Screening of anti-mycobacterial compounds in a naturally infected zebrafish larvae model.

OBJECTIVES: Mycobacterium tuberculosis is a deadly human pathogen that causes the lung disease TB. M. tuberculosis latently infects a third of the world's population, resulting in ∼1.5 million deaths per year. Due to the difficulties and expense of carrying out animal drug trials using M. tuberculosis and rodents, infections of the zebrafish Danio rerio with Mycobacterium marinum have become a useful surrogate. However, the infection methods described to date require specialized equipment and a high level of operator expertise. METHODS: We investigated whether zebrafish larvae could be naturally infected with bioluminescently labelled M. marinum by immersion, and whether infected larvae could be used for rapid screening of anti-mycobacterial compounds using bioluminescence. We used rifampicin and a variety of nitroimidazole-based next-generation and experimental anti-mycobacterial drugs, selected for their wide range of potencies against M. tuberculosis, to validate this model for anti-mycobacterial drug discovery. RESULTS: We observed that five of the six treatments (rifampicin, pretomanid, delamanid, SN30488 and SN30527) significantly reduced the bioluminescent signal from M. marinum within naturally infected zebrafish larvae. Importantly, these same five treatments also retarded the growth of M. tuberculosis in vitro. In contrast, only three of the six treatments tested (rifampicin, delamanid and SN30527) retarded the growth of M. marinum in vitro. CONCLUSIONS: We have demonstrated that zebrafish larvae naturally infected with bioluminescent M. marinum M can be used for the rapid screening of anti-mycobacterial compounds with readily available equipment and limited expertise. The result is an assay that can be carried out by a wide variety of laboratories for minimal cost and without high levels of zebrafish expertise.

A prospective view of animal and human Fasciolosis.

Fasciolosis, a food-borne trematodiasis, results following infection with the parasites, Fasciola hepatica and Fasciola gigantica. These trematodes greatly affect the global agricultural community, infecting millions of ruminants worldwide and causing annual economic losses in excess of US $3 billion. Fasciolosis, an important zoonosis, is classified by WHO as a neglected tropical disease with an estimated 17 million people infected and a further 180 million people at risk of infection. The significant impact on agriculture and human health together with the increasing demand for animal-derived food products to support global population growth demonstrate that fasciolosis is a major One Health problem. This review details the problematic issues surrounding fasciolosis control, including drug resistance, lack of diagnosis and the threat that hybridization of the Fasciola species poses to future animal and human health. We discuss how these parasites may mediate their long-term survival through regulation and modulation of the host immune system, by altering the host immune homeostasis and/or by influencing the intestinal microbiome particularly in respect to concurrent infections with other pathogens. Large genome, transcriptome and proteomic data sets are now available to support an integrated One Health approach to develop novel diagnostic and control strategies for both animal and human disease.

Survival of Mycobacterium avium subsp. paratuberculosis in synthetic human gastric juice and acidified porcine bile.

The bactericidal activities of synthetic gastric juice and acidified porcine bile on Mycobacterium avium subsp. paratuberculosis were assessed using propidium monoazide (PMA)-mediated quantitative reverse transcription-PCR, which allowed rapid relative quantitative analysis of viable M. avium subsp. paratuberculosis cells.

Early and late peritoneal and hepatic changes in goats immunized with recombinant cathepsin L1 and infected with Fasciola hepatica.

