Combined use of two separate but protective vaccine antigens provides protection against Taenia ovis infection in lambs in the presence of protective maternal antibody.

Three recombinant Taenia ovis antigens (To45, To16, To18) each induce protective immunity in lambs or ewes against infection with T. ovis metacestodes. The degree and duration of immunity were assessed in lambs born from vaccinated ewes. Treatment group sizes varied, typically not fewer than 5 animals per group. Ewes were immunised with one T. ovis recombinant protein prior to lambing and the degree and duration of passive immunity in their lambs was assessed by challenge infection up to 18 weeks. Lambs were fully protected up to 6 weeks of age but immunity waned from 6 to 12 weeks and there was no protection when lambs were challenged at 15 weeks. Immunisation of lambs with the homologous recombinant antigen was not effective when vaccinations were given when maternal antibody was high. Lambs were effectively immunised in the presence of passively protective antibody when vaccinated with an antigen that was different to that given to ewes. Vaccination of lambs with a combination of two proteins, To16 and To18, was more effective than giving these single antigens and gave a significant reduction of cyst numbers when lambs were challenged 12 months after immunisation. These results indicate that the use of combinations of T. ovis recombinant antigens could enable complete protection of lambs against infection, if a delivery system becomes available that will maintain antibody at protective levels for 12 months. Alternatively, a third injection given at 6 months may promote the anamnestic response to give long lasting protection.

Immunological responses and potency of the EG95NC- recombinant sheep vaccine against cystic echinococcosis.

Cystic echinococcosis (CE) is a zoonotic disease caused by the cestode parasite Echinococcus granulosus. The disease has an important impact on human health as well as economic costs including the cost of treatment as well as loss of productivity for the livestock industry. In many parts of the world where the disease is endemic, sheep and other livestock play an important role in the parasite's transmission. A vaccine to protect livestock against CE can be effective in reducing transmission and economic costs of the disease. A recombinant antigen vaccine has been developed against infection with E. granulosus (EG95) which could potentially be used to reduce the level of E. granulosus transmission and decrease the incidence of human infections. Further development of the EG95 recombinant vaccine as a combined product with clostridial vaccine antigens is one potential strategy which could improve application of the hydatid vaccine by providing an indirect economic incentive to livestock owners to vaccinate against CE. In this study we investigated the efficacy of the EG95 recombinant vaccine produced in Morocco by vaccination of sheep, including a combined vaccine incorporating EG95 and clostridia antigens. Vaccination with EG95 either as a monovalent vaccine or combined with clostridia antigens, protected sheep against a challenge infection with E. granulosus eggs and induced a strong, long lasting, and specific antibody response against the EG95 antigen.

The first report of hydatid disease (Echinococcus granulosus) in an Australian water buffalo (Bubalus bubalis).

A three year old female water buffalo was slaughtered for human consumption on a dairy buffalo farm in eastern New South Wales, Australia. Gross examination of the offal revealed four small, superficial hydatid cysts in the liver and two larger superficial cysts in one lung. All organs were sliced and no other cysts were found. Histology and PCR confirmed the cysts to be cysts of Echinococcus granulosus senso stricto. None of the cysts contained protoscoleces. The source ofinfection is equivocal, but it is most likely from E. granulosus eggs passed in the faeces of wild dogs (dingoes and dingo-wild dog hybrids). Wild dogs are resident in the bush that abuts the farm boundary and from time to time wild dogs are seen in the buffalo paddocks on the farm. Sylvatic transmission of E. granulosus occurs commonly in eastern Australia through a predator/prey interaction between wild dogs and macropod marsupials.

The HepHIV 2017 Conference in Malta: joining forces for the earlier diagnosis of HIV and viral hepatitis.

