Unbiased random mutagenesis contributes to a better understanding of the virulent behaviour of Paenibacillus larvae.

AIMS: American foulbrood, caused by the Gram-positive bacteria Paenibacillus larvae, is one of the most severe bacterial diseases of the European honey bee. The bacterium has been known for long, but only the last decade the mechanisms used by the pathogen to cause disease in its host are starting to unravel. In this study, the knowledge of this virulent behaviour is expanded and several possible virulence factors are suggested. METHODS AND RESULTS: Identification of possible virulence factors has been done by random mutagenesis to ensure an unbiased approach. A library of mutants was tested for a significant difference in virulence using in vitro exposure assays. Affected loci were characterized and their potential to contribute in virulence of the pathogen was assessed. CONCLUSIONS: The identified mutated loci dacB, dnaK, metN, ywqD, lysC, serC and gbpA are known to encode for virulence factors in other bacteria and are suggested to play a similar role in P. larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: The study identified new possible virulence factors for P. larvae genotype ERIC I in an unbiased way. This contributes to the knowledge and understanding of the possible mechanisms used by this pathogen to colonize and kill its host.

Extending the honey bee venome with the antimicrobial peptide apidaecin and a protein resembling wasp antigen 5.

Honey bee venom is a complex mixture of toxic proteins and peptides. In the present study we tried to extend our knowledge of the venom composition using two different approaches. First, worker venom was analysed by liquid chromatography-mass spectrometry and this revealed the antimicrobial peptide apidaecin for the first time in such samples. Its expression in the venom gland was confirmed by reverse transcription PCR and by a peptidomic analysis of the venom apparatus tissue. Second, genome mining revealed a list of proteins with resemblance to known insect allergens or venom toxins, one of which showed homology to proteins of the antigen 5 (Ag5)/Sol i 3 cluster. It was demonstrated that the honey bee Ag5-like gene is expressed by venom gland tissue of winter bees but not of summer bees. Besides this seasonal variation, it shows an interesting spatial expression pattern with additional production in the hypopharyngeal glands, the brains and the midgut. Finally, our immunoblot study revealed that both synthetic apidaecin and the Ag5-like recombinant from bacteria evoke no humoral activity in beekeepers. Also, no IgG4-based cross-reactivity was detected between the honey bee Ag5-like protein and its yellow jacket paralogue Ves v 5.

Two novel proteins expressed by the venom glands of Apis mellifera and Nasonia vitripennis share an ancient C1q-like domain.

An in-depth proteomic study of previously unidentified two-dimensional polyacrylamide gel electrophoresis spots of honey bee (Apis mellifera, Hymenoptera) venom revealed a new protein with a C1q conserved domain (C1q-VP). BlastP searching revealed a strong identity with only two proteins from other insect species: the jewel wasp, Nasonia vitripennis (Hymenoptera), and the green pea aphid, Acyrthosiphon pisum (Hemiptera). In higher organisms, C1q is the first subcomponent of the classical complement pathway and constitutes a major link between innate and acquired immunity. Expression of C1q-VP in a variety of tissues of honey bee workers and drones was demonstrated. In addition, a wide spatial and temporal pattern of expression was observed in N. vitripennis. We suggest that C1q-VP represents a new member of the emerging group of venom trace elements. Using degenerate primers the corresponding gene was found to be highly conserved in eight hymenopteran species, including species of the Aculeata and the Parasitica groups (suborder Apocrita) and even the suborder Symphyta. A preliminary test using recombinant proteins failed to demonstrate Am_C1q-VP-specific immunoglobulin E recognition by serum from patients with a documented severe bee venom allergy.

Insights into the venom composition of the ectoparasitoid wasp Nasonia vitripennis from bioinformatic and proteomic studies.