The aim of the present study was to study peritoneal and hepatic changes during early [7-9 days postinfection (dpi)] and late [15 weeks postinfection (wpi)] infection of goats immunized with recombinant F. hepatica pro cathepsin L1 (rCL1) in Quil A and challenged with Fasciola hepatica. Despite finding no significant reduction in fluke burdens between the control and immunized group, at 15 dpi the rCL1-vaccinated group showed significantly higher weight gain and reduced severity of hepatic lesions compared with the control group that received only Quil A. In the rCL1-vaccinated group, two of three goats sacrificed at 7-9 dpi had little hepatic damage and had a higher percentage of peritoneal eosinophils and elevated induced nitric oxide synthase (iNOS) expression in peritoneal cells than the goats from the control group. Moreover, while these two goats showed a heavy infiltration of eosinophils surrounding migrating flukes, the remaining animals examined at 7-9 dpi had no inflammatory infiltration surrounding migrating flukes. Two out of seven goats in the rCL1-vaccinated group had low fluke burdens and little hepatic damage at 15 wpi, suggesting an effective protective response in some of the vaccinated goats. This protective response did not correlate with peripheral eosinophilia or with serum titres of anti-rCL1 immunoglobulin (Ig) G. The results of the present work suggest that an eosinophil-mediated immune response plays a crucial role in the early effective host response against F. hepatica in goats. Adjuvants designed to increase cell-mediated immunity should be tested in future vaccine trials against F. hepatica.

Protection of cattle against a natural infection of Fasciola hepatica by vaccination with recombinant cathepsin L1 (rFhCL1).

The liver fluke, Fasciola hepatica causes liver fluke disease, or fasciolosis, in ruminants such as cattle and sheep. An effective vaccine against the helminth parasite is essential to reduce our reliance on anthelmintics, particularly in light of frequent reports of resistance to some frontline drugs. In our study, Friesian cattle (13 per group) were vaccinated with recombinant F. hepatica cathepsin L1 protease (rFhCL1) formulated in mineral-oil based adjuvants, Montanide ISA 70VG and ISA 206VG. Following vaccination the animals were exposed to fluke-contaminated pastures for 13 weeks. At slaughter, there was a significant reduction in fluke burden of 48.2% in the cattle in both vaccinated groups, relative to the control non-vaccinated group, at p

Peroxiredoxin: a central player in immune modulation.

Peroxiredoxins (Prx) are a family of anti-oxidants that protect cells from metabolically produced reactive oxygen species (ROS). The presence of these enzymes in the secretomes of many parasitic helminths suggests they provide protection against ROS released by host immune effector cells. However, we recently reported that helminth-secreted Prx also contribute to the development of Th2-responses via a mechanism involving the induction of alternatively activated macrophages. In this review, we discuss the role helminth Prx may play in modulating the immune responses of their hosts.

Alternative activation of ruminant macrophages by Fasciola hepatica.

The helminth parasite, Fasciola hepatica, has a worldwide distribution and infects a wide variety of mammalian hosts, including ruminants and man. In response to infection, these hosts mount a type 2 helper (Th2) response that is highly polarized and results in the downregulation of type 1 helper (Th1) mechanisms. In a murine macrophage model F. hepatica induces alternative activation of macrophages. These macrophages differ from classically activated cells in that they preferentially use arginase instead of inducible nitric oxide synthase (iNOS) for metabolism of nitrogen. In this study we sought to characterize macrophage phenotype following stimulation of the ovine cell line MOCL7 with recombinant F. hepatica enzymes and crude parasite extracts. An in vitro model using the MOCL7 cell line was established and arginase levels in cells were used to determine the activation status of cells. Stimulation of this cell-line in vitro with F. hepatica products induces alternative activation. We have also found a chitinase-like protein in supernatants which is capable of differentiating alternatively activated from classically activated macrophages.

Comparison of cysteine peptidase activities in Trichobilharzia regenti and Schistosoma mansoni cercariae.

