A new thrombospondin-related anonymous protein homologue in Neospora caninum (NcMIC2-like1).

Neospora caninum is an Apicomplexan protozoan that has the dog as a definitive host and cattle (among other animals) as intermediate hosts. It causes encephalopathy in dogs and abortion in cows, with significant loss in worldwide livestock. As any Apicomplexan, the parasite invades the cells using proteins contained in the phylum-specific organelles, like the micronemes, rhoptries and dense granules. The aim of this study was the characterization of a homologue (denominated NcMIC2-like1) of N. caninum thrombospondin-related anonymous protein (NcMIC2), a micronemal protein previously shown to be involved in the attachment and connection with the intracellular motor responsible for the active process of invasion. A polyclonal antiserum raised against the recombinant NcMIC2-like1 functional core (thrombospondin and integrin domains) recognized the native form of NcMIC2-like1, inhibited the in vitro invasion process and localized NcMIC2-like1 at the apical complex of the parasite by confocal immunofluorescence, indicating its micronemal localization. The new molecule, NcMIC2-like1, has features that differentiates it from NcMIC2 in a substantial way to be considered a homologue.

Immunological responses and cytokine gene expression analysis to Cooperia punctata infections in resistant and susceptible Nelore cattle.

Cellular and humoral immune response, as well as cytokine gene expression, was assessed in Nelore cattle with different degrees of resistance to Cooperia punctata natural infection. One hundred cattle (male, weaned, 11-12 months old), kept together on pasture, were evaluated. Faecal and blood samples were collected for parasitological and immunological assays. Based on nematode faecal egg counts (FEC) and worm burden, the seven most resistant and the eight most susceptible animals were selected. Tissue samples of the small intestine were collected for histological quantification of inflammatory cells and analysis of cytokine gene expression (IL-2, IL-4, IL-8, IL-12p35, IL-13, TNF-alpha, IFN-gamma, MCP-1, MCP-2, and MUC-1) using real-time RT-PCR. Mucus samples were also collected for IgA levels determination. Serum IgG1 mean levels against C. punctata antigens were higher in the resistant group, but significant differences between groups were only observed 14 days after the beginning of the experiment against infective larvae (L3) and 14 and 84 days against adult antigens. The resistant group also presented higher IgA levels against C. punctata (L3 and adult) antigens with significant difference 14 days after the beginning of the trial (P<0.05). In the small-intestine mucosa, levels of IgA anti-L3 and anti-adult C. punctata were higher in the resistant group, compared with the susceptible group (P<0.05). Gene expression of both T(H)2 cytokines (IL-4 and IL-13) in the resistant group and T(H)1 cytokines (IL-2, IL-12p35, IFN-gamma and MCP-1) in the susceptible group was up-regulated. Such results suggested that immune response to C. punctata was probably mediated by T(H)2 cytokines in the resistant group and by T(H)1 cytokines in the susceptible group.

Vaccination against the nematode Haemonchus contortus with a thiol-binding fraction from the excretory/secretory products (ES).

Fractionated excretory/secretory products (ES) of adult Haemonchus contortus were evaluated as protective antigens. The proteins were successively eluted from a Thiol Sepharose column using 25 mM cysteine followed by 25 mM Dl-dithiothreitol (DTT). Sheep were vaccinated three times and challenged with 5000 third stage infective larvae (L3) of H. contortus. Highest level of protection was found in sheep vaccinated with the DTT-eluted fraction in which egg output and worm burden were reduced by 52 and 50%, respectively, compared to the adjuvant control group. There was a positive correlation between fecundity (number of eggs per female) and the cumulative EPG or worm burden. Serum and mucus antibody levels of ES-specific immunoglobulins increased after immunizations and after challenge for IgG, IgA and IgE. The harvesting of H. contortus from animals clustered per group revealed the presence of cysteine protease activity in the ES of all groups but in addition to that, metalloprotease activity was also detected in the groups vaccinated with the DTT-eluted fraction, total ES and adjuvant only, in contrast to previous batches of ES (completely inhibited by E64) obtained from non vaccinated animals.

A family of activation associated secreted protein (ASP) homologues of Cooperia punctata.

