Glyoxysomal malate-dehydrogenase and malate synthase from Soybean cotyledons (Glycine max. L.): Enzyme association, antibody-production and cDNA cloning

In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD(+) oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (M(r)) of 670000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M, 510 000 and of gMDH as a dimer of M, 73 000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X(6))HL motif also found in plant and mammalian thiolases.

A comparative study of the intercellular connections between parasite/host in two Gelidiocolax/Gelidium algal parasitic systems

Comparative ultrastructural study of the intercellular connections between parasite and host cells in two algal parasitic systems, Gelidiocolax christianae Feldmann and Feldmann/Ge/iV/ium spathulatum (Kutz.) Bornet and Gelidiocolax deformans Seoane Camba/Gelidium sesquipedale (Clem.) Thur, shows quantitative and structural differences. The number of free conjunctor cells (before fusión with the adjacent host cells) differs between the two parasitic systems and is inversely related to the number of complex pit connections. The fibrillar cell wall structure of the conjunctor cells and the lamellar structure of the complex pit plugs in the two systems are also different A hypothesis concerning the different activity of the conjuntor cell wall in the two parasitic systems, related with the different structural appearance, is proposed.

Synthetic long peptide booster immunization in rhesus macaques primed with replication-competent NYVAC-C-KC induces a balanced CD4/CD8 T-cell and antibody response against the conserved regions of HIV-1.

The Thai trial (RV144) indicates that a prime-boost vaccine combination that induces both T-cell and antibody responses may be desirable for an effective HIV vaccine. We have previously shown that immunization with synthetic long peptides (SLP), covering the conserved parts of SIV, induced strong CD4 T-cell and antibody responses, but only modest CD8 T-cell responses. To generate a more balanced CD4/CD8 T-cell and antibody response, this study evaluated a pox-vector prime/SLP boost strategy in rhesus macaques. Priming with a replication-competent NYVAC, encoding HIV-1 clade C gag, pol and nef, induced modest IFNγ T-cell immune responses, predominantly directed against HIV-1 Gag. Booster immunization with SLP, covering the conserved parts of HIV-1 Gag, Pol and Env, resulted in a more than 10-fold increase in IFNγ ELISpot responses in four of six animals, which were predominantly HIV-1 Pol-specific. The animals showed a balanced polyfunctional CD4 and CD8 T-cell response and high Ab titres.

Ultrastructure of vitellogenesis and vitellocytes in the trypanorhynch cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax

This is the first TEM examination of vitellogenesis in the cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax and a member of a little-studied trypanorhynch family, the Aporhynchidae. The synthetic activity of vitellocytes plays two important functions in the developmental biology of cestodes: (1) their shell-globules serve in eggshell formation; and (2) their accumulated reserves of glycogen and lipids represent a food source for the developing embryo. In A. menezesi, vitelline follicles consist of cells at various stages of development, from peripheral, immature cells of the gonial type to mature cells towards the centre of the follicle. These stages are: (I) immature; (II) early differentiation; (III) advanced maturation; and (IV) mature. Gradual changes involved in this process occur within each stage. Vitellogenesis involves: (1) an increase in cell volume; (2) the development of a smooth endoplasmic reticulum and an accelerated formation and accumulation of both unsaturated and saturated lipid droplets, along with their continuous enlargement and fusion; (3) the formation of individual β-glycogen particles and their accumulation in the form of glycogen islands scattered among lipid droplets in the cytoplasm of maturing and mature vitellocytes; (4) the rapid accumulation of large, moderately saturated lipid droplets accompanied by dense accumulations of β-glycogen along with proteinaceous shell-globules or shell-globule clusters in the peripheral layer during the advanced stage of maturation; (5) the development of cisternae of granular endoplasmic reticulum that produce dense, proteinaceous shell-globules; (6) the development of Golgi complexes engaged in the packaging of this material; and (7) the progressive and continuous enlargement of shell-globules into very large clusters in the peripheral layer during the advanced stage of maturation. Vitellogenesis in A. menezesi, only to some extent, resembles that previously described for four other trypanorhynchs. It differs in: (i) the reversed order of secretory activities in the differentiating vitellocytes, namely the accumulation of large lipid droplets accompanied by glycogenesis or β-glycogen formation during early differentiation (stage II), i.e. before the secretory activity, which is predominantly protein synthesis for shell-globule formation (stage III); (ii) the very heavy accumulation of large lipid droplets during the final stage of cytodifferentiation (stage IV); and (iii) the small number of β-glycogen particles present in mature vitellocytes. Ultracytochemical staining with PA-TCH-SP for glycogen proved positive for a small number of β-glycogen particles in differentiating and mature vitellocytes. Hypotheses, concerning the interrelationships of patterns of vitellogenesis, possible modes of egg formation, embryonic development and life-cycles, are commented upon.