Cercariae of the bird schistosome Trichobilharzia regenti and of the human schistosome Schistosoma mansoni employ proteases to invade the skin of their definitive hosts. To investigate whether a similar proteolytic mechanism is used by both species, cercarial extracts of T. regenti and S. mansoni were biochemically characterized, with the primary focus on cysteine peptidases. A similar pattern of cysteine peptidase activities was detected by zymography of cercarial extracts and their chromatographic fractions from T. regenti and S. mansoni. The greatest peptidase activity was recorded in both species against the fluorogenic peptide substrate Z-Phe-Arg-AMC, commonly used to detect cathepsins B and L, and was markedly inhibited (> 96%) by Z-Phe-Ala-CHN2 at pH 4.5. Cysteine peptidases of 33 kDa and 33-34 kDa were identified in extracts of T. regenti and S. mansoni cercariae employing a biotinylated Clan CA cysteine peptidase-specific inhibitor (DCG-04). Finally, cercarial extracts from both T. regenti and S. mansoni were able to degrade native substrates present in skin (collagen II and IV, keratin) at physiological pH suggesting that cysteine peptidases are important in the pentration of host skin.

Endemic human fasciolosis in the Bolivian Altiplano.

Fasciolosis, caused by trematodes of the genus Fasciola, is an emerging disease of humans. One of the highest levels of human fasciolosis hepatica is found amongst the indigenous Aymaran people of the Northern Bolivian Altiplano. A meta-analysis of epidemiological surveys from 38 communities in the region demonstrates that fasciolosis has been endemic in the region since at least 1984 and is a zoonosis of rural communities. Human and bovine fasciolosis is associated with the communities lying in the plain from Lake Titicaca to La Paz, predominantly in the Los Andes province. In Los Andes incidences of up to 67% of population cohorts were found, and prevalence is age-related with the highest infection rate in children aged 8-11 years.

Vaccines against the zoonotic trematodes Schistosoma japonicum, Fasciola hepatica and Fasciola gigantica.

Schistosoma japonicum, Fasciola hepatica and F. gigantica are digenetic trematodes and, therefore, possess similar life cycles. While schistosomiasis japonica has for a long time been recognised as a major disease of both humans and animals, infection with fasciolids has only been considered of relevance to animals. However, a number of recent reports indicate that fasciolosis is becoming a serious public health problem, especially in South America, Egypt and Iran (sporadic cases are also on the increase throughout Europe). Vaccines targeted at animals could play an important role in controlling these three diseases in animals and, by blocking transmission of infection, have a concurrent beneficial effect on disease in humans. Approaches towards identifying and producing vaccines against these parasites are similar and are discussed in this review.

Glycosidase activity in the excretory-secretory products of the liver fluke, Fasciola hepatica.

Fasciola hepatica secretes proteolytic enzymes and other molecules that are essential for host penetration and migration. This mixture may include enzymes required for the degradation of supramucosal gels, which defend epithelial surfaces against pathogen entry. These contain hydrated mucins that are heavily glycosylated. Excretory-secretory products (ES) from F. hepatica were examined for a range of glycosidase activities, using synthetic 4-methylumbelliferyl glycosides as substrates. The ES product contained at least 8 different glycosidase activities, the most abundant of which were beta-N-acetylhexosaminidase, beta-galactosidase and beta-glucosidase. Alpha-fucosidase, beta-glucuronidase, alpha-galactosidase, alpha-mannosidase and neuraminidase were also present. Beta-N-acetylhexosaminidase and beta-galactosidase were present in multiple isoforms (at least 4), whereas beta-glucosidase appeared to exist as one isoenzyme with a pI < 3.8. All three enzymes had acidic pH optima (4.5-5.0). Ovine small intestinal mucin was degraded by ES at pH 4.5 or 7.0, with or without active cathepsin L, the major protease found in F. hepatica ES. The ability of F. hepatica ES to degrade mucin in the presence or absence of active cathepsin L suggests that cathepsin L is not essential for mucin degradation. The abundance of beta-galactosidase and beta-hexosaminidase in ES supports a role for these enzymes in mucin degradation.

Helminths at mucosal barriers--interaction with the immune system.

