Abstract

Dogs are pivotal in 00 decals YZF450 Skull Yamaha Graphics 02 YZF250 sticker 450 Kit 250 Shroud YZF Echinococcus granulosus transmission to humans, and dog vaccination provides a very practical and cost-effective prevention strategy. We vaccinated dogs with soluble native proteins isolated from protoscoleces of E. granulosus and induced significant suppression of worm growth and egg production. Accordingly, we tested for vaccine efficacy using recombinant proteins derived from a developmentally regulated gene family (egM) specifically expressed in mature adult E. granulosus worms. Three egM genes—egM4, egM9, and egM123—were subcloned into an expression vector that expressed the molecules as soluble glutathione S-transferase (GST) fusion proteins in Escherichia coli. The 3 fusion proteins were purified for dog vaccination trials (3 doses of 80 μg/protein/dog) in which the dogs were challenged and then necropsied 45 days after infection. Compared with worms in the control dogs that received GST, the 3 recombinant proteins induced a very high level of protection (97%–100%) in terms of suppression of worm growth and, especially, of egg development and embryogenesis. We have thus shown that vaccination of the dog host against E. granulosus is feasible when recombinant proteins are used. Because the egg stage is crucial in the echinococcal life cycle, successful suppression of egg development by vaccination would halt transmission to intermediate hosts, thereby effecting long-term control

Echinococcosis is a zoonosis caused by cestodes of the genus Echinococcus (family Taeniidae). The species of major public-health importance are E. granulosus the agent of cystic echinococcosis (CE), and E. multilocularis the agent of alveolar echinococcosis. In humans and domestic livestock, especially sheep, larval infection is characterized by long-term growth of hydatid cysts that contain brood capsules and protoscoleces (PSCs). Definitive hosts are carnivores, such as dogs, wolves, and foxes; sexual maturity of adult E. granulosus occurs in the small intestine within 4–5 weeks of the ingestion of offal containing viable PSCs. Gravid proglottids or released eggs pass with feces; after ingestion by a human or ungulate host, they hatch to liberate an oncosphere, which forms a hydatid cyst in body tissues

Although substantial efforts have been made to control E. granulosus [1], these have not always been successful. Vaccination can provide an adjunct to improved, integrated control. This area has advanced considerably in recent years with the development and deployment of an effective recombinant vaccine antigen (EG95) that targets infection in the intermediate host [2]. By comparison, dog immunization has received relatively little attention, yet dogs, as definitive hosts, are pivotal in the transmission of CE. For example, monthly treatment of dogs with praziquantel resulted in significant reductions in rates of E. granulosus infection in sheep [3]. However, this high frequency of treatment has to be maintained for a considerable period (decades), because dogs are quickly reinfected. This treatment regimen is costly and difficult to manage. New measures are required to obtain long-term control [4, 5]. Dog vaccination can provide an acceptable and cost-effective complementary control method, because there are far fewer dogs than sheep in areas where the parasite is endemic [6]; hence, far fewer animals need to be vaccinated

Epidemiological evidence has indicated that dogs and other canids can naturally acquire resistance against E. granulosus infection [7–11]. Furthermore, a recent mathematical model has indicated that there is significant herd immunity in dogs under relatively high infection pressure [12]. A series of early studies aimed at inducing immunity in dogs through vaccination with various antigenic sources produced encouraging results. Turner et al., in pioneering vaccination studies [13, 14], induced significant resistance in dogs using antigens prepared from PSCs and membranes. Gemmell [15] injected dogs with freeze-dried, powdered PSCs and found that this suppressed the growth of the terminal segments of worms in a challenge infection. Oral immunization of dogs with irradiated PSCs induced resistance that limited the numbers of established worms that were able to develop to the gravid stage after challenge infection [16–18]. Herd et al. [19, 20] used excretory-secretory antigens of adult E. granulosus worms to vaccinate dogs, which resulted in a reduction in worm numbers and a highly significant suppression of egg production

Here, we describe vaccination and challenge experiments against E. granulosus in dogs using, to our knowledge for the first time, soluble native PSC proteins and recombinant proteins expressed by mature adult worms (MAWs) [21], which resulted in significant suppression of worm growth and egg production