With the Nasonia vitripennis genome sequences available, we attempted to determine the proteins present in venom by two different approaches. First, we searched for the transcripts of venom proteins by a bioinformatic approach using amino acid sequences of known hymenopteran venom proteins. Second, we performed proteomic analyses of crude N. vitripennis venom removed from the venom reservoir, implementing both an off-line two-dimensional liquid chromatography matrix-assisted laser desorption/ ionization time-of-flight (2D-LC-MALDI-TOF) mass spectrometry (MS) and a two-dimensional liquid chromatography electrospray ionization Founer transform ion cyclotron resonance (2D-LC-ESI-FT-ICR) MS setup. This combination of bioinformatic and proteomic studies resulted in an extraordinary richness of identified venom constituents. Moreover, half of the 79 identified proteins were not yet associated with insect venoms: 16 proteins showed similarity only to known proteins from other tissues or secretions, and an additional 23 did not show similarity to any known protein. Serine proteases and their inhibitors were the most represented. Fifteen nonsecretory proteins were also identified by proteomic means and probably represent so-called 'venom trace elements'. The present study contributes greatly to the understanding of the biological diversity of the venom of parasitoid wasps at the molecular level.

Multiplex PCR detection of slowly-evolving trypanosomatids and neogregarines in bumblebees using broad-range primers.

AIMS: The aims of this study were to design universal markers for different protozoan parasites of Bombus spp. based on the phylogenetic position of two important bumblebee parasites Crithidia bombi and Apicystis bombi. METHODS AND RESULTS: Standard PCR and extraction techniques were used to amplify and sequence 18S rDNA. Phylogenetic analysis of the rDNA was performed in order to predict the parasite range of the primers. CONCLUSIONS: Crithidia bombi phylogenetically clusters with the trypanosomatids with slowly-evolving SSU-rRNA sequences (SE), while A. bombi is the closest sister group of Mattesia. A multiplex was designed containing an internal control and two broad-range primer pairs, detecting C. bombi and other SE trypanosomatids and also A. bombi and other neogregarines. SIGNIFICANCE AND IMPACT OF THE STUDY: Sequence data generated will further improve the current systematics of insect trypanosomatids and gregarines that remain troublesome. Broad-range markers for bumblebee parasites are necessary tools enabling the screening of commercially imported colonies and thus controlling their worldwide distribution and to discover related emerging parasites.

Diagnosis of American foulbrood in honey bees: a synthesis and proposed analytical protocols.

Worldwide, American foulbrood (AFB) is the most devastating bacterial disease of the honey bee (Apis mellifera). Because the distinction between AFB and powdery scale disease is no longer considered valid, the pathogenic agent has recently been reclassified as one species Paenibacillus larvae, eliminating the subspecies designations Paenibacillus larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens. The creamy or dark brown, glue-like larval remains of infected larvae continue to provide the most obvious clinical symptom of AFB, although it is not conclusive. Several sensitive and selective culture media are available for isolation of this spore-forming bacterium, with the type of samples that may be utilized for detection of the organism being further expanded. PCR methods for identification and genotyping of the pathogen have now been extensively developed. Nevertheless, biochemical profiling, bacteriophage sensitivity, immunotechniques and microscopy of suspect bacterial strains are entirely adequate for routine identification purposes.

Genomic and transcriptional analysis of protein heterogeneity of the honeybee venom allergen Api m 6.

Several components of honeybee venom are known to cause allergenic responses in humans and other vertebrates. One such component, the minor allergen Api m 6, has been known to show amino acid variation but the genetic mechanism for this variation is unknown. Here we show that Api m 6 is derived from a single locus, and that substantial protein-level variation has a simple genome-level cause, without the need to invoke multiple loci or alternatively spliced exons. Api m 6 sits near a misassembled section of the honeybee genome sequence, and we propose that a substantial number of indels at and near Api m 6 might be the root cause of this misassembly. We suggest that genes such as Api m 6 with coding-region or untranslated region indels might have had a strong effect on the assembly of this draft of the honeybee genome.