Helminth parasites are the cause of very significant morbidity, mortality and economic losses in man and domestic animals. Most parasitic helminths infect their hosts via the oral route, and live either at the mucosal surface of the gastro-intestinal tract (GIT), or cross this mucosal barrier on their way to predilection sites. Many helminths live at mucosal surfaces, typically the gut or respiratory tract, and some cross these barriers, either temporarily, spending a period of time in the mucosa before returning to the mucosal surface, or to access other tissues and sites in the host. Typically, helminths induce strongly polarised Th2 responses, which are often effective in mediating protective immunity against those parasites living at mucosal surfaces, but less so in protecting against tissue-dwelling parasites. Induction of strongly-polarised Th2 responses may impair the ability of parasites hosts to eliminate other pathogens. Control of helminth infections relies largely on chemotherapy, together with management and environmental measures designed to keep hosts away from infective stages. Drug resistance has become a significant problem in some helminth populations, and this has promoted interest in the development of immunoprophylactic strategies. However, despite intensive research efforts, helminth vaccines have not become part of regular control strategies. In addition to the considerable technical difficulties posed in the production of vaccines against these complex organisms, further difficulties in securing acceptance for anti-helminth vaccine by regulatory authorities and by users, will be encountered. Such vaccines need not result in sterile immunity, as is required of anti-bacterial and anti-viral vaccines. Recent evidence indicates that while helminths are responsible for disease, immunopathology and impairment of immunity to other pathogens, a complete absence of helminth infection during early life may be a predisposing factor for the development of auto-immune pathology.

Pre-exposure of cattle to drug-abbreviated Fasciola hepatica infections: the effect upon subsequent challenge infection and the early immune response.

In this study we examined whether juvenile liver flukes are capable of stimulating protective immune responses in cattle. Four experimental groups of cattle were studied as follows: group A, a positive control, received a primary infection on day 0 and a secondary infection 28 days later; group B also received two infections but the primary infection was terminated by drug treatment on day 5; group C, received infections on days 0, 5 and 10 which were terminated by drug treatments on days 1, 6 and 11 and then a secondary infection on day 28; group D received an infection only on day 28. Juvenile flukes appear to induce protective responses because: (a) group B animals had significantly lower levels of gamma-GT (P<0.05) than group D; (b) both groups B and C exhibited lower parenchymal phase GLDH levels (P=0.006 and 0.041, respectively); and (c) both groups B and C had lower secondary phase eosinophilia (P=0.002 and 0.02, respectively) than those in group D. Sera taken from groups A-C contained antibodies reacting to a variety of proteins in adult fluke somatic antigen and excretory-secretory preparations, particularly to proteins of 52-60, 68-72 and 82-96 kDa. After secondary challenge the antibody responses of group A to these proteins declined while reactivity to proteins of 28-30 kDa increased. Antibody responses to the 28-30 kDa proteins were not detected in groups B-D until 3 weeks later than those observed in group A. Antibody responses to Fasciola hepatica cathepsin L proteases, which are known to induce protection, were monophasic, of the IgG1 isotype only and were not observed prior to secondary challenge in any of the four groups. In contrast, the response to another protective antigen fraction, a high molecular sized haem protein, was of a mixed IgG1/IgG2 nature and was detected within 14 days of primary infection. However, no significant difference in antibody titres to either protein preparation was observed after the secondary infection when groups B and C were compared to group D.

Fasciola hepatica cathepsin L cysteine proteinase suppresses Bordetella pertussis-specific interferon-gamma production in vivo.

We have previously demonstrated that Fasciola hepatica infection significantly reduced Bordetella pertussis-specific interferon (IFN)-gamma production in mice coinfected with B. pertussis or immunized with a pertussis whole cell vaccine (Pw). In the present study, we have identified parasite molecules capable of mimicking this suppressive effect of F. hepatica. Parenteral injection of mice with culture medium in which adult F. hepatica were maintained (excretory/secretory, ES, products) suppressed B. pertussis-specific IFN-gamma production in mice immunized with Pw. The suppressive effect of ES was abrogated by coinjecting ES with the cysteine proteinase inhibitor, Z-Phe-Ala-diazomethylketone. Furthermore, purified cathepsin L proteinase (FheCL), a major component of ES products, was capable of suppressing IFN-gamma production. The suppressive effect of FheCL was attenuated in interleukin (IL)-4 defective (IL-4-/-) mice. Therefore, FheCL released by F. hepatica is involved in the suppression of Th1 immune responses and this suppression may be dependent upon IL-4.