Activation-associated secreted proteins (ASP) of nematodes have been studied as potential vaccine components. In this study we report the cloning and analysis of cDNA and genomic sequences of Cooperia punctata and establish the presence of two 75% identical ASP-1 genes in C. punctata. Additional C. punctata ASP paralogues were shown to be present. Analysis of PCR products amplified from genomic DNA from a pool of worms revealed extensive sequence diversity within this family of proteins, reflecting the presence of different ASP paralogues in a single worm as well as extensive polymorphisms between different worms. ASP proteins contain a conserved region called the sperm-coating protein (SCP) domain of unknown function, which is present as a single copy in proteins from yeast and a wide range of multi-cellular organisms. Only in three nematodes has a protein composed of duplicated SCP-domains been identified. C. punctata is the first organism in which at least two such genes are found. Database searches identified similarity of the C-terminal cysteine-rich domain of ASP proteins to a nematode metallothionein motif. Cp-asp-1b was expressed in Escherichia coli and both the N-terminal and C-terminal domain were shown to be recognized by sera of C. punctata infected bovines. The description of the asp gene family of C. punctata provides the basis for more detailed studies into the extent of variation and immunological recognition of this family that may assist in rational vaccine design.

Cooperia punctata trickle infections: parasitological parameters and evaluation of a Cooperia recombinant 14.2 kDa protein ELISA for estimating cumulative exposure of calves.

Three groups of four calves each were trickle infected with three different levels of Cooperia punctata: 310 (group A), 1250 (group B) and 5000 (group C) third stage infective larvae (L3) twice a week over a 17-week period. Group D was the non-infected control group. Parasitological parameters as faecal egg counts (epg), worm burdens, size of worms and number of eggs per female were collected and the differences between the groups compared. Serological analyses were also conducted to investigate the efficiency of a recombinant C. oncophora CoES 14.2kDa protein in an ELISA to detect C. punctata antibodies. Group C had higher faecal egg counts until week 9 when the values decreased to those in group B. Mean faecal egg counts in group A were always lower than in the two other infected groups. The worm burdens were highest in group C, and lowest in group A, although the number of worms as a percentage of total larval intake was higher for the lower group. The mean length of the worms was shorter and the number of eggs per female lower for group C than for both other groups. ELISA using the CoES 14.2kDa proved to be efficient in measuring C. punctata antibodies. For group C it took 4 weeks to get increased levels of antibodies and this was one and 2 months more for groups B and A, respectively. Overall, there was a congruent relation between C. punctata antibodies and the cumulative exposure to the three different levels of trickle infections.

Serum immunoglobulin E response in calves infected with the lungworm Dictyocaulus viviparus and its correlation with protection.

Protection of a primary Dictyocaulus viviparus infection was measured against a homologueous challenge infection in two independent experiments and this was correlated with serum immunoglobulin IgE responses. A primary infection of 30 third stage larvae (L3) of D. viviparus on day 0 protects calves for 70% against a challenge infection of 2000 L3 on day 35 compared to calves with no primary infection. The variation in post mortem worm counts within this group (n = 6) was very large with mean worm counts of 145 (range 3-446) lungworms. Parasite specific IgA, IgE, IgG1 and IgG2 and total IgE levels in serum were measured by ELISA. Parasite specific IgA, IgG1 and IgG2 were elevated after infection, but correlation with protection was only found with IgG1 levels on day 42 and with IgG2 levels on day 70. IgE was measured in a sandwich ELISA using antisheep IgE that cross-reacts with cattle IgE. No parasite specific IgE could be detected. However, total serum IgE was elevated after infection and total serum IgE levels before and on the day of challenge correlated with protection (P < 0.05). Total serum IgE also correlates with peripheral eosinophil counts between days 14 and 28 after primary infection. Western blots with three different parasite antigen preparations, L1, excretory/secretory products and crude worm adult antigens, were used to detect parasite specific IgE in sera depleted of IgG and IgM. These depleted sera from protected calves contained parasite specific IgE, while sera from nonprotected calves were negative. A band of approximately 100 kDa was recognized in all three antigens. In a second experiment, primary doses of 30, 60, 120, 240, 480 and 960 L3 of D. viviparus were used and necropsy was 11 days after challenge. This experiment confirmed the correlation between protection and total IgE levels before and on the day of challenge. The rapid and strong IgE responses in protected animals after such a low infection might be caused by the specific characteristics of the lungworm antigens or by the somatic migration of the worm and might be involved in the rapid development of protection against lungworm reinfections in cattle.

