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The long-term storage of Cryptosporidium life-cycle stages is a prerequisite for in vitro culture of the parasite. Cryptosporidium parvum oocysts, sporozoites, and intracellular forms inside infected host cells were stored for 6-12 mo in liquid nitrogen utilizing different cryoprotectants (dimethyl sulfoxide [DMSO], glycerol and fetal calf serum [FCS]), then cultured in vitro. Performance in vitro was quantified by estimating the total Cryptosporidium copy number with quantitative polymerase chain reaction (qPCR) in 3- and 7-day-old cultures. Although few parasites were recovered either from stored oocysts or from infected host cells, sporozoites stored in liquid nitrogen recovered from freezing successfully. More copies of parasite DNA were obtained from culturing those sporozoites than sporozoites excysted from oocysts kept at 4 C for the same period. The best performance was observed for sporozoites stored in Roswell Park Memorial Institute (RPMI) medium with 10% FCS and 5% DMSO, which generated 240% and 330% greater number of parasite DNA copies (on days 3 and 7 post-infection, respectively) compared to controls. Storage of sporozoites in liquid nitrogen is more effective than oocyst storage at 4 C and represents a more consistent approach for storage of viable infective Cryptosporidium aliquots for in vitro culture.

Infections with Haemonchus contortus are a major constraint on ruminant health world-wide. Young lambs are very sensitive to Haemonchus infection. Older lambs and sheep acquire immunity after a continuous or seasonal exposure to the parasite. The mechanisms underlying immunity are still not completely understood. Antibodies, in particular local IgA and IgE, certainly play a role. The role of IgG is less clear. Lymphocyte proliferation responses seem to correlate to immunity. Sheep that have high antigen-induced lymphocyte responses have a low susceptibility to infection. Furthermore, several studies have demonstrated that immunity against H. contortus is associated with mastocytosis and hypersensitivity reactions. More recently, increasing attention is being paid to the role of cytokines (interleukins and γ- interferon) in the activation of specific defence mechanisms. Reverse transcriptase–polymerase chain reaction (RT–PCR) assays to study cytokine mRNA expression have become available. The inability of young lambs to mount a significant Th2 response, which is normally characterized by high IgE levels, mastocytosis and eosinophilia, may account for the phenomenon of unresponsiveness in these animals.

The IgE response of naive or previously infected sheep to 50,000 infective larvae of Teladorsagia circumcincta was monitored in serum and gastric lymph using a monoclonal antibody generated to recombinant ovine IgE in a dot blot assay. In 4/5 naive sheep, lymph and serum IgE concentrations increased from days 8 and 14 after infection, respectively. In most previously infected sheep, the IgE response to challenge was more rapid, although not necessarily greater than that following a primary infection. IgE concentrations in lymph were some 4-fold higher than in serum indicating that its source was the mucosa or draining nodes.

Two excretory secretory (ES) antigens of adult Haemonchus contortus with molecular weights of 15 and 24 kDa, respectively, were evaluated as protective immunogen against haemonchosis. Sheep were vaccinated three times and subsequently challenged with 20,000 infective larvae. Vaccination induced significant reduction (> 70%) in mean faecal egg counts and abomasal worm burden compared to the non-vaccinated control group or adjuvant control group. Vaccination induced ES-specific antibodies and stimulated infiltration of mast cells in the abomasal tissue.

Partially purified low molecular weight antigens obtained by gel filtration of whole worm homogenates or total adult excretory–secretory (ES) products were tested in a vaccination experiment to determine their ability to induce protective immunity against Haemonchus contortus in sheep. Sheep were challenged with 20000 infective 3rd-stage larvae. One animal in the low molecular weight vaccinated group showed no protection against H. contortus, whereas the 4 other sheep in this group showed a mean reduction of 99·9% in faecal egg counts and of 97·6% in abomasal worm burden compared to the non-vaccinated controls and the adjuvant controls. The ES-vaccinated sheep showed a 32·2% reduction in parasite egg production and a 63·7% reduction in abomasal worm counts. Analysis of the humoral immune responses revealed no significant differences in antibody recognition of putative protective antigens between the protected and non-protected vaccinated animals. However, a marked lower lymphocyte proliferation response was found in non-protected sheep.