Materials and Methods

Preparation of native soluble proteins from PSCsPSCs of E. granulosus were aspirated from hydatid cysts in sheep livers collected from a local slaughterhouse in Urumqi, Xinjiang, China. After 10 washes with PBS (pH 7.2), PSCs were frozen and thawed twice, then subjected to ultrasonication on ice (twice for 4 min each), using a medium pulse (50% duty cycle; Sonifier 250; Branson) to lyse the cells. The homogenate was cleared by ultracentrifugation at 100,000 g for 30 min. After the protein concentration was measured [22], the supernatant was used for injection immediately or stored at −80°C for ELISA

Expression and purification of fusion proteinsIsolation of a gene family (egM) expressed in MAWs that is associated with egg development and the cDNA cloning and expression of 3 constituent members—egM4, egM9, and egM123 (for MINT Mamiya 5 PRO 1000S 408 F 645 NEAR Japan TL from sekor 3 C 35mm N zd5WT accession numbers AF482719, AF482720, and AF482718)—used in the vaccination experiment have been described elsewhere [21]. For serological testing, egM cDNAs were subcloned into the pCal-n vector (Stratagene), which contains a calmodulin-binding protein (CBP) tag. The CBP fusion EgM proteins were purified using CBP affinity purification resin (Stratagene)

Immunization and challenge of dogsAll protocols were approved by the ethics committee of Xinjiang Academy of Animal Science. Dogs (1–3 years-old; average age, 14 months) were purchased from areas in Xinjiang, China, where hydatid control programs using monthly dosing of dogs with baited praziquantel medicine had been in operation for 5 years without any tapeworm infection [23]. The dogs were housed in a quarantine facility at the Veterinary Research Institute, Urumqi, Xinjiang. Dogs were maintained on boiled sheep meat, dog biscuits, and tap water

To test the protection of dogs with native soluble PSC proteins, 6 dogs were given 3 injections with 0.25, 0.125, and 0.125 mg of soluble PSC proteins (low-dose group; L1–L6), and a further 6 dogs were injected with 0.5, 0.25, and 0.25 mg of PSC proteins (high-dose group; H1–H6). The PSC proteins were emulsified with complete Freund’s adjuvant (CFA), incomplete Freund’s adjuvant (IFA), and IFA for the first, second, and third injection, respectively, and administered subcutaneously at an interval of 3 weeks. Five dogs in a control group (C1–C5) received the same schedule of injections, but PBS was used instead of PSC proteins

A total of 5 groups, including 2 control (PBS and glutathione S-transferase [GST]) groups, were used to test recombinant EgM4, EgM9, and EgM123 for protection of dogs against E. granulosus challenge. Dogs were first administered 80 μg of recombinant fusion protein in CFA by subcutaneous injection, followed by 2 subcutaneous injections with 80 μg of protein in IFA at 3-week intervals. Dogs in 2 control groups were administered PBS or 80 μg of GST emulsified with adjuvant on the same schedule as the experimental groups

All dogs were challenged orally with 480,000 PSCs (obtained freshly from sheep livers) 2 weeks after the third vaccination. Forty days after challenge, all dogs were monitored visually every 48 h for proglottids in feces or for single eggs on the anal area using cellophane tape strips and microscopic examination [24]. Dogs were euthanized and necropsied 45–46 days after infection. The small intestine was freed immediately from the mesentery and opened longitudinally. The portion containing E. granulosus worms was cut into 20-cm lengths, which were incubated in PBS at 37°C. Most worms were released naturally within 1 h, and the remainder were scraped carefully from the intestine using a microscope slide. The worms were washed twice with warm PBS and then fixed in 70% (vol/vol) ethanol or 4% (vol/vol) paraformaldehyde in PBS before counting (all worms) and microscopic examination (a subset of 10%–15% of the worms)

Serum collectionSerum samples were collected from dogs weekly from the neck vein. After samples stood at room temperature for 30 min, blood clots were loosened with sterile tips, and the serum was cleared by centrifugation at 2000 g for 30 min at 4°C. Aliquots were stored at −80°C for further use