Analysis of antimalarial synergy between bestatin and endoprotease inhibitors using statistical response-surface modelling.

The pathway of hemoglobin degradation by erythrocytic stages of the human malarial parasite Plasmodium falciparum involves initial cleavages of globin chains, catalyzed by several endoproteases, followed by liberation of amino acids from the resulting peptides, probably by aminopeptidases. This pathway is considered a promising chemotherapeutic target, especially in view of the antimalarial synergy observed between inhibitors of aspartyl and cysteine endoproteases. We have applied response-surface modelling to assess antimalarial interactions between endoprotease and aminopeptidase inhibitors using cultured P. falciparum parasites. The synergies observed were consistent with a combined role of endoproteases and aminopeptidases in hemoglobin catabolism in this organism. As synergies between antimicrobial agents are often inferred without proper statistical analysis, the model used may be widely applied in studies of antimicrobial drug interactions.

The role of aminopeptidases in haemoglobin degradation in Plasmodium falciparum-infected erythrocytes.

Intra-erythrocytic Plasmodium parasites digest host cell haemoglobin and use the liberated amino acids for protein synthesis. Although several endoproteases (aspartic, cysteine, and metallo-) have been shown to be involved in the initial stages of haemoglobin degradation, little is known about the steps immediately before amino acid release. In our studies, fluorometric enzyme assays indicated that the stage of the P. falciparum erythrocytic cycle with highest aminopeptidase activity was the stage at which most haemoglobin degradation occurs, i.e. the trophozoite. Consistent with these results, metabolic growth assays indicated that the late ring/trophozoite stage was most susceptible to aminopeptidase inhibitors. To reconstitute the terminal stages of haemoglobin breakdown in vitro, we synthesised three peptides with amino acid sequences corresponding to known products of the endoproteolytic digestion of haemoglobin and employed them as substrates for aminopeptidases. Both trophozoite cytosolic extract, and partially-purified aminopeptidase, hydrolysed these peptide fragments to amino acids. Hydrolysis appeared to occur sequentially from the amino-termini of the peptides, and was inhibited in a concentration-dependent manner by the aminopeptidase-specific inhibitor nitrobestatin. The results suggest that P. falciparum aminopeptidases could be the enzymes responsible for the hydrolysis of haemoglobin-derived peptides to free amino acids. Lack of ultrastructural change in parasites treated with relevant concentrations of aminopeptidase-specific inhibitors, however, indicated that little feedback exists whereby the inhibition of cytosolic aminopeptidases results in obvious inhibition of initial haemoglobin degradation in the digestive vacuole.

Characterisation of a beta-tubulin gene from the liver fluke, Fasciola hepatica.

This study represents the first beta-tubulin sequence from a trematode parasite, namely, the liver fluke, Fasciola hepatica. PCR of genomic DNA showed that at least one beta-tubulin gene from F. hepatica contains no introns. A number of amino acids in the primary sequence of fluke tubulin are different from those described previously in various nematode species and the cestode, Echinococcus multilocularis. beta-Tubulin is an important target for benzimidazole anthelmintics, although (with the exception of triclabendazole) they show limited activity against F. hepatica. The amino acid differences in fluke beta-tubulin are discussed in relation to the selective toxicity of benzimidazoles against helminths and the mechanism of drug resistance.

Parasite vaccines--a reality?

