Allele-Specific Dual PCRs to Identify Members of the 27a Cluster of PPV.

Porcine Parvovirus (PPV) is one of the most important infectious agents causing severe reproductive failure in pigs. In the last two decades a particular, a novel genotype emerged in Europe and PPV-27a was named as the prototype of this genetic cluster. It was suggested that members of the PPV-27a cluster may adversely influence effective vaccination against PPV. For a reliable updated 27a definition, we aligned 93 databank-deposited partial or full nucleotide and protein sequences of the VP2 of different PPV isolates. We confirmed that the 27a cluster could indeed be distinguished from other members of the species, however, some divergences were identified compared to earlier defined genetic markers. Based on genetic differences, we developed a dual allele-specific polymerase chain reaction for the easy and quick discrimination of members of the 27a cluster from other PPV strains. The detection limit of dual PCR was found <1.66 × 104 copies/reaction. To sensitize and make it more user friendly, the method was further developed for qPCR application with fluorescent probes. Regarding the detection limit of the two PCRs (<1.66 × 104 copies/reaction of the dual PCR versus <2.40 × 102 copy/reaction of the dual qPCR), approximately two log improvement was achieved in the sensitivity of the method.

Maternally Derived Antibody Levels Influence on Vaccine Protection against PCV2d Challenge.

Piglets from a porcine circovirus type 2 (PCV2) stable farm of low and high levels of maternally derived antibodies (MDA) against PCV2 were vaccinated either with a whole virus type or a PCV2 ORF2 antigen-based commercial subunit vaccine at three weeks of age. Two non-vaccinated groups served as low and high MDA positive controls. At four weeks post vaccination, all piglets were challenged with a PCV2d-2 type virus strain and were checked for parameters related to vaccine protection over a four-week observation period. MDA levels evidently impacted the outcome of the PCV2d-2 challenge in non-vaccinated animals, while it did not have a significant effect on vaccine-induced protection levels. The humoral immune response developed faster in the whole virus vaccinates than in the subunit vaccinated pigs in the low MDA groups. Further, high MDA levels elicited a stronger negative effect on the vaccine-induced humoral immune response for the subunit vaccine than for the whole virus vaccine. The group-based oral fluid samples and the group mean viraemia and faecal shedding data correlated well, enabling this simple, and animal welfare-friendly sampling method for the evaluation of the PCV2 viral load status of these nursery piglets.

A Case of Infectious Laryngotracheitis in an Organic Broiler Chicken Farm in Greece.

Infectious laryngotracheitis is an economically significant viral disease of chickens, that mainly affects the upper respiratory tract, and is present worldwide. This case reports the first outbreak of infectious laryngotracheitis in a four-week-old organic broiler farm and surrounding flocks in Greece, with typical clinical symptoms and lesions, allegedly provoked by a wild strain of infectious laryngotracheitis virus. Our findings contradict the general perception indicating that the disease appears mainly in older birds and that vaccine strains are the primary cause of infectious laryngotracheitis outbreaks in most continents. A recombinant vectored vaccine was administered, supplementary to biosecurity measures, containing the viral spread. The responsible strain was potentially circulating in the area; therefore, an industry-wide holistic approach was applied, including the vaccination of neighboring broilers and breeders with the same vaccine, the rapid molecular diagnosis of the disease, and strict biosecurity protocols. The results of this holistic effort were effective because, following the application of vaccine and management protocols, manifestations of the disease in regional flocks dropped significantly, and there was no recurrence to date. These findings suggest that vaccination protocols should be modified, especially for organic broilers, to include vaccination against infectious laryngotracheitis.

A Herpesvirus of Turkey-Based Vector Vaccine Reduces Transmission of Newcastle Disease Virus in Commercial Broiler Chickens with Maternally Derived Antibodies.

