The prevalence of antibodies to morbilliviruses,Brucella, and Toxoplasma in the Black Sea bottlenose dolphin <Emphasis Type="Italic">Tursiops truncatus ponticus</Emphasis> maintained in captivity

The prevalence of antibodies to morbilliviruses, Brucella and Toxoplasma was studied in the Black Sea bottlenose dolphin Tursiops truncatus ponticus maintained in captivity. Serum from 59 dolphins was tested, and in most cases the tests were repeated 2–4 years later. Antibodies to the tested pathogens were detected in 69% of the dolphins. Sixteen Black Sea bottlenose dolphin maintained in captivity (27%) had antibodies to two, or even three, of the pathogens. No apparent signs of infection were found in the dolphins; however, the high prevalence of antibodies to the morbilliviruses, Brucella, and Toxoplasma may indicate an important role of dolphins in the ecology of these pathogens.     

Prevalence of Met-203 type spaA variant in Erysipelothrix rhusiopathiae isolates and the efficacy of swine erysipelas vaccines in Japan

Since 2009, erysipelas infection among pigs in Japan has been increasing. This study investigated the prevalence, and characteristics of Erysipelothrix rhusiopathiae isolates in Japan from 2008 to 2010 and assessed the efficacy of current commercial erysipelas vaccines. Based on polymorphisms in a 432-bp hypervariable region in the surface protective antigen A (spaA) gene, 34 isolates were classified into three groups: (i) Group 1 with methionine at position 203 (Met-203) and isoleucine at position 257 (Ile-257) (18 isolates of serotype 1a and one untypable isolate). (ii) Group 2 with Ile-257 (12 isolates of serotypes 1a, 1b, 2, 10 and 11), and (iii) Group 3 with alanine at position 195 (Ala-195) and Ile-257 (three isolates of serotype 1a). Isolates with Met-203 were highly pathogenic in mice and pigs, causing death in the pig and LD₅₀ values of 0.45–1.45 CFU per mouse. One live and three inactivated commercial E. rhusiopathiae vaccines were evaluated for efficacy against a Met-203 isolate. Almost all mice and pigs that received vaccine survived, while non-vaccinated controls all died within 5 days of the challenge. This indicates that swine erysipelas vaccines might be still effective in protecting animals against the recently prevalent Met-203 isolates in Japan.     

Genetic variation and diversity among loquat accessions

Loquat (Eriobotrya japonica (Thunb.) Lindl., Maloideae, Rosaceae) is a subtropical evergreen fruit tree indigenous of China, where the center of origin of the species is located. Loquat is grown in all subtropical areas and was introduced in the Mediterranean basin in late eighteenth century. In Europe, the largest germplasm bank is located at Instituto Valenciano de Investigaciones Agrarias (IVIA; Valencia, Spain). Thirteen microsatellites and a conserved region of S-allele were used to assess the genetic diversity of 102 accessions of the IVIA collection. A total of 38 SSR alleles and 11 putative S-alleles were used to study the genetic structure of the loquat germplasm bank using the STRUCTURE software, Factorial Correspondence Analysis (FCA), and unweighted pair-group method (UPGMA) cluster analyses. The total diversity was H _T?=?0.5682, the genetic differentiation G _ST?=?0.1660, and the standardized G _ST reached a much higher value of G′_ST?=?0.4948. The Evanno’s test indicated that the most informative number of populations was five, with accessions distributed according to their geographic origin in two, one, and two groups of Spanish, Italo-Spanish, and non-European origin, respectively. Knowledge of the substructure and diversity of the IVIA loquat collection and the self-incompatibility genotype data will allow us to select and incorporate useful materials into the loquat breeding program.     

Successful treatment of suspected erysipelas septicemia in a beluga whale (Delphinapterus leucas)

A 20-year-old male beluga whale (Delphinapterus leucas) was diagnosed with suspected acute septicemic erysipelas. Physical examination was unremarkable and no skin lesions were evident. Hematologic and serum biochemical abnormalities included mild anemia, a marked leukocytosis, an increase in the erythrocyte sedimentation rate, and moderate elevations of total protein, gamma globulins, blood urea nitrogen, and creatinine. Microtitration agglutination testing for Erysipelothrix rhusiopathiae antibodies revealed seroconversion and elevated antibody titers associated with the clinical illness, presumptive evidence of an active E. rhusiopathiae infection. Treatment consisted of oral fluids, cimetidine, and a 25-day course of ciprofloxacin hydrochloride. The whale made a rapid and uneventful recovery. None of the three female beluga whales that shared the pool with the ill male, nor male and female beluga whales maintained in a separate pool, became ill or showed serologic evidence of E. rhusiopathiae exposure. © 1993 Wiley-Liss, Inc.     