OBJECTIVES: The objective of the article is to provide an overview of the results of the HepHIV 2017 Conference organized by the HIV in Europe initiative under the Maltese EU Presidency in January 2017. METHODS: A thourough review of all conference presentations (oral and poster presentations) was performed to retrieve the key outcomes of the conference. RESULTS: The key result from the conference was a call to action summarising key priorities in HIV and viral hepatitis testing and linkage to care. This included improving monitoring of viral hepatitis and HIV, mixing testing strategies and ensuring policy support. The important contribution and outcomes of EU funded projects OptTEST and EuroHIVEdat was highlighted. CONCLUSION: An integrated approach to earlier testing and linkage to care across diseases is needed in Europe and the HepHIV conferences create an important forum to reach this aim.

Vaccination Against Hydatidosis: Molecular Cloning and Optimal Expression of the EG95NC- Recombinant Antigen in Escherichia coli.

Cystic echinococcosis (CE) is a widely distributed zoonosis that is highly endemic in the Mediterranean basin. The disease represents a serious public health threat and causes economic losses. The parasite life-cycle involves dogs and ruminants as definitive and intermediate hosts; humans are accidently infected, causing serious clinical issues. Vaccination of ruminants and dog treatments represent the most efficient measures to prevent parasite transmission. The recombinant protein vaccine, EG95, has been used successfully in sheep vaccine trials against CE in several countries. In this study, we expressed the modified antigen, EG95NC-GST, in Escherichia coli for use as a vaccine against Echinococcus granulosus in ruminants. We tested three different media formulations for E. coli culture and established for each culture conditions for optimal levels of soluble EG95 expression. The results demonstrate that SOC and TB media provided high yields in cell density and EG95 protein expression. Purification of the recombinant protein with affinity chromatography (using FPLC) was also performed to increase the purity of the EG95NC--GST antigen.

Microdiversity of Echinococcus granulosus sensu stricto in Australia.

Echinococcus granulosus (sensu lato) is now recognized as an assemblage of cryptic species, which differ considerably in morphology, development, host specificity (including infectivity/pathogenicity for humans) and other aspects. One of these species, E. granulosus sensu stricto (s.s.), is now clearly identified as the principal agent causing cystic echinococcosis in humans. Previous studies of a small section of the cox1 and nadh1 genes identified two variants of E. granulosus s.s. to be present in Australia; however, no further work has been carried out to characterize the microdiversity of the parasite in its territory. We have analysed the sequence of the full length of the cox1 gene (1609 bp) from 37 isolates of E. granulosus from different hosts and geographic regions of Australia. The analysis shows that seven haplotypes of E. granulosus s.s. not previously described were found, together with five haplotypes known to be present in other parts of the world, including the haplotype EG01 which is widespread and present in all endemic regions. These data extend knowledge related to the geographical spread and host range of E. granulosus s.s. in a country such as Australia in which the parasite established around 200 years ago.

Monitoring the outcomes of interventions against Taenia solium: options and suggestions.

There is an increasing interest in reducing the incidence of human neurocysticercosis, caused by infection with the larval stage of Taenia solium. Several intervention trials are currently assessing various options for control of T. solium transmission. A critical aspect of these trials will be the evaluation of whether the interventions have been successful. However, there is no consensus about the most appropriate or valuable methods that should be used. Here, we undertake a critical assessment of the diagnostic tests which are currently available for human T. solium taeniasis and human and porcine cysticercosis, as well as their suitability for evaluation of intervention trial outcomes. Suggestions are made about which of the measures that are available for evaluation of T. solium interventions would be most suitable, and which methodologies are the most appropriate given currently available technologies. Suggestions are also made in relation to the most urgent research needs in order to address deficiencies in current diagnostic methods.

Sensitivity of partial carcass dissection for assessment of porcine cysticercosis at necropsy.

Many interventions against Taenia solium are evaluated by assessing changes in the prevalence of porcine cysticercosis ascertained by carcass dissection. Financial and logistical difficulties often prohibit dissection of entire pig carcasses. We assessed 209 pigs from rural areas of Cameroon and Peru for the presence of T. solium cysticerci and determined the distribution of parasites within the musculature of infected animals. Considering the presence of cysts in the tongue, masticatory muscles and heart, 31 of the 38 (81%) naturally infected animals were identified as having cysts. Dissection of only the tongue, masticatory muscles and heart provides a relatively sensitive and highly specific method for diagnosis of porcine cysticercosis.