Extracts of infective larvae and adults of the trichostrongylid Haemonchus contortus were studied for the presence of carbohydrate moieties. Several different lectin-binding sites were demonstrated in both stages using a panel of nine lectins. The carbohydrate specificity of the lectins used strongly suggests that alpha-D-mannose, alpha-D-glucose, and D-N-acetylglucosamine are the most important carbohydrate epitopes present on H. contortus proteins. Thus, N-linked oligosaccharides form the major part of the carbohydrate moieties on these glycoproteins. Treatment with sodium periodate was performed to investigate the immunoreactivity towards the carbohydrate moieties. This treatment resulted in a reduction in the immunoreactivity of these antigens as demonstrated by enzyme-linked immunosorbent assay (ELISA) and immunoblotting, suggesting that a substantial part of the host immune response against H. contortus is directed against the carbohydrate epitopes on the parasite antigens.

The immune response of calves against excretory secretory (ES) products of adult Haemonchus placei was studied. The ES products of adult H. placei comprise at least ten polypeptides with molecular weights ranging from 10 to over 100 kDa. A primary infection of calves with H. placei did not result in a marked elevation of IgG serum antibodies reactive to ES antigens as demonstrated by ELISA. Immunoblotting experiments revealed that sera of primary infected calves recognised a 50 kDa antigen in most cases. An increase in the level of serum IgG antibodies was observed after trickle infections. The secondary immune response coincided with the recognition of a group of three ES antigens of around 50–55 kDa and one protein with a molecular weight of 24 kDa.

Adult specimens of Haemonchus contortus were microdissected into four body fragments: oesophagus, cuticle of oesophagus, gut and cuticle with adjacent muscle layer. The antigenicity of these different body fragments was analysed in comparison to total (whole) worm extracts with immunoblotting and ELISA using sera of H. contortus-infected sheep. In particular, oesophagus-derived antigens appeared to be specifically recognized and may prove valuable in diagnostic assays.

The primary and secondary serum antibody responses of Texel sheep to infective larvae (L3) and adult worms of Haemonchus contortus were studied. Ten-month-old sheep were infected with 20,000 H contortus L3, treated with ivermectin seven weeks later and, after four weeks, reinfected with 10,000 L3 once a week for six weeks. Faecal egg counts were significantly lower during the secondary infection than during the primary infection, but both infections induced antibody responses, as demonstrated by an enzyme-linked immunosorbant assay ( ELISA ). The primary antibody response developed rather slowly, but the secondary response developed more rapidly and the IgA responses against L3 antigens and the IgG 1 and IgG 2 responses against adult antigens were twice those observed during the primary infection. These accelerated and enhanced responses after the reinfection suggest an immunological memory for H contortus antigens.

Leishmania (Kinetoplastida: Trypanosomatidae) are protozoan parasites of significant medical and veterinary importance. Over the last decade, visceral leishmaniasis (VL) has emerged as a major opportunistic infection associated with HIV/AIDS in North Western Ethiopia. This paper reports on serological evidence of possible Leishmania donovani (L. donovani) infection in dogs using two serological tests: direct agglutination test (DAT) and Kalazar detect rapid test (KDRT). Two hundred and seventeen asymptomatic local breed dogs were examined for L. donovani antibodies. Performance of the DAT and KDRT was assessed in 162 matching samples of blood collected on filter paper and serum, respectively. Using DAT and KDRT testing in parallel, the overall seroprevalence of L. donovani infection was 27.7% and 14.8%, respectively. The degree of agreement was found to be fair (68.8%, k = 0.234). Univariable logistic regression analysis of some risk factors for L. donovani infection in dogs using DAT indicates that place of residence, sex, age, dog keeping purpose and dog housing condition were not significantly associated with seropositivity. The high proportion of positive dogs suggests the exposure of these animals to L. donovani infection and needs further investigation. Isolation and typing of the parasite aiming at confirming the role of these animals in maintenance and transmission of kala-azar is advocated.