Disinfection of wooden structures contaminated with Paenibacillus larvae subsp. larvae spores.

AIMS: The aim of the study is to examine the disinfection of wood contaminated with Paenibacillus larvae subsp. larvae spores, in order to find a practical decontamination method for hive materials. METHODS AND RESULTS: The number of viable spores recovered after the treatment, on the surface by swabbing, and in the deeper parts of the wood by scraping, was used to test the efficiency of the disinfection. Our results indicate that chemical disinfection is only complete when high concentrations (> 50%) of the disinfectant are used. Heat treatment in general was found to be very effective. The scorching of wood was not satisfactory as it only killed spores at the surface. CONCLUSION: Complete disinfection is only possible with some heat treatments or by using high concentrations of chemical disinfectants. SIGNIFICANCE AND IMPACT OF THE STUDY: This study puts forward some methods that can provide complete decontamination, which is necessary for an effective control of American foulbrood disease.

Speculation on whether a vaccine against cryptosporidiosis is a reality or fantasy.

In this paper the authors question whether the development of a vaccine against cryptosporidiosis could be taken into consideration. The necessity and feasibility of such a vaccine for human and veterinary application is discussed. Developmental stages within the life cycle of the parasite that might act as possible targets for vaccine development are summarised, as well as the target antigens offered by molecular biology and immunology studies. Vaccination trials against cryptosporidiosis carried out so far, including the active and passive immunisation approach, are also overviewed. It seems that with respect to a Cryptosporidium vaccine two target groups can be considered: children of the developing world and neonatal ruminants. Antigens representing possible candidates for a subunit vaccine were identified based on their function, location and/or the immune response they evoke. While the active vaccination of newborn calves, lambs and goat kids has to face a number of important limitations, the passive immunisation approach, where dams were immunised to protect their progeny by colostral transfer, was proven to be a valuable alternative. Finally, a number of points of action for the near future are put forward.

A review of the importance of cryptosporidiosis in farm animals.

Cryptosporidium species are coccidian parasites with a large capacity to reproduce and to disseminate. Several species are known to infect farm animals, although the economic importance of cryptosporidiosis is highly host species dependent. This paper reviews the impact of cryptosporidial infections in livestock and poultry. For different farm animals, the Cryptosporidium spp. that occur, as well as their clinical and pathological features, and their interactions with other pathogens, are described. In addition, data concerning the prevalence, the transmission and the epidemiology of the disease are mentioned and a description of the economic losses associated with cryptosporidiosis in each of the hosts is given. Cryptosporidiosis seems to be mainly a problem in neonatal ruminants. Cryptosporidium parvum is considered to be an important agent in the aetiology of the neonatal diarrhoea syndrome of calves, lambs and goat kids, causing considerable direct and indirect economic losses. Avian cryptosporidiosis is an emerging health problem in poultry, associated with respiratory disease in chickens and other Galliformes, and with intestinal disease in turkeys and quails. Because of limited availability of effective drugs, the control of cryptosporidiosis relies mainly on hygienic measures and good management.

Serological differences in Neospora caninum-associated epidemic and endemic abortions.

A sandwich enzyme-linked immunosorbent assay (ELISA) for the sensitive and specific detection of bovine antibodies to Neospora caninum was developed and evaluated using sera from cattle experimentally infected with N. caninum, Toxoplasma gondii, Sarcocystis cruzi, Sarcocystis hominis, Sarcocystis hirsuta, Eimeria bovis, Cryptosporidium parvum, Babesia divergens, and field sera from naturally exposed animals. Field sera were classified using a gold standard that included the results from an indirect fluorescent antibody test (IFAT) and an immunoblot (IB). Based on these gold standard results, i.e., IFAT-IB results, an equal relative sensitivity and specificity of 94.2%(theta0) was reached when a cutoff of 0.034 (d0) was employed. The analysis of IFAT-IB-positive field sera showed that within groups of aborting and nonaborting dams, the animals from herds with endemic N. caninum-associated abortions had significantly higher ELISA indices than animals from herds with N. caninum-associated epidemic abortions. By contrast, IFAT-IB-positive aborting dams from herds with endemic N. caninum-associated abortions had significantly lower IFAT titers than IFAT-IB-positive aborting dams from herds with epidemic N. caninum-associated abortions. This is the first time that statistically significant serological differences between herds exhibiting epidemic and endemic N. caninum-associated abortions are described.