Newcastle Disease is one of the most important infectious poultry diseases worldwide and is associated with high morbidity, mortality, and economic loss. In several countries, vaccination is applied to prevent and control outbreaks; however, information on the ability of vaccines to reduce transmission of ND virus (NDV) is sparse. Here we quantified the transmission of velogenic NDV among 42-day-old broilers. Chickens were either vaccinated with a single dose of a vector vaccine expressing the F protein (rHVT-ND) at day-old in the presence of maternally derived antibodies or kept unvaccinated. Seeders were challenged 8 h before the co-mingling with the corresponding contacts from the same group. Infection was monitored by daily testing of cloacal and oro-nasal swabs with reverse transcription-real-time PCR and by serology. Vaccinated birds were completely protected against clinical disease and virus excretion was significantly reduced compared to the unvaccinated controls that all died during the experiment. The reproduction ratio, which is the average number of secondary infections caused by an infectious bird, was significantly lower in the vaccinated group (0.82 (95% CI 0.38-1.75)) than in the unvaccinated group (3.2 (95% CI 2.06-4.96)). Results of this study demonstrate the potential of rHVT-ND vaccine in prevention and control of ND outbreaks.

Characterization of a PCV2d-2 isolate by experimental infection of pigs.

Porcine circovirus type 2 (PCV2), a highly prevalent, economically important swine pathogen is classified into different genotypes (PCV2a-f) based on phylogenetic analysis. Since the introduction of extensive vaccination programs, at least two major shifts have been observed in the prevalence of PCV2 genotypes. The first genotype shift from 2a towards 2b occurred around 2003, while in recent years, we are witnessing the second change in genotype prevalence from the predominant 2b towards 2d.In this study, a PCV2d-2 isolate was characterized as a potential challenge virus for the evaluation of PCV2 vaccine efficacy. Ten-week-old pigs carrying low to moderate levels of maternally derived antibodies to PCV2 were infected with the isolate by the nasal route. Over the next 4 weeks post-infection, the pigs were monitored for the presence of viremia, fecal virus excretion, and humoral immune responses. At the end of the post-infection observation period, samples were taken from the mediastinal and mesenteric lymph nodes of the animals and tested for viral load. The gradual depletion of maternally derived antibodies in the sera of piglets was demonstrated by ELISA and virus neutralization tests. Following experimental infection by PCV2d-2, specific IgM antibodies were first detected at 14 days post challenge (dpch), while IgG class antibodies were first detected at 21 dpch. Both viremia and virus shedding could be detected at 7 dpch, in 36 and 50% of the pigs, respectively. The proportion of shedders reached 100% by 14 dpch and remained at this level, while viremia was demonstrated in 86, 100, and 100% of the pigs at 14, 21, and 28 dpch, respectively. Both the mediastinal and mesenteric lymph nodes contained high levels of virus (7.6 and 8.5 log10 copies/mg tissue, respectively).

Investigation of Archaeal and Bacterial community structure of five different small drinking water networks with special regard to the nitrifying microorganisms.

Total microbial community structure, and particularly nitrifying communities inhabiting five different small drinking water networks characterized with different water physical and chemical parameters was investigated, using cultivation-based methods and sequence aided Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis. Ammonium ion, originated from well water, was only partially oxidized via nitrite to nitrate in the drinking water distribution systems. Nitrification occurred at low ammonium ion concentration (27-46µM), relatively high pH (7.6-8.2) and over a wide range of dissolved oxygen concentrations (0.4-9.0mgL(-1)). The nitrifying communities of the distribution systems were characterized by variable most probable numbers (2×10(2)-7.1×10(4) MPN L(-1)) and probably originated from the non-treated well water. The sequence aided T-RFLP method revealed that ammonia-oxidizing microorganisms and nitrite-oxidizing Bacteria (Nitrosomonas oligotropha, Nitrosopumilus maritimus, and Nitrospira moscoviensis, 'Candidatus Nitrospira defluvii') were present in different ratios in the total microbial communities of the distinct parts of the water network systems. The nitrate generated by nitrification was partly utilized by nitrate-reducing (and denitrifying) Bacteria, present in low MPN and characterized by sequence aided T-RFLP as Comamonas sp. and Pseudomonas spp. Different environmental factors, like pH, chemical oxygen demand, calculated total inorganic nitrogen content (moreover nitrite and nitrate concentration), temperature had important effect on the total bacterial and archaeal community distribution.