Antigenic differences between the EG95-related proteins from Echinococcus granulosus G1 and G6 genotypes: implications for vaccination.

Cystic echinococcosis caused by Echinococcus granulosus remains an important and neglected issue in public health. The study of the likely efficacy of the currently available EG95 vaccine against other genotypes of the parasite is important to improve the vaccine as a potential tool to be used in control programmes. The recombinant vaccine EG95-1G1 was developed based on the G1 genotype of E. granulosus. Characterization of the eg95 gene family in the G6 genotype by genomic DNA cloning previously produced the first unequivocal information about the composition of the gene family in a different genotype. The information was used in this study to predict and express two EG95-related proteins from the G6 genotype as recombinants, for assessment of their capacity to bind antibodies raised in sheep vaccinated with the EG95-1G1 vaccine. The proteins (EG95-1G6 and EG95-5G6) from the G6 genotype of E. granulosus were unable to bind all the antibodies raised by sheep vaccinated with EG95-1G1. Differences in the amino acid sequence of EG95-related proteins from G6 and likely the differences in the encoded FnIII domain may be responsible for changes in the conformation of these epitopes.

Oncospheral penetration glands are the source of the EG95 vaccine antigen against cystic hydatid disease.

Immunohistochemistry and immunogold labelling techniques were used to localize the EG95 vaccine antigen in Echinococcus granulosus oncospheres. In non-activated oncospheres, the cytoplasm of 2 pairs of bilateral cells exhibited specific positive labelling for the presence of EG95. No surface localization was seen in non-activated or recently activated oncospheres. Besides the staining of 2 pairs of bilateral cells, there was also a generalized distribution of specific staining for EG95 throughout the parenchyma of activated oncospheres. Immunogold labelling of non-activated oncosphere revealed specific reactivity for EG95 involving 2 pairs of bilateral cells and the ultrastructural characteristics of these cells were consistent with them being penetration gland cells. No other oncospheral structures stained specifically for the presence of EG95. The absence of surface location of EG95 in oncospheres suggests that the parasite may not be susceptible to vaccine-induced antibody and complement mediated attack until some post-oncospheral development has occurred. Further studies would be required to determine when the EG95 antigen associates with the parasite's surface, thus making them susceptible to immune attack.

Variation in the cellular localization of host-protective oncospheral antigens in Taenia saginata and Taenia solium.

Immunohistochemistry and immunofluorescence with confocal microscopy were used to localize the host-protective antigens of Taenia saginata (TSA9 and TSA18) and Taenia solium (TSOL16, TSOL18 and TSOL45). In nonactivated oncospheres, TSA9 and TSOL45 antigens were found primarily in the cytoplasm of the penetration gland type one (PG1) cell. A similar pattern of staining was seen for TSOL45 in oncospheres of T. solium that remained within the oncospheral membrane. In addition, there was less intense staining of TSA9 and TSOL45 in the quadri-nucleate penetration gland type 2 (PG2) cell. TSA18, TSOL16 and TSOL18 were predominantly found in the PG2 cell. In activated oncospheres that had escaped the oncospheral membrane, the antigens (other than TSA9) were seen both in the penetration gland cell locations and throughout the oncospheral parenchyma. Co-localization analyses revealed that only TSOL16 and TSOL18 antigens were co-localized in the PG2 cell of oncospheres that had not escaped the oncospheral membrane. However, in activated oncospheres that escaped the oncospheral membrane, all three antigens of T. solium were co-localized as they were present throughout the parenchyma. No positive staining was observed on the surface of nonactivated or recently activated oncospheres of T. saginata or T. solium.

Antibody responses to the host-protective Taenia solium oncosphere protein TSOL18 in pigs are directed against conformational epitopes.