A Cryptosporidium parvum oocyst low molecular mass fraction evokes a CD4+ T-cell-dependent IFN-gamma response in bovine peripheral blood mononuclear cell cultures.

T-Cell antigens that induce the in-vitro interferon-gamma response during Cryptosporidium parvum infection of neonatal calves were identified. A total oocyst extract was separated into a high and a low Mr fraction by a microfiltration technique. Both the high and low Mr fractions evoked an in-vitro interferon-gamma response in naturally infected animals, although strong individual differences between the hosts were observed. Using a complement-mediated technique CD4+ T-cells or WC1+gammadelta T-cells were depleted, whereupon the remaining lymphocyte cultures were stimulated with the different antigen preparations. It was shown that the in-vitro interferon-gamma response of Cryptosporidium-infected calves is CD4+ T-cell-dependent.

Specific interferon-gamma, IgA and IgM responses after experimental infection of neonatal calves with Cryptosporidium parvum.

The in vitro interferon-gamma production by peripheral blood mononuclear cells and the local antibody build up was monitored in neonatal calves experimentally infected with Cryptosporidium parvum and in negative controls of the same age. From day 6 p.i. on, an infection-induced interferon-gamma response was observed in lymphocyte cultures after stimulation with Cryptosporidium oocyst antigen preparation. Only the Cryptosporidium-infected calves developed local IgA and IgM responses from day 6 p.i. on, with peak values at day 10 p.i. These antibodies disappeared quickly, perhaps due to the strict hygienic measures and consequently the absence of a continuous antigenic stimulation.

Isolation, characterization and immunolocalization of a globin-like antigen from Ostertagia ostertagi adults.

Western blot analysis using an anti-globin rabbit serum Rb94 revealed a major band of 17 kDa in extracts of Ostertagia ostertagi adults and 4th-stage larvae. The adult stage globin-like antigen (OoAdGlb) was purified from total worm extracts by liquid chromatography. The protein has an estimated molecular mass of 36 kDa under non-reducing conditions, suggesting a dimeric structure containing 2 non-covalently linked 17 kDa monomers. Tryptic peptides were sequenced and showed strong similarities with the globins of free-living and parasitic nematodes. Immunolocalization revealed the presence of this globin-like antigen in the body wall musculature and/or the cuticle of O. ostertagi adults. An enzyme-linked immunosorbent assay based on the purified OoAdGlb showed no differences in response between calves infected by O. ostertagi and/or Cooperia oncophora and the negative controls.

Immunisation of calves with Ostertagia ostertagi fourth stage larval antigens failed to protect calves from infection.

Excretory-secretory and somatic antigens of Ostertagia ostertagi fourth stage larvae, emulsified with complete Freund's adjuvant, were intraperitoneally administered to calves on three occasions. Two weeks after the last immunisation all calves were infected with a single dose of 130,000 O. ostertagi third stage larvae. All animals were necropsied 25 days after infection. The immunisation procedure resulted in IgG1 and IgG2 antibodies, however no protective immunity was induced as O. ostertagi worm burdens and worm length were similar in all groups.

Interactions between Ostertagia ostertagi and Cooperia oncophora in calves.

