' disease are endemic parasitosis provoked by the protozoa Leishmania spp and Trypanosoma cruzi, respectively. Due to the scarce chemotherapy, to the high toxicity of the available drugs and to their low effectiveness, mainly in the treatment of intracellular replicant forms of those parasites, the search for new chemotherapeutic alternatives is extremely important. Hydroxymethylnitrofurazone, a nitrofurazone Mannich basis, has proven to be active against trypanomicide before and its activity in cultures of L. amazonensis, L. chagasi and L. braziliensis promastigotes was determined in this work. With the purpose of obtaining prodrugs potentially active in Chagas\' disease and visceral and mucocutaneous leishmaniases, hydrosoluble hydroxymethylnitrofurazone prodrugs have been designed and synthesized using chitosan, a polysaccharide showing immunomudulatory activity, as the carrier. Membranes from chitosan linked with hydroxymethylnitrofurazone have been synthesized for topical administration in cutaneous leishmaniasis. Membranes were obtained by graft copolymerization of hydroxyethy/methacrylate and acrylic acid onto chitosan and their biocompatibility - trombogenicity, citotoxicity and hemolysis potential - was evaluated. Those membranes with higher content of hydroxyethylmethacrylate showed to be neither cytotoxic nor hemolytic; those with higher content of acrylic acid showed good swelling properties, although a certain level of cytotoxicity and haemolysis has been detected, due to the presence of non-reacted monomers. The linkage of hydroxymethylnitrofurazone to chitosan by a succinyl spacer group led to a prodrug with filmogenic properties for topic administration. The derivatives obtained - prodrugs and carriers (modified chitosans) - were analyzed by infrared, nuclear magnetic resonance (1H NMR and 13C NMR) and by thermal analysis - DMTA, TG and DSC. Tests of trypanomicide and leishmanicide activity with polymer prodrugs and membranes will be further developed.

The dusky grouper Epinephelus marginatus (Lowe 1834), is a protogynous sequential hermaphrodite and is considered to be one of the most important fish species in the Mediterranean Sea. It is a K-strategist, being slow growing and late maturing, and this, coupled with its reproductive biology and relatively sedentary behaviour, has made it extremely sensitive to overexploitation, leading it to be classified by the IUCN as an endangered fish species. Wild dusky grouper have suffered from disease outbreaks in the past decade, leading to mass mortalities across the Mediterranean Sea, including Libyan coastal waters. These mortalities have mostly been attributed to Nodavirus infections. In Europe and Brazil, efforts are in place to culture this fish for commercial grow-out and stock enhancement programmes. In Libya, the dusky grouper is consumed regularly and is considered a prime-eating fish. Its importance for the Libyan internal market, as well as its potential for export, makes it an ideal candidate for future Libyan aquaculture activities. Given the scarce literature regarding the dusky grouper in Libya, this study aimed first to assess dusky grouper fisheries, spawning seasons and to identify the main threats that the fishing sector poses for wild stocks. Second this study aimed to determine the health status of wild dusky grouper offered at a local fish market in the capital Tripoli, in order to identify pathogens, pathologies or other health issues that might pose a hazard to cultured populations but also to remaining wild dusky grouper stocks. To achieve these aims, twelve field surveys spanning the period of 2013-2015 were conducted. From these surveys, it was established that the dusky grouper is captured throughout the year, including the spawning season. Fish sizes offered for sale ranged between 20-92 cm total length (TL), with the fish being sold from local fishing grounds around Tripoli, but also from as far as Benghazi, 1300 km to the east of Tripoli. The dusky grouper is principally caught in artisanal fisheries and by spearfishing, with approximately 300 spear-fishermen serving one particular fish market in Tripoli that was a focus in this study, and with dusky grouper being one of their main targets. Over the period of the survey, 267 landed dusky grouper were inspected for visible lesions prior to sampling. A total of 50 dusky grouper with sizes ranging from 27- 66 cm TL including the gonads from a further five fish measuring 66-92 cm TL that were sampled separately and examined to assess the stage of sexual maturity and to look for the presence of parasitic infections mainly affecting the gills, skin and gonads. The spawning season was found to extend from May to early September, with females ranging between 39-68 cm TL, males measuring 57-92 cm TL, and transient fish measuring 