ELISAELISA was performed to measure levels of immunoglobulin and IgG subclasses in dog serum. Microtiter plate wells were coated overnight at 4°C with 100 μL of 5 μg/mL of soluble PSC proteins or with 0.5 μg/mL of recombinant CBP-EgM protein in 0.05 mol/L bicarbonate buffer (pH 9.6). After PBS washes and blocking in 5% (wt/vol) skim milk/PBS for 1 h at 37°C, wells were exposed to 100 μL of dog serum diluted 1:200 with 5% (wt/vol) skim milk in PBS that contained 0.1% Tween-20 (PBST), and plates were incubated at 4°C overnight. After 3 PBST washes, plates were incubated with goat anti–dog immunoglobulin (IgG, IgG1, IgG2, IgGa, IgE, and IgM) horseradish peroxidase conjugates (Bethyl Laboratory) diluted in accordance with the manufacturer’s instructions with PBST/5% skim milk for 1 h at 37°C. The color reaction was developed by adding 100 μL of 2,2-azino-bis (ethylbenz-thiazoline-6-sulfonic acid) substrate solution to each wall after the plates were washed 3 times with PBST. The absorbance was read using an automatic microplate reader at 405 nm after incubating the plates at room temperature for 15 min in a dark box

Western-blot analysisNative purified PSC proteins and recombinant proteins were separated by 12% SDS-PAGE. The proteins were then transferred to nitrocellulose membrane (NCM). The NCM was blocked with 5% (wt/vol) skim milk in PBS for 1 h at 37°C, incubated with dog serum that had been preabsorbed with Escherichia coli lysate or recombinant GST, as described elsewhere [25], diluted 1:100, and then incubated with anti–dog IgG conjugate (diluted 1:1000; Sigma) at 37°C. After 3 washes with PBST, recognized bands were developed by soaking the NCM in 4-chloro-1-naphal substrate solution

In situ hybridizationParaffin-embedded 4% (vol/vol) paraformaldehyde–fixed parasite tissues were cut to 5-μm thickness and mounted on Colorfrost/Plus microscope slides (Fisher Scientific). For RNA probe hybridization, the egM123 cDNA fragment was subcloned into the pSPT19 vector (Roche Molecular Biochemicals) and transformed into JM109 cells. Plasmid was purified using a QIAprep Spin Miniprep Kit (QIAGEN) and then digested with EcoRI or XbaI to obtain linear plasmid DNA. Digoxigenin (DIG)–labeled sense and antisense cRNA were synthesized by in vitro transcription from the flanking T7 and T3 RNA polymerase. The probes were hydrolyzed into 100 nt using alkaline solution (60 mmol/L sodium carbonate and 40 mmol/L sodium hydrocarbonate) for 30 min and then precipitated with 50% isopropanol. The pellet was washed with 75% ethanol and stored at −80°C

Tissue section pretreatment, prehybridization, and hybridization were performed in accordance with online protocols from Roche Molecular Biochemicals. Color was developed using anti-DIG Fab fragments (from sheep) conjugated to alkaline phosphatase and the nitroblue tetrazolium/bromochloroindolyl phosphate colorimetric detection system (Roche)

Statistical analysisThe Mann-Whitney YZF450 450 00 Skull 02 Graphics Kit YZF250 YZF decals Yamaha 250 sticker Shroud U test was used to compare worm burdens and ELISA optical densities in experimental and control groups using SPSS software (release 10.0; SPSS). The χ2 test was used to compare the percentage of worms recovered in the experimental and control groups. Spearman’s&amp;rank correlation was used to analyze the correlation between worm burden and serum optical-density values in ELISA. P<.05 was taken to indicate statistically significant differences