Development of immunity to Ostertagia ostertagi and Cooperia oncophora and interactions between both species in primed calves were investigated after homologous, heterologous and concurrent challenge infections. Worm counts, faecal egg output, IgG1 and IgG2 antibodies and the presence of globule leucocytes were used to evaluate the possible interactions. Results show that immunity build-up against O. ostertagi is slow in comparison with C. oncophora. The presence of early fourth-stage larvae and globule leukocytes in the O. ostertagi primed groups was significantly different to that of a previously uninfected control group. Ostertagia ostertagi and C. oncophora IgG1 antibodies were high in the previously exposed groups compared with uninfected controls and C. oncophora antibodies cross-reacted strongly with O. ostertagi antigens. There was no conclusive evidence for an interaction between C. oncophora and O. ostertagi. Globule leukocytes, IgG1 antibodies and early fourth-stage larvae seem to be related to development of immunity to O. ostertagi.

Identification and isolation of a 19.7 kDa Ostertagia ostertagi specific antigen and evaluation of its potential for immunodiagnosis.

Ostertagia ostertagi adult worm extracts were analysed by Western blotting using sera from calves experimentally infected with O. ostertagi and Cooperia oncophora. Strong differences in antigen recognition were noticed, even between animals from the same group. Two Ostertagia specific antigens with apparent molecular mass of 19.7 (OoA19.7) and 20.7 kDa (OoA20.7) were identified. One of them (OoA19.7) was purified by three subsequent chromatographic steps, i.e. gelfiltration, ion-exchange and reversed phased chromatography. It was demonstrated that this antigen does not show any cross-reactions with heterologous sera from C. oncophora, Dictyocaulus viviparus, Nematodirus helvetianus and Fasciola hepatica-infected animals. It was found that only IgG1 antibodies reacted against OoA19.7. The application of this antigen in an ELISA resulted in a highly species-specific test when compared to crude worm extracts. However, strong individual differences in anti-OoA19.7 response could be noticed between calves which received the same number of O. ostertagi larvae. These individual differences can hinder the application of the ELISA as a diagnostic tool, since the anti-OoA19.7 response does not seem to reflect the level of exposure to L3 larvae. This was supported by the absence of a clear infection-dose-related effect. It was shown that the anti-OoA19.7 response started from week 6 to 8, and reached its highest level at week 15.

Partial characterization of proteolytic enzymes in different developmental stages of Ostertagia ostertagi.

Proteolytic enzymes present in extracts of third (L3) and fourth (L4) stage larvae and adults of the cattle nematode Ostertagia ostertagi were defined on the basis of pH optima and proteinase inhibitor sensitivity in spectrophotometric assays using azocasein and elastin-orcein as protein substrates. Evidence that different classes of proteinases are expressed in a stage specific manner was provided by the contrasting pH optima and inhibitor sensitivities shown by the enzymes in the different parasite stages. Stage specificity was confirmed by gelatin-substrate analysis. In addition, proteolytic activity was sought in the excretory/secretory products (ES) of the L4 following simple in vitro culture. Contrasting pH and inhibitor sensitivities as well as gelatin-substrate analysis showed that different proteinases were present in somatic L4 extracts and L4 ES products. The secreted proteinases may be useful targets for serodiagnosis or vaccination.

Characterisation of Ostertagia ostertagi antigens by the different bovine immunoglobulin isotypes.

Antigenic differences between the developmental stages of Ostertagia ostertagi were studied by SDS-PAGE and Western blotting. Gel electrophoresis showed a complex protein pattern different for every stage with the O ostertagi fourth stage larvae (L4) showing an intermediate protein pattern between the third stage larvae (L3) and the adult stage. Immunoblotting showed that IgG1, IgG2 and IgM immunoglobulins present in serum from uninfected calves identified several O ostertagi antigens at every stage. When using serum from O ostertagi infected calves, O ostertagi specific IgG1 was the predominant bovine immunoglobulin. Specific IgG2 and IgM responses were also observed, while specific IgA antibodies were hardly detectable. Severe IgG1 cross reactivity was demonstrated when using anti-Cooperia oncophora serum.