Signs of a vector's adaptive choice: on the evasion of infectious hosts and parasite-induced mortality

Laboratory and field experiments have demonstrated in many cases that malaria vectors do not feed randomly, but show important preferences either for infected or non-infected hosts. These preferences are likely in part shaped by the costs imposed by the parasites on both their vertebrate and dipteran hosts. However, the effect of changes in vector behaviour on actual parasite transmission remains a debated issue. We used the natural associations between a malaria-like parasite Polychromophilus murinus, the bat fly Nycteribia kolenatii and a vertebrate host the Daubenton's bat Myotis daubentonii to test the vector's feeding preference based on the host's infection status using two different approaches: 1) controlled behavioural assays in the laboratory where bat flies could choose between a pair of hosts; 2) natural bat fly abundance data from wild-caught bats, serving as an approximation of realised feeding preference of the bat flies. Hosts with the fewest infectious stages of the parasite were most attractive to the bat flies that did switch in the behavioural assay. In line with the hypothesis of costs imposed by parasites on their vectors, bat flies carrying parasites had higher mortality. However, in wild populations, bat flies were found feeding more based on the bat's body condition, rather than its infection level. Though the absolute frequency of host switches performed by the bat flies during the assays was low, in the context of potential parasite transmission they were extremely high. The decreased survival of infected bat flies suggests that the preference for less infected hosts is an adaptive trait. Nonetheless, other ecological processes ultimately determine the vector's biting rate and thus transmission. Inherent vector preferences therefore play only a marginal role in parasite transmission in the field. The ecological processes rather than preferences per se need to be identified for successful epidemiological predictions.

Trophic structure in a seabird host-parasite food web: insights from stable isotope analyses

Ecological studies on food webs rarely include parasites, partly due to the complexity and dimensionality of host-parasite interaction networks. Multiple co-occurring parasites can show different feeding strategies and thus lead to complex and cryptic trophic relationships, which are often difficult to disentangle by traditional methods. We analyzed stable isotope ratios of C (13C/12C, δ13C) and N (15N/14N, δ15N) of host and ectoparasite tissues to investigate trophic structure in 4 co-occurring ectoparasites: three lice and one flea species, on two closely related and spatially segregated seabird hosts (Calonectris shearwaters). δ13C isotopic signatures confirmed feathers as the main food resource for the three lice species and blood for the flea species. All ectoparasite species showed a significant enrichment in δ15N relatively to the host tissue consumed (discrimination factors ranged from 2 to 5 depending on the species). Isotopic differences were consistent across multiple host-ectoparasite locations, despite of some geographic variability in baseline isotopic levels. Our findings illustrate the influence of both ectoparasite and host trophic ecology in the isotopic structuring of the Calonectris ectoparasite community. This study highlights the potential of stable isotope analyses in disentangling the nature and complexity of trophic relationships in symbiotic systems.

Spermiogenesis and spermatozoon ultrastructure of the dilepidid cestode Molluscotaenia crassiscolex (von Linstow, 1890), an intestinal parasite of the common shrew Sorex araneus

Spermiogenesis in Molluscotaenia crassiscolex begins with the formation of a differentiation zone containing two centrioles. One of the centrioles develops a flagellum directly into the cytoplasmic extension. The nucleus elongates and later migrates along the spermatid body. During advanced stages of spermiogenesis, a periaxonemal sheath appears in the spermatid. Spermiogenesis finishes with the appearance of a single helicoidal crested body at the base of the spermatid and, finally, the narrowing of the ring of arched membranes causes the detachment of the fully formed spermatozoon. The mature spermatozoon of M. crassiscolex exhibits a partially detached crested body in the anterior region of the spermatozoon, one axoneme, twisted cortical microtubules, a periaxonemal sheath, and a spiralled nucleus. The anterior spermatozoon extremity is characterized by the presence of an electron-dense apical cone and a single spiralled crested body, which is attached to the sperm cell in the anterior and posterior areas of region I, whereas in the middle area it is partially detached from the cell. This crested body is described for the first time in cestodes. The posterior extremity of the male gamete exhibits only the disorganizing axoneme. Results are discussed and compared particularly with the available ultrastructural data on dilepidids sensu lato.