58-68 cm TL. From otolith readings of 8 fish, the youngest fish was a 3 year old juvenile of 28 cm TL and the oldest was an 8-9 year old 56 cm TL female. Whilst the highest prevalence of parasitic infection was found to be monogenean infection of the gills, with 100% prevalence, followed by gnathiid isopods infecting the oral cavity with 92% prevalence, it was the nematode Philometra sp. infecting post-spawning ovaries at 52% prevalence, that gave the highest apparent pathological impact. Necrosis potentially attributed to Philometra sp. in one particular ovary, was at a level likely to have caused complete parasitic castration, while others showed varying levels of probable functional reduction. The pathologies described need further investigation, especially in relation to possible synergies between Philometra sp. and bacteria in causing the necrosis. From the 267 inspected dusky grouper, 55 fish ranging in size from 42-92 cm TL were observed to be affected by external skin lesions of unknown aetiology. Twenty-six of these fish were sampled, having lesions at various stages of severity, and 5 further unaffected fish were used for histological assessment of the skin as negative controls. Histopathologically, the lesions comprised a multifocal, unilateral or bilateral dermatitis, involving the epidermis, superficial dermis and scale pockets, and sometimes, in severe cases, the hypodermis. Severe lesions had marked epidermal spongiosis progressing to ulceration. Healing was observed in some fish. Bacteria and fungi could be isolated from severe lesions, although they were not seen histopathologically in early-stage lesions. By contrast, metazoan parasite eggs were observed in the dermis and epidermis of some fish with mild and moderate dermatitis. Unidentified gravid digenean trematodes, carrying similar eggs, were also seen within the blood vessels of the deep and superficial dermis. The newly described condition was termed dusky grouper dermatitis (DGD). DGD’s geographical distribution along the Libyan coastline was investigated using a novel application of the social media network Facebook. Using Facebook, it was possible to document skin lesions of dusky grouper in Libyan waters from images attached to the entries of spear-fishermen. Thirty two Facebook accounts and 8 Facebook groups posting from 23 Libyan coastal cities provided a retrospective observational dataset comprising a total of 382 images of dusky grouper caught by spearfishing from December 2011-December 2015. Skin lesions were observable on 57 / 362 fish, for which images were of sufficient quality for analysis, giving a minimal prevalence for lesions of 15.75%. Only dusky grouper exceeding an estimated 40 cm total length exhibited lesions. The ability to collect useful data about the occurrence and geographical distribution of pathological conditions affecting wild fish using social media networks, demonstrates their potential utility as a tool to support epidemiological studies and monitor the health of populations of aquatic animals. The gravid digenean trematode described from mild lesions of five fish was identified using reconstruction through histological sectioning as belonging to the Family Aporocotylidae Odhner, 1912. This is the first description of a blood fluke from the dusky grouper, as well as from dermal blood vessels. The parasite was relatively long; the longest section of the parasite that could be measured was 1500 µm and 20-80 µm in width, while the total length of the parasite was estimated at 1500-2000 µm. Minute tegumental spines, possibly covering only a few parts of the parasite, were seen from some cross-sections. The parasite had one post-testicular ovary, which might overlap the testis, a pre-ovarian ascending uterus, and a post-ovarian descending uterus. It also possessed an oesophagus surrounded by oesophageal glandular cells and a pre-ovarian and pre-testicular extension of the vitelline cells, mostly at the level of the ascending uterus. The parasite was observed to be intra-vascular, the uterine lumen varies in size to accommodate between 1-7 eggs. The uterine eggs were embryonated and observed to span several stages of maturation. Eggs were also found in the dermal blood vessels, in the dermis, and in the epidermis, with the latter appearing to provide a potential route of egress of eggs into the environment. The extra-uterine eggs were 23.5 to 37.52 µm long and contained a ciliated miracidium. The eggs seemed to elicit a mixed inflammatory reaction, with degranulation of eosinophilic granular cells attached to the external surface of some of the eggs within the blood vessels but also the dermis. From observations made in the current study, this parasite appears to be a new species, most closely allied to none of the currently described Aporocotylidae genera.