Results

Vaccination of Dogs with Soluble Native PSC Proteins

Challenge infection in the control group resulted in ∼55% of PSCs developing into adult worms (Music Player Lossless Playback Supports to CFZC up 64GB 8GB 70 Sound Hours MP3 qRS0Aw4). In contrast, vaccination induced a significant suppression of growth and egg production. The burden of worms in the high-dose group was significantly lower than that in the control group (P<.01) (Music Player Lossless Playback Supports to CFZC up 64GB 8GB 70 Sound Hours MP3 qRS0Aw4). The protection generated was dose dependent. Only 1.8% of worms were gravid (eggs present) in dogs given the high-dose vaccine, with 4 of 6 dogs having no gravid proglottids, whereas 22.6% of worms were gravid in the control group. Dogs given the low-dose vaccine also had a significant, although lower, reduction (P<.02) (Music Player Lossless Playback Supports to CFZC up 64GB 8GB 70 Sound Hours MP3 qRS0Aw4) in the number of worms developing to the egg-producing stage. The inhibition of maturation [(% of gravid worms in control group-% of gravid worms in vaccinated group)/% of gravid worms in control group] was 58.0% and 92.0% for the low- and high-dose vaccine groups, respectively. Typical morphological characteristics of adult worms obtained from dogs vaccinated with the high-dose vaccine and those in the control group are shown in figure 1

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Table 1

Vaccination of dogs (n=17) against Echinococcus granulosus using soluble native proteins from protoscoleces

Table 1

Vaccination of dogs (n=17) against Echinococcus granulosus using soluble native proteins from protoscoleces

Figure 1

Typical morphological characteristics of Echinococcus granulosus adult worms obtained from the intestines of groups of dogs vaccinated with PBS control (A) or with soluble native proteins isolated from protoscoleces (B)

Figure 1

Typical morphological characteristics of Echinococcus granulosus adult worms obtained from the intestines of groups of dogs vaccinated with PBS control (A) or with soluble native proteins isolated from protoscoleces (B)

Serum samples collected during the vaccination regime and after challenge infection were tested using ELISA to measure levels of specific immunoglobulin raised to PSC antigens (figure 2). The total levels of IgG increased gradually from week 1 to week 8 and then decreased 10 weeks after the first injection in both the low- and high-dose vaccine groups. Levels of IgG antibodies were boosted by the second and the third vaccinations. IgG2 predominated and followed the same pattern as the total IgG response. Levels of IgG, especially IgG2, were vaccine dose dependent, whereas IgG1 responses were low in both vaccine groups. Levels of IgE and IgA were also low, with minimal changes throughout the whole course of the trial at both vaccine doses. Levels of IgM showed a slight increase after the first vaccination and decreased at week 4 thereafter. Serum levels of IgA, IgE, and IgM did not increase after challenge with PSC in either vaccine group. Levels of IgG2 were specifically stimulated by vaccination with the low and high vaccine doses and were significantly higher than those in control dogs, but there was no correlation between worm burden and levels of IgG2 antibody (P>.05)

Figure 2

Serum levels of immunoglobulins and subclasses of IgG from groups of dogs vaccinated with soluble native proteins isolated from protoscoleces (PSCs) of Echinococcus granulosus in ELISA. Triangles control dogs vaccinated with PBS; squares dogs vaccinated with a low dose of PSC proteins; circles dogs vaccinated with a high dose of PSC proteins. Error bars show SDs of optical-density values

Figure 2

Serum levels of immunoglobulins and subclasses of IgG from groups of dogs vaccinated with soluble native proteins isolated from protoscoleces (PSCs) of Echinococcus granulosus in ELISA. Triangles control dogs vaccinated with PBS; squares dogs vaccinated with a low dose of PSC proteins; circles dogs vaccinated with a high dose of PSC proteins. Error bars show SDs of optical-density values

Vaccination of Dogs with Recombinant Proteins

SDS-PAGE analysis showed that the 3 purified soluble recombinant fusion proteins (EgM4-GST, 50.5 kDa; EgM9-GST, 49.5 kDa; and EgM123-GST, 52.3 kDa) used as vaccines were of the expected molecular size (figure 3A). As with the native PSC proteins, recombinant proteins induced significant protection both in terms of the reduction in worm burdens and inhibition of worm development, compared with PBS and GST control dogs (table 2). Dogs given the GST control had 7–13 times more worms that those in the vaccination groups (table 2). In the GST control dogs, 26.2% of worms had developed to the gravid stage, whereas only 0.7% and 0.5% of worms in the EgM4- and EgM9-vaccinated groups, respectively, were gravid. No eggs were present in any of the worms from the group vaccinated with EgM123 at 45 days after infection (table 2)