Spermiogenesis and spermatozoon ultrastructure of the diphyllidean cestode Echinobothrium euterpes (Neifar, Tyler and Euzet 2001) Tyler 2006, a parasite of the common guitarfish Rhinobatos rhinobatos

Spermiogenesis and the ultrastructural characters of the spermatozoon of Echinobothrium euterpes are described by means of transmission electron microscopy, including cytochemical analysis for glycogen. Materials were obtained from a common guitarfish Rhinobatos rhinobatos caught in the Gulf of Gabès (Tunisia). Spermiogenesis in E. euterpes is characterized by the orthogonal development of two unequal flagella followed by the flagellar rotation and the proximodistal fusion of these flagella with the median cytoplasmic process. The most interesting pattern characterizing the diphyllidean cestodes is the presence of a triangular body constituted by fines and dense granules without visible striation and assimilated at the striated rootlets. This pattern, only related in the Diphyllidea cestodes may be a synapomorphy of this order. Spermiogenesis is also characterized by the presence of a very short flagellum (around 1 μm long), observed in all the stages of spermiogenesis. This type of flagellum has never been commented in the diphyllidean cestodes and should be considered as an evolved character in this group. In the latest stage of spermiogenesis, this short axoneme probably degenerates. Thus, the mature spermatozoon of E. euterpes possesses only one axoneme of 9 + '1' trepaxonematan pattern. It also exhibits a single helical electron-dense crested body, a spiraled nucleus, few parallel cortical microtubules, and α-glycogen granules. Similitudes and differences between spermatozoa of diphyllideans are discussed.

Spermiogenesis and spermatozoon ultrastructure of the diphyllidean cestode Echinobothrium euterpes (Neifar, Tyler and Euzet 2001) Tyler 2006, a parasite of the common guitarfish Rhinobatos rhinobatos

Spermiogenesis and the ultrastructural characters of the spermatozoon of Echinobothrium euterpes are described by means of transmission electron microscopy, including cytochemical analysis for glycogen. Materials were obtained from a common guitarfish Rhinobatos rhinobatos caught in the Gulf of Gabès (Tunisia). Spermiogenesis in E. euterpes is characterized by the orthogonal development of two unequal flagella followed by the flagellar rotation and the proximodistal fusion of these flagella with the median cytoplasmic process. The most interesting pattern characterizing the diphyllidean cestodes is the presence of a triangular body constituted by fines and dense granules without visible striation and assimilated at the striated rootlets. This pattern, only related in the Diphyllidea cestodes may be a synapomorphy of this order. Spermiogenesis is also characterized by the presence of a very short flagellum (around 1 μm long), observed in all the stages of spermiogenesis. This type of flagellum has never been commented in the diphyllidean cestodes and should be considered as an evolved character in this group. In the latest stage of spermiogenesis, this short axoneme probably degenerates. Thus, the mature spermatozoon of E. euterpes possesses only one axoneme of 9 + '1' trepaxonematan pattern. It also exhibits a single helical electron-dense crested body, a spiraled nucleus, few parallel cortical microtubules, and α-glycogen granules. Similitudes and differences between spermatozoa of diphyllideans are discussed.

Immunolocalisation of secreted-excreted products of Meloidogyne spp. using polyclonal and monoclonal antibodies

Molecules expressed at the surface cuticle (SC) of plant parasitic nematodes represent the primary plant-nematode interface, and together with secreted-excreted (S-E) products are probably the first signals perceived by the host. These molecules, which are released into plant tissue, probably play important roles in the host-parasite interactions. Characterisation of these antigens will help in the identification of nematode targets useful for novel control strategies, which interfere with the nematode infection of plants. Three monoclonal (MAbs) and three polyclonal (PAbs) antibodies produced to S-E products of Meloidogyne spp. and Heterodera avenae were used to examine their reactivity towards M. incognita and/or M. arenaria second stage juveniles and adult females. The three PAbs showed cross-reactivity with M. incognita and M. arenaria. Antibody Roth-PC 373 strongly recognised molecules present in the SC, amphids and intestine, antibody Roth-PC 389 recognised the nematode amphids and metacorpus, while antibody Roth-PC 419 bound to molecules present in the subventral glands. Reactivity of the MAbs was only tested against M. arenaria. Monoclonal antibody Roth-MAb T116C1.1 showed intense reactivity with molecules present in the amphidial and phasmidial glands. Monoclonal antibodies Roth-MAb T46.2 and T42D.2 labeled the nematode amphids and molecules present in the nematode oesophagus (metacorpus), respectively.