In summary, the present study has demonstrated that the dusky grouper is extensively fished in Libya without discrimination to sizes and season, by both artisanal and spearfishing, with the latter as one of the main fishing methods, posing treats to the spawning potential and conservation of dusky grouper in Libya. The philometrid infecting the ovaries has a potential to reduce fecundity or to result in parasitic castration of wild broodstock. Gill-infecting monogeneans might represent a hazard for all stages of dusky grouper production. Dusky grouper dermatitis is a skin lesion, although there are no indications that infections may result in mortalities. Under culture conditions, however, this might change due to increase bacterial loads, which might lead to secondary bacterial infection. The presence of skin lesions would undoubtedly reduce the market value of whole fish. These findings are important for existing wild stocks, and for future plans regarding the aquaculture of dusky grouper. Future studies need to focus on the pathology of DGD, describing the disease process and aetiology using laboratory techniques such as TEM and virology as well as using morphology and molecular-based tools to describe the blood fluke and to determine their potential role in the initiation the disease. The novel approach to disease surveillance using social media Facebook posts could be further expanded by attracting citizen scientists, for future research assessing disease in wild fish, for sightings of mortality events and/or the appearance of disease outbreaks, or, for mapping marine mammal stranding’s and/or turtle nesting activity.

Detection and treatment of bacterial kidney disease (BKD) was investigated. Experiments were conducted to evaluate the quantitative, fluorescent antibody technique (QFAT) that is used to detect, identify, and quantify both typical and 'bar form' Renibacterium salmoninarum cells. Smears of kidney tissue from naturally and artificially infected salmonids, both with and without chemotherapy, were quantitatively examined throughout the course of R. salmoninarum infections. Detection and quantification by QFAT has been reported to provide assessments of prevalence and severity of R. salmoninarum of individual fish. These assessments and the occurrence of 'bar forms' of R. salmoninarum have been used as an indication of recovery within a population. 'Bar forms' were observed in kidney tissue smears of fish that survived bacterial challenge when treated with erythromycin. The 'bar form' was also detected when rainbow trout were artificially infected with lower doses of live R . salmoninarum and in fish that were injected with irradiation-inactivated R. salmoninarum cells. By examining R. salmoninarum cultures in vitro by QFAT, it was determined that 'bar forms' did not occur on artificial media even when antibiotics were incorporated into the agar. When QFAT was compared to direct fluorescent antibody technique (DFAT) and quantitative enzyme linked immunosorbent assay (ELISA), it was determined that QFAT had similar sensitivity as ELISA but was more sensitive than DFAT. QFAT was also used to predict minimum mortality. Experiments were also conducted to evaluate drug regimes to treat both artificial and natural R. salmoninarum infections. Erythromycin was administered by intraperitoneal injection in different doses and at selected days post infection. Erythromycin decreased percent mortality and increased mean day to death, but did not completely eradicate R. salmoninarum from infected test animals. Sarafloxacin and erythromycin were incorporated into daily ration of artificially infected test animals. Contrary to erythromycin, sarafloxacin did not decrease mortality or increase mean day to death when tested in vivo against R. salmoninarum. A new drug, A-77143, was tested in vitro to determine if it was bactericidal and its minimum inhibitory concentration. When A- 77143 was compared to other antibiotics, it had a relatively low minimum inhibitory concentration and was shown to be bactericidal against the eight strains of R. salmoninarum tested.