Antibodies to selected pathogens in wild boar (Sus scrofa) from Catalonia (NE Spain)

From 2004 to 2007, blood samples from 273 healthy wild boars (Sus scrofa), culled during the hunting season, were obtained in three areas of Catalonia (NE Spain): Pyrenees, Sant Llorenç del Munt i l’Obac Natural Park (SLM), and Ports de Tortosa i Beseit National Hunting Reserve (PTB). We investigated the presence of antibodies against classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine vesicular disease virus (PVDV), porcine respiratory and reproductive syndrome virus (PRRSV), Aujeszky’s disease virus (ADV), porcine influenza A virus (PIV), porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), Mycoplasma hyopneumoniae, Erysipelothrix rhusiopathiae, Salmonella spp., and Toxoplasma gondii. Four wild boars were suspicious for CSFV, but the infection was discarded with a virus neutralization test, and infection with a border disease virus was confirmed. Negative results were obtained against ASFV and PVDV. Antibodies were detected against PRRSV (3%), ADV (0.8%), PIV (6.4%), PCV2 (64.6%), PPV (54.7%), M. hyopneumoniae (26.6%), E. rhusiopathiae (5.3%), Salmonella spp. (11.3%), and T. gondii (43.5%). In SLM, we detected a higher seroprevalence for PIV and M. hyopneumoniae and a lower seroprevalence for E. rhusiopathiae than in the other two areas. In PTB, seroprevalence was higher for PPV, Salmonella spp., and PCV2. Adult wild boar displayed higher seroprevalence for PPV, PIV, and M. hyopneumoniae, whereas presence of antibodies for Salmonella spp. was higher in juveniles compared with adults and piglets.

     

Possible Effects of Glyphosate on Mucorales Abundance in the Rumen of Dairy Cows in Germany

Glyphosate (N-phosphonomethyl glycine) is registered as a herbicide for many food and non-food crops, as well as non-crop areas where total vegetation control is desired. Glyphosate influences the soil mycobiota; however, the possible effect of glyphosate residues in animal feed (soybean, corn, etc.) on animal mycobiota is almost unknown. Accordingly, the present study was initiated to investigate the mycological characteristics of dairy cows in relationship to glyphosate concentrations in urine. A total of 258 dairy cows on 14 dairy farms in Germany were examined. Glyphosate was detected in urine using ELISA. The fungal profile was analyzed in rumen fluid samples using conventional microbiological culture techniques and differentiated by MALDI-TOF mass spectrometry. LPS-binding protein (LBP) and antibodies (IgG1, IgG2, IgA, and IgM) against fungi were determined in blood using ELISA. Different populations of Lichtheimia corymbifera, Lichtheimia ramosa, Mucor, and Rhizopus were detected. L. corymbifera and L. ramosa were significantly more abundant in animals containing high glyphosate (>40 ng/ml) concentrations in urine. There were no significant changes in IgG1 and IgG2 antibodies toward isolated fungi that were related to glyphosate concentration in urine; however, IgA antibodies against L. corymbifera and L. ramosa were significantly lower in the higher glyphosate groups. Moreover, a negative correlation between IgM antibodies against L. corymbifera, L. ramosa, and Rhizopus relative to glyphosate concentration in urine was observed. LBP also was significantly decreased in animals with higher concentrations of glyphosate in their urine. In conclusion, glyphosate appears to modulate the fungal community. The reduction of IgM antibodies and LBP indicates an influence on the innate immune system of animals.     

Recombinant DNA Technology for Melanoma Immunotherapy: Anti-Id DNA Vaccines Targeting High Molecular Weight Melanoma-Associated Antigen

Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V _H) and light (V _L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V _H/V _L, and V _L/V _H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy.     

The predatory mirid Dicyphus maroccanus as a new potential biological control agent in tomato crops