Recombinant Turkey Herpesvirus-AI Vaccine Virus Replication in Different Species of Waterfowl.

Waterfowl play a key role in the epidemiology of the H5N1 subtype of highly pathogenic avian influenza (HPAI) virus; therefore, efficient immunization of domesticated ducks and geese to maximize the impact of other control measures is of great importance. A recombinant (r)HVT-AI, expressing the HA gene of a clade 2.2 H5N1 HPAI strain had been developed and proved to be efficient against different clades of H5N1 HPAI virus in chickens after a single vaccination at 1 day old and could provide long-term immunity. We investigated whether rHVT-AI applied at 1 day old is able to replicate in different species and crossbreeds of ducks and in geese with the aim of collecting data on the possible application of rHVT-AI vaccine in different species of waterfowl for the control of H5N1 HPAI. We tested the possible differences among different waterfowl species, i.e., between geese (Anser anser, domesticated greylag goose), Muscovy ducks (Cairina moschata forma domestica), Pekin ducks (Anas platyrhynchos forma domestica), and mule ducks (Muscovy duck × Pekin duck), in their susceptibility to support the replication of rHVT-AI. Vaccine virus replication was followed by real-time PCR in spleen, bursa, and feather tip samples. Humoral immune response to vaccination was tested using the hemagglutination inhibition (HI) test and H5-specific commercial ELISA. Significant differences among the different waterfowl species regarding the rate of rHVT-AI replication was detected that were not reflected by the same difference in the immune response to vaccination. Replication of the rHVT-AI vaccine was very limited in Pekin ducks, somewhat better in mule ducks, and the vaccine virus was replicating significantly better in Muscovy ducks and geese, reaching 100% detectability at certain time points after administration at 1 day old. Results indicated that the vaccine virus could establish different levels of persistent infection in these species of waterfowl. No humoral immune response could be detected either by HI test or ELISA during the tested postvaccination period (5 wk).

Survey indicates circulation of 4/91 and QX-type infectious bronchitis viruses in Hungary in 2014 - Short communication.

Understanding the epidemiology and improving vaccinal protection against the highly variable chicken infectious bronchitis virus (IBV) requires the knowledge of circulating IBV serotypes/genotypes in defined geographic areas. Accordingly, the authors initiated a survey among the major poultry producers in Hungary in order to reveal the prevailing IBV serotypes in the country. Tracheal swabs and organ samples (caecal tonsils, kidneys, and trachea) were collected from broiler, layer, and meat-type breeder flocks, and were subjected to IBV detection by virus isolation and polymerase chain reaction (PCR). The IBV-positive samples were further characterised by nucleotide sequencing and phylogenetic analysis of a portion of the S1 IBV gene. Seventeen out of the 26 submitted samples proved to be positive for IBV. Sequence analyses revealed ten 4/91 and six QX serotypes, and a single D274 type IB virus. One sample contained a mixture of QX and Massachusetts serotype viruses. Presumably most of the 4/91 and D274 type viruses were vaccine strains. The proportion of QX type viruses and their observed variation are in good agreement with the situation in a few other European countries. The detected viruses clustered largely according to their geographic origin, with a few exceptions. If updated regularly, the preliminary 'virus map' will be useful for the adjustment of vaccination protocols.

Arenimonas subflava sp. nov., isolated from a drinking water network, and emended description of the genus Arenimonas.