Graduation date: 1993

碩士
國立臺灣大學
獸醫學研究所
88
The detection of parasitic and bacterial diseases which affect the fishes and shrimps raised in culture ponds in Yunlin, Chia-Yi and Tainan has been made from September of 1998 to August of 1999. 596 samples were taken out of 259 ponds, 42 kinds of fishes and shrimps. In the part of parasitical diseases, 14 species of parasites in 420 infectious samples (70.47%) were found. The parasites with the morbidity are as followed: Trichodina spp. ( 28.81 % ), Pseudodactylogyrus spp. ( 22.86 % ), Centrocetus spp. ( 14.52 % ), Oodinium spp. ( 11.67 % ), Ambiphyra spp. ( 10.95 % ), Epistylis spp. ( 2.14 % ), Cryptocaryon irritans ( 1.90 % ), Anguillicola spp. ( 1.90 % ), Caligus spp. ( 1.90 % ), Myxidium spp. ( 0.95 % ), Lernara spp. ( 0.95% ), Ergasilus spp. ( 0.71 % ), Gyrodactylus spp. ( 0.48 % ), and Leeches ( 0.24 % ). Complicated infections were found in 258 cases ( 61.43 % ), Trichodina spp. infection complicated with Ambiphyra spp. was mostly commonly seen, followed by Trichodina spp. with Pseudodactylogyrus spp., and Trichodina spp. with Centrocetus spp.10 types of complicated infections were detected. In the part of bacterial diseases, by the use of biochemical identification, 201 bacterial strains were isolated ( 33.72 % ), including Aeromonas hydrophila ( 44.28 % ), Streptococcus spp. ( 20.90 % ), Edwardsiella tarda ( 17.42 % ), Cytophaga columnaris ( 6.97 % ), Vibrio vulnificus ( 4.98 % ), Vibrio parahaemolyticus ( 1.99 % ), Pseudomonas aeruginosa ( 1.00 % ), Pseudomonas fluorescens ( 1.00 % ), Shigella sonnei ( 1.00 % ), and Yersinia rohdei ( 0.50 % ). By agar dilution susceptibility test, bacterial isolates were studied for their sensitivity to 10 chemotherapeutic agents: Oxytetracycline ( OTC ), Chloramphenicol ( CM ), Flumequine ( Fq ), Oxolinic acid ( OA ), Nalidixic acid ( NA ), Norfloxacin ( Nor ), Streptomycin ( SM ), Sulfamethazine ( SA ), Amonxicillin ( AM ), and Erythromycin ( EM ). The result showed that among 201 bacterial strains isolated, 156 strains ( 77.61 % ) were resistant to one or more chemotherapeutic agents. 6.47 % of bacterial strains were resistant to Nor ( 13 / 201 ), 8.81 % to OA ( 14 / 201 ), 12.94 % to OTC ( 26 / 201 ), 15.92 % to CM ( 32 / 201 ), 19.50 % to Fq ( 31 / 201 ), 30.82 % to NA ( 49 / 201 ), 47.26 % to SM ( 95 / 201 ), and 73.63% to SA ( 148 / 201 ). Among 10 chemotherapeutic agents, the susceptibility of all strains to Nor was the highest, followed by OA, OTC, and CM, and the SA was the lowest. Furthermore, susceptibility against AM and EM were examined for Streptococcus spp., and the results presented dramatically different, the susceptibility to EM was 97.32 %, while the sensitivity to AM was 100 %. In the analysis for resistance pattern of various bacterial strains showed that the resistance pattern of 67 ( 33.33 % ) A. hydrophila with drug resistance were mainly anti-SA, anti-OTC, NA, SA, SM, anti-CM, Fq, NA, SA, and anti-SM, and that of 42 ( 20.90 % ) Streptococcus spp. were mainly anti-AM, SA, SM, anti-AM, SM, and anti-AM, CM, SA, and that of 16 ( 7.96 % ) E. tarda were mainly anti-CM, Fq, NA, SA, SM, anti-OTC, SA, SM, and anti-SA, and that of 12 ( 5.97 % ) C. columnaris were mainly anti-NA, SA, anti-CM, Fq, NA, Nor, SA, and anti-NA, Nor, SA, ant that of 10 ( 4.98 % )Vibrio vulnificus were maily anti-SA, anti-OTC, SA, and anti-CM, Fq, OA, NA, Nor, SA, SM.