Genetic and antigenic analysis of Babesia bigemina isolates from five geographical regions of Brazil

A molecular epidemiological study was performed with Babesia bigemina isolates from five geographical regions of Brazil. The genetic analysis was done with random amplification of polymorphic DNA (RAPD), repetitive extragenic palindromic elements-polymerase chain reaction (REP-PCR) and enterobacterial repetitive intergenic consensus sequences-polymerase chain reaction (ERIC-PCR) that showed genetic polymorphism between these isolates and generated fingerprinting. In RAPD, ILO872 and ILO876 primers were able to detect at least one fingerprinting for each B. bigemina isolate. The amplification of B. bigemina DNA fragments by REP-PCR and ERIC-PCR gave evidence for the presence in this haemoprotozoan of the sequences described previously in microorganisms of the bacterial kingdom. For the first time it was demonstrated that both techniques can be used for genetic analysis of a protozoan parasite, although the ERIC-PCR was more discriminatory than REP-PCR. The dendogram with similarity coefficient among isolates showed two clusters and one subcluster. The Northeastern and Mid-Western isolates showed the greatest genetic diversity, while the Southeastern and Southern isolates were the closest. The antigenic analysis was done through indirect fluorescent antibody technique and Western blotting using a panel of monoclonal antibodies directed against epitopes on the merozoite membrane surface, rhoptries and membrane of infected erythrocytes. As expected, the merozoite variable surface antigens, major surface antigen (MSA)-1 and MSA-2 showed antigenic diversity. However, B cell epitopes on rhoptries and infected erythrocytes were conserved among all isolates studied. In this study it was possible to identify variable and conserved antigens, which had already been described as potential immunogens. Considering that an attenuated Babesia clone used as immunogen selected populations capable of evading the immunity induced by this vaccine, it is necessary to evaluate more deeply the cross-protection conferred by genetically more distant Brazilian B. bigemina isolates and make an evaluation of the polymorphism degree of variable antigens such as MSA-1 and MSA-2.

Evaluation of the indirect fluorescent antibody test and modified agglutination test for detection of antibodies against Toxoplasma gondii in experimentally infected pigs

The study determined the sensitivity and specificity of the indirect fluorescent antibody test (IFAT) and modified agglutination test (MAT) for anti-Toxoplasma gondii antibody detection by analyzing sera from 46 experimentally infected pigs. Values for sensitivity were 95.7% (confidence interval 95%: 84.0-99.2%) and for specificity 97.8% (confidence interval 95%: 87.0-99.9%) in both tests. There was an optimum agreement of results between IFAT and MAT evidenced by a Kappa test of 0.86. These results validate these tests for the detection of T. gondii infection in pigs. IFAT and MAT despite methodologies with different characteristics and readings have similar accuracy in pig serum samples.

Toxoplasma gondii in experimentally infected Bos taurus and Bos indicus semen and tissues

Eighteen young steers were inoculated with Toxoplasma gondii and randomly distributed into three groups of six animals each: GI, 2.5x10(5) "P" strain oocysts, GII, 5.0x10(6) "RH" strain tachyzoites, and GIII (Control). Clinical, serological and parasitemia exams were realized. Parasite investigation by bioassay and PCR was realized on semen and fragments of skeletal musculature, lymph nodes, brain, retina, spleen, liver, lung, testicle, epididymis and seminal vesicle. Blood and semen samples were collected on days -2, -1, 1, 3, 5, 7, 14 and weekly thereafter, up to postinfection day (PID) 84. The inoculated steers (GI and GII) presented hyperthermia from PID 3 to 16. Antibodies against T. gondii were detected through the indirect fluorescence antibody test (IFAT) on PID 5 (1:16) in both inoculated groups (oocysts and tachyzoites), reaching peaks of 1:4096 on PID 7. Parasitemia outbursts occurred in all infected bovines, principally from PID 7 to 28, independent of the strain and inoculate used. Bioassays revealed the presence of parasites in semen samples of animals infected with oocysts (GI) and tachyzoites (GII) on several experimental days between PID 7 and 84. Tissue parasitism by T. gondii was diagnosed by bioassay and the PCR technique in several organ and tissue fragments. These findings suggest the possibility of sexual transmission of T. gondii in the bovine species.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgM antibodies against Leishmania chagasi in dogs

Visceral leishmaniasis is an emergent zoonosis with an increasing number of new cases in Brazil where the domestic dog is an important parasite reservoir in the infectious cycle of Leishmania chagasi. An enzyme-linked immunosorbent assay (ELISA), based upon the use of a total soluble antigenic preparation of L. chagasi, was adapted for the detection of IgM antibodies in the serum of infected dogs. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkboard titrations. The specificity and sensitivity of the ELISA were 100 %. A total of 110 serum samples were taken from dogs in Belo Horizonte, Minas Gerais, Brazil, and examined for anti-L. chagasi IgM antibody by ELISA and indirect fluorescent antibody test (IFAT). About 25% (n=27) of all the dogs tested were found serologically positive for L. chagasi by IFAT, while 89.09% (n=98) were seropositive by ELISA. The results obtained by ELISA and IFAT were significantly different (P<0.01). The combined use of ELISA and IFAT is recommended in order to enable veterinary services to more efficiently detect canine visceral leishmaniasis.