SUMMARYInfection with digenetic trematodes causes an inhibition or complete cessation of fecundity in their intermediate hosts, freshwater snails. It has been demonstrated in the host–parasite combination Lymnaea stagnalis–Trichobilharzia ocellata that the action of the female gonadotropic hormones upon their target organs is inhibited by the peptide schistosomin. Schistosomin is produced in the central nervous system of the snail and released upon parasitic infection. In order to study the in vitro release of schistosomin, a bioassay was developed. Central nervous systems were incubated with either an acetic acid or a methanolic extract of larval stages of Trichobilharzia ocellata (miracidia, mother sporocysts, cercariae). The incubation media were chromatographed using HPLC and released schistosomin (-like material) was tested for bioactivity in the calfluxin bioassay. The in vitro release of schistosomin was only induced with a methanolic extract of cercariae. The nature of the cercarial factor is discussed.

Of gastropod molluscs, only in the Aplysiidae and the Lymnaeidae have the genes encoding the respective egg-laying hormones been cloned and the neurons controlling egg laying and egg-laying behavior been identified. Immunocytochemistry, using antibodies raised against alpha-CDCP (one of the neuropeptides encoded on the egg-laying hormone gene of Lymnaea stagnalis), identified neurons in various species of gastropods. In the basommatophoran snail, Biomphalaria glabrata, large and small neurons were observed in areas of the central nervous system similar to where immunoreactive cells exist in L. stagnalis, i.e., in the cerebral and pleural ganglia. In the stylommatophoran snail (Helix aspersa) and the slug (Limax maximus), large immunopositive neurons occur in the visceral and right parietal ganglia. In L. maximus, small immunoreactive neurons were found in the cerebral ganglia while in H. aspersa similar cells were observed intermingled with the large cells in the visceral and right parietal ganglia. Similar to the situation in L. stagnalis, in the female part of the reproductive tract of B. glabrata, L. maximus, and A. californica, but not in H. aspersa, neurons and/or fiber tracts are present. The results indicate that egg-laying hormone precursor molecules of gastropod molluscs are phylogenetically closely related. The alpha-CDCP antiserum may allow the identification of hitherto unknown egg-laying regulating systems of gastropod molluscs.

Extracts of cercariae of the avian schistosomeTrichobilharzia ocellata were analysed for the presence of ecdysteroids by radioimmunoassay, high-performance liquid chromatography monitoring fractions by radioimmunoassay, and gas chromatography/mass spectrometry (selected ion monitoring). Both free ecdysteroids and polar conjugated ecdysteroids were detected in the cercarial extracts. The free ecdysteroid fraction, as well as the hydrolysed polar conjugated ecdysteroid fraction, contained both ecdysone and 20-hydroxyecdysone in approximately equal amounts. The amount of ecdysteroids detected is comparable to those found in other platyhelminths. A possible role for the ecdysteroids in the development of the parasite and/or the interactions between the parasite and its intermediate host, the freshwater snailLymnaea stagnalis, is discussed.

SUMMARYSubadult and adult specimens of the pond snail Lymnaea stagnalis were infected with the schistosome Trichobilharzia ocellata. Egg production and growth of the snails were monitored over an 8-week period post-infection (p.i.). Snail haemolymph was collected and analysed for the presence of schistosomin, a neuropeptide which antagonizes the action of the snails' female gonadotropic hormones. Snails infected as subadults showed an increase in fecundity during the first 4 weeks p.i. compared with non-infected controls. The possibility is discussed that this increase is caused by an accelerated maturation of the female sex organs due to elevated levels of Dorsal Body Hormone, a female gonadotropic hormone. No difference in fecundity was found between snails infected as adults and control snails during the first 4 weeks p.i. Snails infected as subadults and as adults showed a decrease in fecundity from week 5 p.i. and onwards. This decrease coincided with the appearance of schistosomin in the haemolymph of the snails and with that of differentiating cercariae in the daughter sporocysts. Cercariae are probably involved in the induction of schistosomin release from the snails' CNS into the haemolymph. Snails infected as subadults or as adults grew at approximately the same rate as uninfected snails.