TSOL18 is a recombinant protein that has been shown in repeated experimental trials to be capable of protecting pigs against challenge infection with the cestode parasite Taenia solium. Antibodies raised by the vaccine are capable of killing the parasite in an in vitro culture and it is believed that antibody and complement-mediated killing of invading parasites is the major protective immune mechanism induced by vaccination with TSOL18. Investigations were undertaken to characterize whether the principal antibody specificities raised by TSOL18 in pigs were against linear or conformational determinants. TSOL18 was expressed in two truncated forms representing either the amino terminal portion or the carboxy terminal portion, with the two truncations overlapping in sequence by 25 amino acids. The original protein (designated TSOL18N(-)) and the two truncations (TSOL18N(-)-1 and TSOL18N(-)-2) were used in inhibition ELISA. TSOL18N(-) was shown to be capable of completely inhibiting the binding of pig anti-TSOL18N(-) antibodies to TSOL18N(-) in ELISA. However, neither TSOL18N(-)-1 nor TSOL18N(-)-2, either alone or when combined together, was capable of inhibiting any detectable amount of reactivity of pig anti-TSOL18N(-) antibodies with TSOL18N(-). It is concluded that the dominant antibody specificities, and probably the host-protective specificities, of TSOL18 are conformational epitopes.

Preliminary field trial of a vaccine against coenurosis caused by Taenia multiceps.

Taenia multiceps is a taeniid cestode that in its adult stage lives in the small intestine of dogs and other canids. In the intermediate hosts, the larval stage of T. multiceps causes coenurosis, a common disease in the CNS of ruminants, which typically leads to the death of the infected animals. Recent research into new methods for control of coenurosis and other taeniid cestode infections such as hydatidosis has identified vaccination as a potentially valuable new tool. In order to test the applicability of vaccination as an approach for control of T. multiceps infection in sheep, a field trial was carried out against natural infection in Sardinian farms (Italy) with recombinant proteins of T. multiceps. The recombinant proteins with Quil A as adjuvant were injected subcutaneously, the first administered to lambs at 10-12 weeks of age and a booster dose given after 2-4 weeks. A total of 632 sheep were selected, belonging to the "replacement quota" of six different farms, of which 424 were used as controls (unvaccinated) and 208 were vaccinated. After a period of more than 40 months from the beginning of the field trial, 33 episodes of cerebral coenurosis occurred in the monitored farms, including 32 cases in control sheep and l case in a vaccinated animal. Statistical analysis revealed a significant reduction in the number of coenurosis cases in the vaccinated animals (chi(2)=14.08, P<0.001). This is the first successful field test of a practical vaccine against T. multiceps and, considering the high degree of effectiveness achieved, could be a prelude to routine application in field situations of particular risk, such as Sardinia.

Purification of polyclonal anti-conformational antibodies for use in affinity selection from random peptide phage display libraries: a study using the hydatid vaccine EG95.

The use of polyclonal antibodies to screen random peptide phage display libraries often results in the recognition of a large number of peptides that mimic linear epitopes on various proteins. There appears to be a bias in the use of this technology toward the selection of peptides that mimic linear epitopes. In many circumstances the correct folding of a protein immunogen is required for conferring protection. The use of random peptide phage display libraries to identify peptide mimics of conformational epitopes in these cases requires a strategy for overcoming this bias. Conformational epitopes on the hydatid vaccine EG95 have been shown to result in protective immunity in sheep, whereas linear epitopes are not protective. In this paper we describe a strategy that results in the purification of polyclonal antibodies directed against conformational epitopes while eliminating antibodies directed against linear epitopes. These affinity purified antibodies were then used to select a peptide from a random peptide phage display library that has the capacity to mimic conformational epitopes on EG95. This peptide was subsequently used to affinity purify monospecific antibodies against EG95.

Efficacy of the EG95 hydatid vaccine in a macropodid host, the tammar wallaby.