Figure 3

SDS-PAGE and immunoblot analysis of the purified recombinant EgM proteins used in the vaccine trial. A SDS-PAGE showing the purity and molecular sizes of EgM4–glutathione S-transferase (GST) (lane 2) EgM9-GST (lane 3) EgM123-GST (lane 4) and recombinant GST (lane 1). Proteins were stained with Coomassie blue. B Western blot showing recognition, by pooled serum samples from dogs vaccinated with EgM4-GST (lane 2) EgM9-GST Shroud YZF250 YZF Skull 02 decals Kit YZF450 450 00 250 Graphics Yamaha sticker (lane 3) and EgM123-GST (lane 4) of the SDS-separated recombinant EgM proteins shown in panel A, after anti-GST antibodies were preabsorbed from the serum pools using an Escherichia coli lysate expressing recombinant GST [25]. Recombinant GST was recognized by pooled serum samples from the 3 vaccinated groups before (lane 1) but not after (lane 5) absorption of anti-GST antibodies using the Escherichia coli lysate expressing recombinant GST. M, molecular-weight markers (in kilodaltons)

Figure 3

SDS-PAGE and immunoblot analysis of the purified recombinant EgM proteins used in the vaccine trial. A SDS-PAGE showing the purity and molecular sizes of EgM4–glutathione S-transferase (GST) (lane 2) EgM9-GST (lane 3) EgM123-GST (lane 4) and recombinant GST (lane 1). Proteins were stained with Coomassie blue. B Western blot showing recognition, by pooled serum samples from dogs vaccinated with EgM4-GST (lane 2) EgM9-GST (lane 3) and EgM123-GST (lane 4) of the SDS-separated recombinant EgM proteins shown in panel A, after anti-GST antibodies were preabsorbed from the serum pools using an Escherichia coli lysate expressing recombinant GST [25]. Recombinant GST was recognized by pooled serum samples from the 3 vaccinated groups before (lane 1) but not after (lane 5) absorption of anti-GST antibodies using the Escherichia coli lysate expressing recombinant GST. M, molecular-weight markers (in kilodaltons)

Table 2

Vaccination of dogs (n=18) against Echinococcus granulosus using recombinant EgM proteins derived from mature adult worms

Table 2

Vaccination of dogs (n=18) against Echinococcus granulosus using recombinant EgM proteins derived from mature adult worms

Serum samples from vaccinated dogs were used in Western blots to determine the specificity of antibodies raised against the recombinant EgM proteins after the serum samples were preabsorbed by bacterial lysates that contained GST, to remove antibodies generated against GST and E. coli proteins [25]. The preabsorption completely removed anti-GST antibodies (figure 3B lane 5), and the recombinant proteins were strongly and specifically recognized by serum from dogs vaccinated with the 3 recombinant proteins (figure 3B lanes 2–4)

Serum samples collected during the course of the vaccine trial with the 3 recombinant proteins were tested for levels of immunoglobulin antibody by ELISA using both native PSC proteins and recombinant EgM-CBP proteins. As with the serum obtained from dogs vaccinated with native PSC antigens, IgG2 was the predominant antibody; other antibody isotypes tested (IgG1, IgA, IgE, and IgM) were detectable at low levels (data not shown)

Expression of EgM Proteins in E. granulosus

Western blottingWe showed previously, using reverse-transcription polymerase chain reaction (RT-PCR) and Northern analysis [21], that the egM genes are expressed specifically in MAWs. In the present study, we probed native worm proteins (figure 4A) with a hyperimmune mouse serum raised against recombinant EgM4 and confirmed that this protein is expressed exclusively in MAWs and not in immature adult worms (IAWs; figure 4B)

Figure 4

Hyperimmune serum raised in mice against recombinant EgM4 binding to native proteins from mature but not immature adult worms. A Separation by SDS-PAGE and staining by Coomassie blue of purified recombinant glutathione S-transferase (GST) expressed in Escherichia coli (lane 1); native proteins isolated from immature adult worms (35 days old without eggs) (lane 2); and native proteins isolated from mature adult worms (62 days old with eggs) (lane 3). B Proteins separated by SDS-PAGE in panel a probed in a Western blot with an anti-EgM4 serum preabsorbed against an E. coli lysate expressing recombinant GST [25]. Note the binding of the serum to native proteins in mature (lane 3) but not immature (lane 2) adult worms or to recombinant GST (lane 1). M, molecular-weight markers (in kilodaltons)