The first record of the omnivorous predator Dicyphus maroccanus Wagner (Hemiptera: Miridae) inhabiting tomato crops in the Valencia region (East Coast of Spain) was in 2009. Since then, D. maroccanus has often been found preying on the eggs of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) in this area. To evaluate this predator’s potential as a biological control agent, its life-history traits in the presence and absence of prey [(eggs of Ephestia kuehniella Zeller (Lepidoptera: Pyralidae)] on tomato plants were studied under laboratory conditions. Immature stages that preyed on eggs of E. kuehniella developed successfully. However, no nymph completed development on the plant without the addition of E. kuehniella eggs. To reach adulthood, male and female D. maroccanus nymphs consumed 267 and 312 E. kuehniella eggs, respectively. The net reproductive rate (R₀) was estimated to be 34.52 female eggs per female, the generation time (T) was 40.48 days, and the estimated intrinsic rate of increase (r_m) was 0.0868 females per female per day at 25 °C. In a second experiment, the capacity to detect plants infested or not infested with T. absoluta was studied using a Y-tube olfactometer. Female D. maroccanus were strongly attracted to the odor of T. absoluta-infested plants. In a third experiment, the capacity of D. maroccanus to control T. absoluta on tomato plants was investigated under extended laboratory conditions. Dicyphus maroccanus significantly reduced the number of T. absoluta-infested leaves in over 90 % of cases relative to control conditions. These results suggest that D. maroccanus could play a significant role in T. absoluta management. The potential of this zoophytophagous predator as a biocontrol agent on tomato crops is discussed.     

In-vitro synthesis of phycobiliproteids and ribulose-1,5-bisphosphate carboxylase by non-poly-adenylated-RNA of Cyanidium caldarium and Porphyridium aerugineum

Polyadenylated and nonpolyadenylated mRNa from the red algae Cyanidium caldarium Geitler and Porphyridium aerugineum Geitler were translated in a cell-free system. The α-and β-subunits of phycocyanin and allophycocyanin and also both subunits of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase) were identified with the help of specific antibodies as translation products of non-polyadenylated mRNA. Both subunits of RuBPCase were synthesized with molecular weights in the range of the mature forms. This is in contrast to the findings with green algae and higher plants where the small subunits of RuBPCase are always made by polyadenylated mRNA in the form of a larger precursor molecule. We discuss our findings with regard to the systematic position of the algae used and the evolution of plastids.     

Wild boars: A potential source of Erysipelothrix rhusiopathiae infection in Japan

The potential role of wild boars as a source of erysipelas infection was investigated. An ELISA test of wild boar serum samples from 41 prefectures in Japan revealed that proportions of the Erysipelothrix rhusiopathiae‐positive samples were very high in all the prefectures, and the mean positive rate was 95.6% (1312/1372). Serovars of E. rhusiopathiae isolates from wild boars were similar to those of previously reported swine isolates, and all serovar isolates tested were found to be pathogenic to mice. These results suggest that wild boars in Japan constitute a reservoir of E. rhusiopathiae and may pose risks to other animals.     

Flora y vegetation briofítica higro-hidrófila de la comunidad Valenciana (Este de España)

The bryophyte flora and vegetation have been studied in aquatic and wet environments of Valencia County (eastern Spain). A catalogue of 74 taxa is given, 3 species are new for eastern Spain and 6 for provinces of Valencia County (Valencia = 4, Castellón = 2). Aquatic communities have been studied according to the Braun-Blanquet (1952) method. Nine associations belonging to the classes Adiantetea, Montio-Cardaminetea and Platyhypnidio-Fontinalietea have been recognized. One association has been described as new, Fontinaletum hypnoidis ass. nova, which always grows submerged in calcareous, unpolluted and flowing water. Ecological and syntaxonomical data on problematical communities in the Mediterranean region (EucladioAdiantetum, Eucladietum and Eucladio-Didymodonetum) are given, as well as information on the dynamics of the communities.     

Genetic basis for the polymorphism of rat liver cytosolic aldehyde dehydrogenase

Liver aldehyde dehydrogenase (ALDH), the enzyme involved in the oxidation of aldehydes such as acetaldehyde derived from ethanol, exists in multiple forms in most mammals. Up to five separable forms have been identified from the cytosolic fraction of Wistar rat liver. We investigated the genetic basis of a particular set of three enzyme forms by selective breeding and analysis of electrophoretic patterns of liver ALDH by isoelectric focusing. The forms of liver ALDH investigated were at pI 5.8 or 6.2, or a triple form with enzymes at pI 5.8, 6.0, and 6.2. There are two alleles found at the ALDH locus which encode in homozygotes for one of two electrophoretically separable ALDH forms. A rat heterozygous at the locus forms both ALDH types plus a hybrid. The alleles are expressed codominantly, found at an autosomal locus, and remain constant postpartum. The activities associated with the triplet enzyme form were statistically indistinguishable from a 1:2:1 ratio. This suggests that the enzymes hybridize to form a set of dimers or tetramers of the form A₂, AB, B₂ or A₄, A₂B₂, B₄, respectively.     