The relative contribution of third (L3), fourth (L4) or adult stages of Haemonchus contortus to the development of immunity was evaluated in three groups of sheep subjected to infections terminated by oxfendazole treatments at the L3, L4 or adult stage. A control group did not receive immunising infections. All the groups were challenged with 5000 L3, to evaluate the protection provided by the different protocols. All sheep were necropsied at the end of the experiment to count the abomasal worm burdens. A marked reduction in egg counts after challenge infection was only observed in sheep in which the infection was terminated in the adult stage (Group 4). A significant reduction in worm burden was also observed in Group 4. The immunising infections and/or the challenge infection resulted in moderately elevated IgG antibody levels against L3, L4 and adult somatic antigens in all the groups. In contrast, a strong IgG response against H. contortus excretory/secretory (ES) antigens was observed in the groups in which the immunising infection was terminated in the L4 and the adult stage. An elevated lymphocyte proliferation response against Haemonchus ES antigens was found only in the group that had their immunising infection terminated at the adult stage. The combined data suggest that exposure to and elicited immunological responses to ES antigens are important for the development of immunity against H. contortus.

A monoclonal antibody to ovine IgE was employed in an ELISA to investigate the IgE antibody responses in serum and gastric lymph to a primary infection of Teladorsagia circumcincta, and following challenge in previously infected sheep. During a primary response, IgE antibody to antigens derived from the infective third stage (L3) and adult (L5) worms were negligible, with low levels of IgE antibody detected in serum and lymph. In contrast, there was a pronounced IgE antibody response in 2/4 sheep to L3 antigens during 2-8 days after challenge of previously infected animals but low levels of IgE antibody to L5 antigens. This response was confirmed in a second but similar experiment, where relatively high levels of IgE antibody was detected to antigens from L3. Antibody levels were higher in lymph than in serum from the same animals, and Western blots of L3 antigen following SDS-PAGE under reducing conditions revealed several bands of MW26-96KD which reacted with the IgE antibody from gastric lymph. Immunohistochemical staining indicated that these IgE antibodies may be reacting with allergens associated with the surface cuticle of the worms.

The potential of Cooperia oncophora excretory/secretory (ES) proteins as antigens in a serological assay which aims to establish exposure levels in cattle was assessed. ES proteins were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis and immunoblotting. The N-terminal domains of two ES proteins were sequenced, and the corresponding cDNAs were cloned. Two cDNAs, designated CoES14.0 and CoES14.2, were expressed in Escherichia coli. The recombinant proteins were tested in an indirect enzyme-linked immunosorbent assay (ELISA) in which crude worm antigen (CWA) was used as a reference standard. In total, 67 reference serum samples were used: 27 negative serum samples, 29 C. oncophora-specific serum samples, 7 Dictyocaulus viviparus-specific serum samples, and 4 Ostertagia ostertagi-specific serum samples. This showed respective sensitivities and specificities of 17 and 84%, 0 and 100%, and 100 and 100% by the ELISAs with the three different types of proteins (CWA, CoES14.0, and CoES14.2, respectively). Since the CoES14.2 ELISA had the best sensitivity and specificity with reference sera, its specificity was further validated in an antigen inhibition ELISA. In this assay CoES14.2 and CWA preparations of C. oncophora, Cooperia curticei, O. ostertagi, Nematodirus helvetianus, Fasciola hepatica, D. viviparus, Haemonchus placei, and Trichostrongylus colubriformus were used as competitor antigens. This experiment showed that only the homologous antigens C. oncophora CWA and CoEs14.2 resulted in 100% inhibition. The CWA preparations of all other nematodes did not affect the ELISA, even if concentrations of 250 times the 50% inhibitory concentration of C. oncophora CWA were used. These results indicate that CoES14.2 does not share cross-reactive epitopes with heterologous CWAs. Finally, we tested the CoES14.2 ELISA with sequential serum samples from naturally infected groups of animals. The optical density values that were obtained correlated well with exposure levels based on cumulative egg excretion. Thus, the CoES14.2 ELISA seems to be a very sensitive tool for estimating exposure levels in cattle.