In vitro interaction of Brazilian strains of the nematode-trapping fungi Arthrobotrys spp. on Panagrellus sp. and Cooperia punctata.

In vitro tests were carried out to verify the activity of 26 Brazilian isolates of predatory fungi of the genus Arthrobotrys on a free-living nematode (Panagrellus sp.) and on infective larvae of Cooperia punctata, a parasitic gastrointestinal nematode of cattle. The results showed that the free-living nematode Panagrellus sp. was the most preyed upon, compared to C. punctata, for all the fungal treatments. Also, variable predatory capacity was observed for different fungal isolates belonging to the same genus when applied to different nematode species.

In vitro interaction of Brazilian strains of the Nematode-trapping fungi Arthrobotrys spp. on Panagrellus sp. and Cooperia punctata

In vitro tests were carried out to verify the activity of 26 Brazilian isolates of predatory fungi of the genus Arthrobotrys on a free-living nematode (Panagrellus sp.) and on infective larvae of Cooperia punctata, a parasitic gastrointestinal nematode of cattle. The results showed that the free-living nematode Panagrellus sp. was the most preyed upon, compared to C. punctata, for all the fungal treatments. Also, variable predatory capacity was observed for different fungal isolates belonging to the same genus when applied to different nematode species

A Cooperia punctata gene family encoding 14 kDa excretory-secretory antigens conserved for trichostrongyloid nematodes.

A polymorphic set of 14 kDa excretory-secretory (E-S) antigen-encoding cDNAs, with similarity to a previously characterized 15 kDa E-S antigen of Haemonchus contortus, was cloned from Cooperia punctata. Five cDNAs encoding predicted proteins of 70-80% identity were sequenced. Genomic analyses of individuals proved the existence of three 14 kDa E-S antigen-encoding genes, excluding that the differences reflected polymorphisms between individuals in a population. Southern blots indicated the presence of additional members of this gene family. Thus, despite the fact that heterologously expressed C. punctata 14 kDa E-S products are shown to be recognized by immune sera, potential pitfalls in the development of a recombinant vaccine are presented by this genetic diversity. Vaccine design could be further rationalized by knowledge of the function, and possible redundancy in function, of the E-S products which is presently lacking. The limitations encountered in assigning a function to the 14/15 kDa family of E-S proteins that is thus far unique to the trichostrongyloid nematodes are discussed.

Dynamics of the humoral immune response of calves infected and re-infected with Cooperia punctata.

The dynamics of the humoral immune response of calves were analysed after primary infection and re-infection with the intestinal nematode Cooperia punctata. 12 male 5 month-old Holstein-Friesian calves were randomly divided into two groups A and B. At the beginning of the experiment Group A animals were each infected experimentally with a single oral dose of 130,000 infective third stage larvae (L3) of C. punctata. The animals of Group B were kept as non-infected controls. The two calves from Group A with the highest infections died of cooperiosis at 32 and 44 days after infection (DAI), respectively. On DAI 100 the calves were treated with the recommended dose of oxfendazole. On DAI 180 the remaining four calves of Group A and three animals of Group B (B1) were infected with 260,000 L3 of C. punctata, while the other three calves of Group B (B2) served as non-infected controls. Monitoring of the humoral immune response predominantly demonstrated an IgG1 response against both adult and L3 antigen of C. punctata. Moreover, re-infections increased the levels of these immunoglobulins. IgA levels were less increased than IgG1 and no significant increase was observed in IgG2 and IgM levels. Immunoblotting analysis showed that total IgG present in the serum of the primary infected animals mainly reacted against adult proteins of 12-14 and 17-20 kDa and against L3 proteins of 33 and 43 kDa. After re-infection total IgG reacted with the same adult proteins but also with an adult 29 kDa protein.