A virosomal malaria peptide vaccine elicits a long-lasting sporozoite-inhibitory antibody response in a phase 1a clinical trial

Objectives

Peptides delivered on the surface of influenza virosomes have been shown to induce solid humoral immune responses in experimental animals. High titers of peptide-specific antibodies were also induced in a phase 1a clinical trial in volunteers immunized with virosomal formulations of two peptides derived from the circumsporozoite protein (CSP) and the apical membrane antigen 1 (AMA-1) of Plasmodium falciparum. The main objective of this study was to perform a detailed immunological and functional analysis of the CSP-specific antibodies elicited in this phase 1a trial.

Methodology/Principal Findings

46 healthy malaria-naïve adults were immunized with virosomal formulations of two peptide-phosphatidylethanolamine conjugates, one derived from the NANP repeat region of P. falciparum CSP (designated UK-39) the other from P. falciparum AMA-1 (designated AMA49-C1). The two antigens were delivered in two different concentrations, alone and in combination. One group was immunized with empty virosomes as control. In this report we show a detailed analysis of the antibody response against UK-39. Three vaccinations with a 10 µg dose of UK-39 induced high titers of sporozoite-binding antibodies in all volunteers. This IgG response was affinity maturated and long-lived. Co-administration of UK-39 and AMA49-C1 loaded virosomes did not interfere with the immunogenicity of UK-39. Purified total IgG from UK-39 immunized volunteers inhibited sporozoite migration and invasion of hepatocytes in vitro. Sporozoite inhibition closely correlated with titers measured in immunogenicity assays.

Conclusions

Virosomal delivery of a short, conformationally constrained peptide derived from P. falciparum CSP induced a long-lived parasite-inhibitory antibody response in humans. Combination with a second virosomally-formulated peptide derived from P. falciparum AMA-1 did not interfere with the immunogenicity of either peptide, demonstrating the potential of influenza virosomes as a versatile, human-compatible antigen delivery platform for the development of multivalent subunit vaccines.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00400101","term_id":"NCT00400101"}}NCT00400101     

Characterization of mitochondrial bioenergetic functions between two forms of Leishmania donovani – a comparative analysis

Leishmaniasis is a growing health problem in many parts of the world partly due to drug resistance of the parasite. This study reports on the fisibility of studying mitochondrial properties of two forms of wild-type L. donovani through the use of selective inhibitors. Amastigote forms of L. donovani exhibited a wide range of sensitivities to these inhibitors. Mitochondrial complex II inhibitor thenoyltrifluoroacetone and F_oF₁-ATP synthase inhibitors oligomycin and dicyclohexylcarbodiimide were refractory to growth inhibition of amastigote forms, whereas they strongly inhibited the growth of promastigote forms. This result indicated that complex II and F_oF₁-ATP synthase were not functional in amastigote forms suggesting the presence of attenuated oxidative phosphorylation in the mitochondria of amastigote forms. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited cellular multiplication and substrate level phosphorylation in amastigote forms, suggesting the role of complex I and complex III for the survival of amastigote forms. Further we studied the mitochondrial activities of both forms by measuring oxygen consumption and ATP production. In amastigote form, substantial ATP formation by substrate level phosphorylation was observed in NADPH-fumarate, NADH-fumarate, NADPH-pyruvate and NADH-pyruvate redox couples. None of the redox couple generated ATP formation was inhibited by F_oF₁-ATP synthase inhibitor oligomycin. Therefore, we may conclude that there are significant differences between these two forms of L. donovani in respect of mitochondrial bioenergetics. Our results demonstrated bioenergetic disfunction of amastigote mitochondria. Therefore, these alterations of metabolic functions might be a potential chemotherapeutic target.     

Genetics of the anti-dextran B512 and the autoanti-idiotypic response: Codominant expression in F1 hybrids and dichotomy of response and allotype-linked idiotype

The IgM plaque-forming response to the alpha 1–6 epitope of dextran B512 is linked to the Ig-1 heavy chain allotypes j and b characteristic of CBA and C57BL strains, respectively, and the response typically induces the formation of autoanti-idiotypic antibodies that can distinguish between anti-dextran antibodies of CBA and C57BL origin. Nevertheless, some substrains of Balb/c mice (allotype a) and some Bailey recombinant stains give a PFC response although they do not possess allotypes j or b. The anti-dextran antibodies in these strains lack the idiotypes characteristic of either CBA and C57BL antibodies to dextran, but they possess their own particular idiotype. F₁ hybrids between two responder strains possessing different idiotypes on their antibodies against dextran, produce both idiotypes and two different autoanti-idiotypic antibodies. CBA(Ig-1^b) mice were high responders to dextran and possessed the idiotype of C57BL, whereas C57BL/6(Ig-1^a) mice were low responders. The V_H recombinant strains BAB.14 and CB-8KN that possess the Ig-1^b allotype of C57BL, but have some of theV _H genes from Balb/c and the rest from C57BL/6 were high responders to dextran, but did not possess the C57BL idiotype, suggesting that the genes determining the response against dextran and the idiotype may have different locations in the heavy chain locus.     