A strain designated PYM3-14T was isolated from the drinking water network of Budapest (Hungary) and was studied by polyphasic taxonomic methods. The straight-rod-shaped cells stained Gram-negative, were aerobic and non-motile. Phylogenetic analysis of the 16S rRNA gene sequence of strain PYM3-14T revealed a clear affiliation with members of the family Xanthomonadaceae within the class Gammaproteobacteria. The 16S rRNA gene sequence of strain PYM3-14T showed the closest sequence similarities to Arenimonas daechungensis CH15-1T (96.2 %), Arenimonas oryziterrae YC6267T (95.2 %) and Lysobacter brunescens UASM DT (94.4 %). The DNA G+C content of strain PYM3-14T, measured by two different methods (52.0 mol% and 55.9 mol%, respectively), was much lower than that of any member of the genus Arenimonas. The predominant fatty acids (>8 %) were iso-C16:0, iso-C15:0, iso-C14:0, iso-C17:1ω9c and C16:1ω7c alcohol. Strain PYM3-14T contained Q-8 as the major ubiquinone and phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylmonomethylethanolamine as the major polar lipids. According to phenotypic and genotypic data strain PYM3-14T represents a novel species of the genus Arenimonas, for which the name Arenimonas subflava sp. nov. is proposed. The type strain is PYM3-14T ( = NCAIM B 02508T = DSM 25526T). On the basis of new data obtained in this study, an emended description of the genus Arenimonas is also proposed.

Bacterial communities in the collection and chlorinated distribution sections of a drinking water system in Budapest, Hungary.

Bacterial communities of a bank-filtered drinking water system were investigated by aerobic cultivation and clone library analysis. Moreover, bacterial communities were compared using sequence-aided terminal restriction fragment length polymorphism (T-RFLP) fingerprinting at ten characteristic points located at both the collecting and the distributing part of the water supply system. Chemical characteristics of the samples were similar, except for the presence of chlorine residuals in the distribution system and increased total iron concentration in two of the samples. Assimilable organic carbon (AOC) concentration increased within the collection system, it was reduced by chlorination and it increased again in the distribution system. Neither fecal indicators nor pathogens were detected by standard cultivation techniques. Chlorination reduced bacterial diversity and heterotrophic plate counts. Community structures were found to be significantly different before and after chlorination: the diverse communities in wells and the collection system were dominated by chemolithotrophic (e.g., Gallionella and Nitrospira) and oligocarbophilic-heterotrophic bacteria (e.g., Sphingomonas, Sphingopyxis, and Bradyrhizobium). After chlorination in the distribution system, the most characteristic bacterium was related to the facultative methylotrophic Methylocella spp. Communities changed within the distribution system too, Mycobacterium spp. or Sphingopyxis spp. became predominant in certain samples.

Bacterial communities in the gut of the freshwater copepod Eudiaptomus gracilis.

Eudiaptomus gracilis is the most abundant member of the zooplankton, plays a key role in the food web of Lake Balaton (Hungary). In the present study the composition of bacterial communities of this copepod was investigated based on cultivation and molecular cloning. The cultivated bacterial strains from the gut homogenate samples of Eudiaptomus gracilis belonged to four different clades: Firmicutes, Actinobacteria, Bacteriodetes and Proteobacteria. Clone library showed high species diversity, Firmicutes, Actinobacteria, Proteobacteria, representatives of Deinococcus-Thermus lineage and Cyanobacteria were detected. The isolated strains were very effective in degradation of different biopolymers. Many of the detected bacteria are known as opportunistic human or fish pathogens (Pseudomonas spp., Aeromonas spp., Chryseobacterium sp. and Staphylococcus sp.).

Tahibacter aquaticus gen. nov., sp. nov., a new gammaproteobacterium isolated from the drinking water supply system of Budapest (Hungary).