Over the last decade, the anti-parasitics market has been the fastest growing sector of the overall $18 billion animal health market. While drugs for the treatment of parasites of livestock still dominate this sector and will continue to be developed or re-formulated, because of consumer demands for chemical-free food and of concerns regarding the environment and animal welfare there is a growing interest in the development of safe and effective vaccines. There is also a call for vaccines in the lucrative $3 billion-plus companion animal market. These demands for vaccines will add a greater impetus to an area that has seen tremendous success in the last 15 years. A number of anti-parasite vaccines have been developed, e.g. the recombinant 45w and EG95 oncosphere proteins against Taenia ovis and Echinococcus granulosis, respectively, and the Bm86 vaccine against Boophilus microplus. In addition, the cathepsin L vaccines against the liver fluke, Fasciola hepatica, and the H11 vaccine against Haemonchus contortus are progressing well. There are also many additional vaccine candidates for H. contortus and for other nematodes such as Ostertagia and Trichostrongylus spp. that may ultimately lead to broad-spectrum gastrointestinal worm vaccines. Live or attenuated-live vaccines are available for the control of avian coccidiosis, toxplasmosis in sheep and anaplasmosis in cattle, although molecular vaccines against protozoans are still proving elusive. The wealth of information in genomics, proteomics and immunology that has been forthcoming together will new methods of vaccine production and delivery should see many new vaccines reach the marketplace in the near future.

Gut-associated immunolocalization of the Schistosoma mansoni cysteine proteases, SmCL1 and SmCL2.

Transcripts encoding discrete, cathepsin L-like cysteine proteases, known as SmCL1 and SmCL2, have been reported from the adult stages of the human blood fluke Schistosoma mansoni. However, the physiological roles of these 2 enzymes and their natural substrates remain uncertain and controversial. To determine their localization in adult S. mansoni by immunocytochemical procedures, and thereby to gain insight into their likely functions, polymerase chain reaction-based cDNAs encoding mature SmCL1 and SmCL2 were ligated into Escherichia coli. The bacterially expressed recombinant proteins (bSmSL1, bSmCL2) were used to generate monospecific rabbit antisera. For light microscopy, paraffin-embedded sections were visualized with the fluorophore Cy3. For transmission electron microscopy (TEM), LR White-embedded tissue was visualized with 15 nm gold. Under light microscopy, fluorescence was visible on the luminal surface of the gastrodermis in both sexes for both proteins. For bSmCL1 and bSmCL2, TEM revealed gold particles primarily associated with amorphous deposits within superficial digestive vacuoles on the gastrodermal surface of males and females. Some bSmCL1 reaction product was observed in vesicles within the gastrodermis, and very sparse gastrodermal activity was observed with bSmCL2. By contrast, neither enzyme was immunolocalized in the reproductive organs, vitelline glands, nor gynecorphoric canal. The gut-associated immunolocalization of SmCL1 and SmCL2 indicates that both these endopeptidases participate in hemoglobin proteolysis.

Cathepsin L proteinases as vaccines against infection with Fasciola hepatica (liver fluke) in ruminants.

Fasciolosis, caused by trematodes of the genus Fasciola, is one of the most important diseases of farmed ruminants in temperate and tropical zones. The appearance of Fasciola hepatica populations that are resistant to common flukicidal drugs means that new methods of treatment will soon be required. The future prospect for the development of anti-liver fluke vaccines is optimistic and given their consumer acceptability and environmental friendliness, offer the best way forward. Cathepsin L proteases (Fhe CL 1 and Fhe CL 2), secreted by liver flukes at all stages of their development in the mammalian host, are believed to play important roles in facilitating parasite migration (tissue degradation), feeding and immuno-evasion. The authors consider them prime targets for which new vaccines can be developed. Vaccine studies in cattle and sheep have shown that protection levels of up to 72 and 79 per cent, respectively, can be obtained with immunisations of cathepsin Ls in Freunds' adjuvant. The vaccine also exhibited high anti-embryonation/anti-fecundity effects on parasites that survived in vaccinated animals and thus could have a major impact on the transmission of disease to the intermediate host. While natural infections in sheep and cattle appear to elicit non-protective Th2 immune responses, the authors' studies indicate that the protection induced by vaccination involves elements of a Th1 response.