Figure 4

Hyperimmune serum raised in mice against recombinant EgM4 binding to native proteins from mature but not immature adult worms. A Separation by SDS-PAGE and staining by Coomassie blue of purified recombinant glutathione S-transferase (GST) expressed in Escherichia coli (lane 1); native proteins isolated from immature adult worms (35 days old without eggs) (lane 2); and native proteins isolated from mature adult worms (62 days old with eggs) (lane 3). B Proteins separated by SDS-PAGE in panel a probed in a Western blot with an anti-EgM4 serum preabsorbed against an E. coli lysate expressing recombinant GST [25]. Note the binding of the serum to native proteins in mature (lane 3) but not immature (lane 2) adult worms or to recombinant GST (lane 1). M, molecular-weight markers (in kilodaltons)

In situ hybridizationTo determine whether egM transcripts were present in E. granulosus worms in different stages of development, histological sections of PSC and IAWs and MAWs were subjected to in situ hybridization, using sense and antisense RNA probes specific for egM123 mRNA. Because the egM123 sequence overlaps the majority of the egM9 sequences missing just 1 repeat sequence [21], hybridization experiments were undertaken only with egM123. The cDNA generated 2 probes (antisense and sense), and serial sections of worm tissue were probed for direct comparison of labeling distribution. Sections of MAWs hybridized very strongly with the antisense probe of egM123 (figure 5B and 5D). egM123 mRNA was present mostly in testes and differentiating uterine epithelium, and to a lesser extent, in the subtegument tissues of the penultimate proglottid. No signal was detected when the egM123 sense probe was used as a negative control with mature worm tissue sections (figure 5A and 5C), and neither the antisense nor sense egM123 probes hybridized to sections of PSC (not shown) or IAWs (not shown)

Figure 5

Localization of transcription of egM123 in the mature adult worm of Echinococcus granulosus. In situ hybridization to probe egM123 mRNA in the terminal (A and B) and penultimate (C and D) proglottids of the mature adult worm. Hybridization with the sense probe of egM123 was negative (A and C) whereas that of the antisense probe (B and D) showed positive hybridization, as indicated by dark purple staining (arrows). CS, cirrus sac; T, testes; U, uterus

Figure 5

Localization of transcription of egM123 in the mature adult worm of Echinococcus granulosus. In situ hybridization to probe egM123 mRNA in the terminal (A and B) and penultimate (C and D) proglottids of the mature adult worm. Hybridization with the sense probe of egM123 was negative (A and C) whereas that of the antisense probe (B and D) showed positive hybridization, as indicated by dark purple staining (arrows). CS, cirrus sac; T, testes; U, uterus

Discussion

We have demonstrated, to our knowledge for the first time, protection against E. granulosus in dogs vaccinated with recombinant proteins derived from MAWs. Although a number of groups had previously used whole PSC extracts/membranes in vaccination experiments with dogs (see above), none had tested the potency of native, soluble PSC proteins. We initially tested the vaccine efficacy of native PSC proteins in dog vaccinations. Such vaccines induced significant protection, as measured by the suppression of worm growth and, importantly, of egg development. Because released eggs are crucial for the maintenance of the parasite life cycle, reductions in infection patency would help to block or minimize parasite transmission to intermediate hosts, including humans

In earlier work, we used differential-display PCR to differentiate gene-expression profiles in 2 populations of adult worms: IAWs collected from dogs at 35 days after infection (at the commencement of egg production) and MAWs collected at 62 days after infection (fully embryonated eggs present in the uterus) [21]. Because proglottid formation occurs continuously, we reasoned that MAWs would contain mRNA associated with embryogenesis, egg formation, and production of gravid proglottids. The genes represented were reasoned to be vaccine candidates both for intermediate (genes expressed in eggs) and definitive (genes associated with egg development) hosts. Three differentially expressed sequences were isolated and shown to belong to a novel egM family of proteins [21] expressed exclusively by MAWs. Recombinant egM proteins were expressed in Shroud sticker Skull 00 YZF450 Graphics decals Kit 250 450 YZF 02 Yamaha YZF250 E. coli. In ELISA, only the soluble form of the proteins was recognized by serum from infected dogs [21], which suggests that conformational epitopes play a role in antigen-antibody responses. Here, soluble recombinant egM proteins used as vaccines induced significant protection, comparable with soluble PSC protein. No eggs were present 45 days after infection in any worms in the group vaccinated with EgM123