Three Gram-stain negative, aerobic, non-motile, non-spore-forming, rod-shaped bacterial strains, PYM5-11(T), RaM5-2 and PYM5-8, were isolated from the drinking water supply system of Budapest (Hungary) and their taxonomic positions were investigated by a polyphasic approach. All three strains grew optimally at 20-28°C and pH 5-7 without NaCl. The G+C content of the DNA of the type strain was 65.4mol%. On the basis of 16S rRNA gene sequence analysis, the isolates showed 94.5-94.9% sequence similarity to the type strain of Dokdonella koreensis and a similarity of 93.0-94.1% to the species of the genera Aquimonas and Arenimonas. The major isoprenoid quinone of the strains was ubiquinone Q-8. The predominant fatty acids were iso-C(15:0), iso-C(17:1)ω9c, C(16:1)ω7c, and C(16:0). Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine, as well as several unidentified aminolipids and phospholipids were present. The 16S rRNA gene sequence analysis, the predominant fatty acids, the polar lipid composition, RiboPrint patterns, physiological and biochemical characteristics showed that the three strains were related but distinct from the type strains of the four recognized species of the genus Dokdonella, and indicated that the strains represented a new genus within the Gammaproteobacteria. The strain PYM5-11 (=DSM 21667(T)=NCAIM B 02337(T)) is proposed as the type strain of a new genus and species, designated as Tahibacter aquaticus gen. nov., sp. nov.

Nocardioides hungaricus sp. nov., isolated from a drinking water supply system.

Three Gram-positive, rod-shaped bacterial strains were isolated from the drinking water supply system of the Hungarian capital, Budapest. Phylogenetic analysis on the basis of 16S rRNA gene sequence comparison revealed that the isolates represented a distinct cluster within the clade of the genus Nocardioides and were most closely related to Nocardioides pyridinolyticus OS4(T), Nocardioides aquiterrae GW-9(T), Nocardioides sediminis MSL-01(T) and N. hankookensis DS-30(T). The peptidoglycan based on LL-2,6-diaminopimelic acid, the major menaquinone MK-8(H4), the cellular fatty acid profile with iso-C16:0 and anteiso-C17:0 as predominating components and the DNA G+C content of 71.4 mol% (strain 1RaM5-12(T)) were consistent with the affiliation of the isolates to the genus Nocardioides. Because of differences in physiological characteristics, matrix-assisted laser-desorption/ionization time-of-flight mass spectra of protein extracts, PvuII RiboPrinter patterns and 96.1 % 16S rRNA gene sequence similarity between strain 1RaM5-12(T) and its closest phylogenetic neighbour, N. pyridinolyticus OS4(T), a novel species, Nocardioides hungaricus sp. nov., is proposed. The type strain is 1RaM5-12(T) (=DSM 21673(T) =NCAIM 02330(T)).

Nocardioides daphniae sp. nov., isolated from Daphnia cucullata (Crustacea: Cladocera).

A Gram-positive, rod-shaped or coccoid, yellow-pigmented bacterial strain, D287(T), was isolated from the water flea Daphnia cucullata (Crustacea: Cladocera) collected from Lake Balaton in Hungary. Phylogenetic analysis on the basis of 16S rRNA gene sequence comparisons revealed that the strain represented a distinct lineage within the cluster of the genera Nocardioides and Marmoricola. The following characteristics were consistent with the affiliation of strain D287(T) to the genus Nocardioides: peptidoglycan based on LL-2,6-diaminopimelic acid, MK-8(H(4)) as the major menaquinone, iso-C(16:0) as the predominant cellular fatty acid, the presence of phosphatidylglycerol and diphosphatidylglycerol and a DNA G+C content of 69.9 mol%. Owing to characteristic differences in physiological traits and levels of 16S rRNA gene sequence similarity to its phylogenetically closest neighbours that were below 97%, strain D287(T) is considered to represent a novel species of the genus Nocardioides, for which the name Nocardioides daphniae sp. nov. is proposed. The type strain is D287(T) (=DSM 18664(T)=CCM 7403(T)).