Production of IgM monoclonal antibodies in DG44 cells

Two-bicistronic vectors for the production of recombinant IgM monoclonal antibodies in the DG44 DHFR-negative cell line have been designed. We used tandem vectors, in which one bicistronic unit encoded the immunoglobulin light chain and DHFR and the other encoded the heavy chain and EGFP. The construct structure presumes that green cells surviving selection would be capable of producing both immunoglobulin chains. We found that the agglutinating IgM antibodies could be secreted in the absence of J-peptide. It was shown that the germinal leader peptide plays a key role in the expression of the genes for the light and heavy chains. A comparison of the chromatin regulatory elements demonstrated that construct-flanking 2xHS4 insulators stabilized the biosynthesis of the recombinant antibodies, whereas the 5′-MARLyz matrix attachment region proved to be less efficient. The strategy for obtaining a DG44-based producer cell line should include the following consecutive steps: selection on the medium without nucleoside → amplification of the inserted gene → cloning of transfectants → selection of high-productive clones. An attempt to clone before amplification and to amplify individual clones failed to result in effective producers. Cloning on a medium without selection pressure allows a more adequate assessment of the stability of the antibody production.     

Counterimmunoelectrophoresis as a routine mycoserological procedure

Counterimmunoelectrophoresis (CIE) has been compared in a diagnostic laboratory with agar gel double diffusion (DD) as a routine procedure for detection of antibodies to pathogenic and allergenic fungi and actinomycetes. It was shown to be of particular value in detecting antibodies to Aspergillus fumigatus. Thus 72 of 106 sera in which precipitins were detected were positive by CIE alone. Some sera were positive only by CIE to antigens prepared from Histoplasma capsulatum, Allescheria boydii, Candida albicans and C. parapsilosis.     

Production and purification of monoclonal antibodies to Pisum and Avena phytochrome

At least nine monoclonal antibodies against phytochrome from Pisum sativum L. and 20 against phytochrome from Avena sativa L. have been obtained from mouse hybridomas that were produced by fusion of spleen cells with SP 2/O-Ag14 myeloma cells. Hybridomas were selected and cloned in a single step by plating on a semisolid methylcellulose medium. Eight antibodies to Pisum and one to Avena phytochrome were immunopurified from hybridoma medium or ascitic fluid. When necessary, secreted antibodies were verified to be against phytochrome by demonstrating to be against phytochrome by demonstrating immunoadsorption of phytochrome, detected as loss of photoactivity and-or by appearance of the approx. 120,000-dalton phytochrome band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Effect of bovine apo-lactoferrin on the growth and virulence of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae (App) is a Gram-negative bacterium that causes porcine pleuropneumonia, leading to economic losses in the swine industry. Due to bacterial resistance to antibiotics, new treatments for this disease are currently being sought. Lactoferrin (Lf) is an innate immune system glycoprotein of mammals that is microbiostatic and microbicidal and affects several bacterial virulence factors. The aim of this study was to investigate whether bovine iron-free Lf (BapoLf) has an effect on the growth and virulence of App. Two serotype 1 strains (reference strain S4074 and the isolate BC52) and a serotype 7 reference strain (WF83) were analyzed. First, the ability of App to grow in iron-charged BLf was discarded because in vivo, BapoLf sequesters iron and could be a potential source of this element favoring the infection. The minimum inhibitory concentration of BapoLf was 14.62, 11.78 and 10.56 µM for the strain BC52, S4074 and WF83, respectively. A subinhibitory concentration (0.8 µM) was tested by assessing App adhesion to porcine buccal epithelial cells, biofilm production, and the secretion and function of toxins and proteases. Decrease in adhesion (24–42 %) was found in the serotype 1 strains. Biofilm production decreased (27 %) for only the strain 4074 of serotype 1. Interestingly, biofilm was decreased (60–70 %) in the three strains by BholoLf. Hemolysis of erythrocytes and toxicity towards HeLa cells were not affected by BapoLf. In contrast, proteolytic activity in all strains was suppressed in the presence of BapoLf. Finally, oxytetracycline produced synergistic effect with BapoLf against App. Our results suggest that BapoLf affects the growth and several of the virulence factors in App.