Part of the Cε3–Cε4 region of the ovine immunoglobulin E (IgE) gene (nucleotides 1111–1575) was amplified by PCR. The recombinant protein (recIgE1-2) was expressed in E. coli and both monoclonal and polyclonal antibodies were produced. These antibodies recognized recIgE1-2 and native IgE on Western blots and in ELISA. The polyclonal serum showed cross-reactivity with other sheep immunoglobulin classes. The monoclonal antibody was specific for ovine IgE and goat IgE. Infection of sheep with the abomasal nematode Haemonchus contortus resulted in elevated IgE levels in serum 2–4 weeks after infection, as measured by sandwich ELISA using the rabbit polyclonal as capture antibody and the monoclonal antibody against ovine IgE as second antibody. A negative correlation between worm counts and total serum IgE levels at the end of the experiment was found in repeatedly infected sheep. Significant increased levels of excretory–secretory antigens specific IgE levels were found after H. contortus infection. In contrast, no significant changes in 3rd-stage larvae (L3) antigen-specific IgE titre in sera could be detected after infection.

Two enzyme-linked immunosorbent assays (ELISA) using either excretory/secretory (ES) products or crude somatic antigens (CSA) of adult Haemonchus contortus were compared for their ability to detect antibodies against H. contortus in sheep. Serum samples obtained from a group of 32 H. contortus mono-infected sheep were tested in the two ELISAs and the obtained data were compared with the results of the faecal examinations of these sheep. The first sheep became patent 3 weeks post infection (p.i.) and all sheep had positive egg counts at week 5 p.i. The first antibodies against H. contortus were detected 1 week p.i. and all sheep were found positive by both ELISAs at week 4 p.i. Using sera from a large number of H. contortus-infected sheep a difference in sensitivity between the ES ELISA (97.7%) and the CSA ELISA (89.2%) was found. The specificity of each assay was determined by testing sera obtained from sheep with mono-infections of H. contortus, Trichostrongylus colubriformis, Trichostrongylus vitrinus, Ostertagia circumcincta, Nematodirus battus, Cooperia curticei, Fasciola hepatica, Taenia ovis or Eimeria spp. The specificity of the ES ELISA was 87.2%, whereas the specificity of the CSA ELISA was 82.7%.

In total, 19 8-month-old Texel sheep were used to study the isotype-specific serum antibody responses against infective larvae and adult worms of Haemonchus contortus. Group 1 sheep (n = 7) were infected with 20 000 L3 larvae (Week 0), treated with ivermectin 6 weeks post-infection and subsequently challenged at Week 10 of the experiment. This challenge consisted of a trickle infection of 10 000 L3 larvae per week for 5 weeks. Group 2 sheep (n = 7) received a single infection at Week 10 of the experiment, and Group 3 (n = 5) served as a non-infected control group throughout the entire experiment. Individual blood and faeces samples were collected at weekly intervals. The immune responses were monitored by ELISA and Western blotting. The secondary immune response coincided with a significant reduction of the Haemonchus egg output and reduction of worm counts. Both primary and challenge infections induced humoral immune responses, and ELISA revealed that the most dominant serum antibody responses belong to the IgG1 isotype and to a lesser extent to IgG2. IgM and IgA responses were less dominant. Western blotting experiments demonstrated that many antigens were commonly recognized by antibodies from both primary and challenge infected animals. However, sera of immune animals specifically reacted with low molecular weight proteins. In particular, a 24 kDa antigen present in adult worms appeared to be specifically recognized.

SUMMARYThe excretory/secretory (E/S) products of adult Haemonchus contortus comprise of at least 15 polypeptides with molecular weights ranging from 10 to > 100 kDa. These E/S products induce an immune response in infected Texel sheep, as demonstrated by specific IgGI levels and a significant lymphocyte proliferation index. Moreover, immunoblotting analysis revealed that sera of primary H. contortus-infected sheep specifically recognize a 24 kDa E/S product. In addition, sera of challenged sheep react strongly with a 15 kDa E/S product. The other E/S products of H. contortus showed immunoreactivity with serum samples of Haemonchus-infected sheep as well as with samples of sheep harbouring other trichostrongylid infections. These cross-reacting epitopes are the main cause of the lack of specificity of an E/S material- based ELISA. This ELISA can differentiate Haemonchus infections from Nematodirus battus infections, but not from Ostertagia circumcincta or Trichostrongylus colubriformis infections.