In Australia, macropodids are common intermediate hosts for the cestode Echinococcus granulosus, and sylvatic transmission is maintained via wild dogs. The parasite causes mortality in a number of macropodid species and the sylvatic cycle provides a source of infection to domestic livestock and humans. We determined the efficacy of the hydatid vaccine, EG95 in the tammar wallaby, Macropus eugenii, challenging either 1 or 9 months post-vaccination. EG95 provides similar protection to that seen in sheep (96-100%). Control tammars were significantly more likely to become infected (odds ratio 29.44; CI 4.13, 209.97; P=0.001) and to develop more cysts (count ratio 26.69; CI 5.83, 122.19; P<0.001). The vaccination may be beneficial if administered pre-release in captive breeding programmes for endangered macropodids. Further work to develop oral delivery methods may enable vaccine administration of wild animals and thereby a reduction in sylvatic transmission.

Isolation of antibodies specific to a single conformation-dependant antigenic determinant on the EG95 hydatid vaccine.

EG95 is a recombinant vaccine protein that elicits protection against hydatid disease in sheep. Previous studies have shown that the host-protective epitopes on EG95 depend on correct conformation and cannot be represented by simple "linear" peptides. By screening random peptide phage display libraries with polyclonal antibodies directed against conformation-dependant epitopes of EG95, we have selected a number of peptides that mimic these epitopes. The selected peptides did not show sequence homology to EG95. Antigen binding assays involving these peptides have provided evidence of at least four conformationally-dependant epitope regions on EG95. One of the selected peptides, E100, has been used to purify antibodies from anti-sera raised in sheep vaccinated with EG95. This yielded monospecific antibodies capable of recognizing recombinant EG95 in ELISA and native EG95 in Western blot assays. This antibody was demonstrated to be effective in antibody-dependant complement-mediated in vitro killing of Echinococcus granulosus oncospheres. Peptide E100 may represent the basis for a quality control assay for EG95 production, and has the potential to become a component of a synthetic peptide-based vaccine against E. granulosus.

A common, symptom-based case definition for gastroenteritis.

National studies determining the burden of gastroenteritis have defined gastroenteritis by its clinical picture, using symptoms to classify cases and non-cases. The use of different case definitions has complicated inter-country comparisons. We selected four case definitions from the literature, applied these to population data from Australia, Canada, Ireland, Malta and the United States, and evaluated how the epidemiology of illness varied. Based on the results, we developed a standard case definition. The choice of case definition impacted on the observed incidence of gastroenteritis, with a 1.5-2.1 times difference between definitions in a given country. The proportion of cases with bloody diarrhoea, fever, and the proportion who sought medical care and submitted a stool sample also varied. The mean age of cases varied by <5 years under the four definitions. To ensure comparability of results between studies, we recommend a standard symptom-based case definition, and minimum set of results to be reported.

Kawasaki disease presenting as hepatitis.

Kawasaki disease (KD) presenting with an acute hepatitic picture is exceedingly rare. A 3.5-year-old girl presented with acute hepatitis and went on to satisfy the criteria for the diagnosis of KD for which she was treated. There were no cardiac abnormalities or sequelae.

General practitioners role in the notification of communicable diseases - study in Malta.

General practitioners (GPs) have an essential role in notification of communicable diseases. The main aim of the study described here was to assess the GPs' awareness of and attitudes towards the notification system in Malta, with special focus on infectious intestinal disease (IID). A questionnaire collecting demographic data, information on reporting practices, opinions on the existing notification system and suggestions for improvement was sent to 256 GPs working in either private or public health sector. In all, 150 GPs took part in the survey (response rate 58.6%). The responses revealed that Maltese GPs were aware of their obligations to notify communicable diseases but often did not report them, relying on the hospitals or laboratories to do so. The Disease Surveillance Unit (DSU) website and medical school training were the main sources of information on notification. Notification forms were obtained from health centres and usually kept at the place of work. Most GPs reported filling in the forms during the patients' visits. Private GPs tended to notify earlier than GPs working in public health centers. Among IID, food-borne illness was reported more frequently than person-to-person transmitted gastroenteritis and was considered to be of a higher priority with regard to public health importance (p<0.001). The survey highlighted also some areas for improvement, including need of feedback especially by direct communication or a newsletter.