The mechanisms of protection are unclear at this point. Protection does not correlate with predominant levels of IgG2, although dogs in experimental groups generated higher antibody responses than did control dogs. Statistically, levels of IgG1 had an inverse association with worm burden, but levels of antibodies were very low. It could be that the protection involves mucosal immune responses or T cell responses—this is an area that requires future research. Use of immunostimulating complexes made from tegumental antigens of E. granulosus PCSs, such as intranasal immunogens, in dogs evoked significant secretory IgA antibody responses detected in saliva and serum, but no challenge infection data were reported [26]

In both trials presented here, all dogs were challenged with a large number (480,000) of PSCs, because our pilot experiments showed that control dogs challenged with this number of PSCs had a high and consistent number (>50%) of larvae developing into adult worms. Challenge with fewer (100,000) PSCs resulted in <1% of larvae developing into adult worms (data not shown), for reasons that are unclear. It is not uncommon to find naturally infected dogs and other carnivores harboring large numbers of adult E. granulosus; dogs with infections of >300,000 and >375,000 worms have been reported from Australia [MC Case 5 With 3 Zoom 5 Auto Sunagor F 72 4 80mm 1 Lens 28 xwRTWUqa] and western China [28]

We have shown, by Northern-blot analysis and RT-PCR, that the egM genes are exclusively transcribed in MAWs [21]. In the present article, we have further confirmed the specificity of expression by Western-blot analysis, which showed that the EgM4 protein occurs exclusively in MAWs (figure 4B). In situ hybridization results showed that egM123 was abundantly expressed in the testes of the penultimate proglottid of MAWs (Special Invicta LeSabre Custom Bezel Buick 1959 Taillight Electra pxABEXEqw). It is notable that this was quite different from the IAWs at 35 days after infection. At this stage, testes are fully developed in the terminal segment [29], but they stained negative (not shown), which suggests that the expression of the egM family may be regulated by some regulatory mechanisms in mature worms. It has been demonstrated that, on day 35 after infection, fertilization is completed [29, 30]. Therefore, the products of the egM family, or at least that of egM123, are expressed after spermatogenesis and fertilization, which suggests that some members of the gene family may function in the maturation of eggs

The deduced protein sequences of EgM proteins share no similarity with other proteins in GenBank, and there is no clear indication of their function. When vaccination and localization results are combined, the proteins are likely to have multiple functions and to be especially associated with worm maturation and egg development. Evidence for involvement in these predicated functions was indirectly obtained by the dog vaccination experiments, which showed that these processes were affected by the vaccine-induced host immune response. Given that relatively little is known about gene expression during egg formation, biochemical studies of the EgM proteins and elucidation of their function in maturing eggs will provide insights into the control mechanisms and metabolic pathways underlying this critically important biological process

The present work provides proof of principle that vaccination of the dog host is feasible. The prepatency period in E. granulosus infections in dogs is 50–80 days [29]. Vaccination with soluble PSC antigens and recombinant proteins can induce either total inhibition of egg production or delayed embryogenesis. Because worms can survive in the dog intestine for 1–2 years [31], delayed or reduced egg production might be sufficient to reduce transmission in areas where the parasite is endemic. We have shown that the antigens directly involved in protection can be produced in recombinant form for wide-scale application, either on their own or in combination with immunostimulating complexes [26] or live attenuated Salmonella typhimurium [32]

Acknowledgment

We thank Zu Gu (Queensland Institute of Medical Research), for help with statistical analysis

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Potential conflicts of interest: none reported
Financial support: Wellcome Trust (grant GR057212 to W.B.Z. and D.P.M.); National Institutes of Health (grant 1R03 AI063367-01A1 to W.B.Z.); National Health and Medical Research Council of Australia (support to D.P.M.)