Evidence of <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> and <Emphasis Type="Italic">Rickettsia helvetica</Emphasis> infection in free-ranging ungulates in central Slovakia

The purpose of this study was to investigate the role of wild animals for Anaplasma phagocytophilum, other ehrlichiae/anaplasmae, Rickettsia helvetica and other rickettsiae and whether different genetic variants of A. phagocytophilum in central Slovakia exist. A total of 109 spleen samples from 49 red deer (Cervus elaphus), 30 roe deer (Capreolus capreolus), 28 wild boar (Sus scrofa) and two mouflon (Ovis musimon) were collected from June 2005 to December 2006. Polymerase chain reaction (PCR) amplification of the16S rRNA gene was used for detection of ehrlichiae/anaplasmae. A nested PCR targeting part (392 bp) of groESL gene was applied for the specific detection of A. phagocytophilum. Fragments of the gltA and ompA genes (381 bp and 632 bp, respectively) were amplified to detect rickettsiae, followed by sequencing. A. phagocytophilum and R. helvetica were detected in wild animals. The prevalence of A. phagocytophilum was 50.0 ± 18.2% in roe deer and 53.1 ± 14.1% in red deer. None of the 28 wild boar was PCR positive for ehrlichiae/anaplasmae. A. phagocytophilum was detected in one mouflon. R. helvetica was found in one roe deer. Our study suggests a role of cervids as a natural reservoir of A. phagocytophilum in Slovakia. However, the role of cervids and wild boars in the circulation of R. helvetica remains unknown. The analysis of sequence variation in the msp4 coding region of A. phagocytophilum showed the presence of different variants previously described in ruminants.     

Pigmentation in Black-boned sheep (Ovis aries): association with polymorphism of the MC1R gene

Variations in vertebrate skin and hair color are due to varied amounts of eumelanin (brown/black) and phaeomelanin (red/yellow) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eumelanin and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many vertebrates. We have sequenced the entire coding region of the MC1R gene in Black-boned, Nanping indigenous and Romney Marsh sheep populations and found two silent mutation sites of A12G and G144C, respectively. PCR-RFLP of G144C showed that frequency of allele G in Black-boned, Nanping indigenous and Romney Marsh sheep was 0.818, 0.894 and 0, respectively. Sheep with GG genotype had significantly higher (P < 0.05) tyrosinase activity than sheep with CC genotype in the all investigated samples. Moreover, there was significant effect of MC1R genotype on coat color, suggesting that MC1R gene could affect coat color but not black traits. There would be merit in further studies using molecular techniques to elucidate the cause of black traits in these Black-boned sheep.     

Microbiological survey for selected bacterial pathogens in European storm petrel (<Emphasis Type="Italic">Hydrobates pelagicus</Emphasis>, Linnaeus 1758) from Grosa Island (Murcia,Southeastern Spain)

The current work shows the first step in the knowledge on the health status of European storm petrel (Hydrobates pelagicus) colony inhabiting Grosa Island (Murcia, SE Spain). We performed a screening about the bacterial pathogens carried by them (among the infectious agents checked, bacteria of the orders Mollicutes and Chlamydiales, and the genera Salmonella are of main interest) and compare these results with similar works performed in Larus species because most of the breeding colonies of storm petrel share habitats with gull colonies, and these could become pathogen reservoirs to petrels. Our results show the European storm petrels sampled have absence of pathogens of main interest and low levels of opportunistic pathogens. No Mycoplasma species were isolated, and no Chlamydophila psittaci were demonstrated by lipopolysaccharide antigen immunodetection. The commensal bacteria were isolated in higher frequencies than the previous [Staphylococcus epidermidis (5/15), Staphylococcus hominis (2/15) and Staphylococcus aureus (1/15)]. The rate of isolation of Gram-negative was lower than in the previous Gram-positive bacteria [Pasteurella sp. (1/15) and Pseudomonas aeruginosa (1/15)], and no Enterobacteriaceae were isolated. The absence of pathogen carriers on European storm petrel is the main conclusion of this survey; it is an evidence that the bacterial infectious pathogens described in gulls may not be an important selective force on their survival.     

The sequence and phylogenesis of the α-globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon) and Cyprus mouflon (Ovis aries ophion)

In order to investigate the polymorphism of α-globin chain of hemoglobin amongst caprines, the linked ^Iα and ^IIα globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon), and Cyprus mouflon (Ovis aries ophion) were completely sequenced, including the 5′ and 3′ untranslated regions. European and Cyprus mouflons, which do not show polymorphic α globin chains, had almost identical α globin genes, whereas Barbary sheep exhibit two different chains encoded by two nonallelic genes. Four different α genes were observed and sequenced in goat, validating previous observations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromosomal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequences at the level of molecular evolution of the ^Iα-globin gene family in the caprine taxa suggests a closer relationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon, goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively.     

Toxin profiles of natural populations and cultures ofAlexandrium minutum Halim from Galician (Spain) coastal waters

The toxin profiles of three isolates and natural populations of the PSP agentAlexandrium minutum from several Galician rías (NW Spain) was obtained by HPLC. The toxin content of cultures ofA. minutum is dominated by GTX4 (80–90%) and GTX4 (10–15%) with small amounts of GTX3 and GTX2 (less than 3% of each); similar results were obtained for natural populations ofAlexandrium from three different Galician rías, where a mixture ofA. lusitanicum Balech andA. minutum can occur. Important quantitative differences were found between the three isolates, one being highly and two weakly toxic. The results obtained from these isolates and natural populations ofAlexandrium were very similar to those obtained from HPLC analyses of mussels intoxicated during a PSP outbreak in Ría de Ares (Rías Altas) in 1984, confirming thatA. minutum (previously identified asGonyaulax tamarensis Lebour andAlexandrium lusitanicum) was the PSP agent during the toxic outbreak in May 1984. Toxin profiles obtained from natural populations during different PSP outbreaks in different rías and from cultures are fairly consistent and suggest that at least from the toxin point of view,A. lusitanicum andA. minutum are identical, and that the toxin profile ofA. minutum from Galicia can be used as a biochemical marker.     

The key role of lamb presence in affecting flight response in Sardinian mouflon (Ovis orientalis musimon)

Observations of the flight response in mammals are commonly used to test the behavioural response to predation risk. Such a response is likely to be stronger in more sensitive individuals such as females, and females with offspring in particular, as well as during such periods as the birth season. From August 2005 to July 2006 we recorded the flight distances of different mouflon group types in a natural population in Sardinia. The comparison of the flight distances of the groups (n groups=375, n mouflons=1303) provided evidence for the key role of lamb presence. Female groups with lambs fled at greater distances than male groups and female groups without lambs. This difference was linked to the females' priority to secure the survival of their offspring. Indeed, the uniformity of the flight distances of males and non-reproductive females was in contrast to the general expectations regarding ungulate species. In fact, smaller females were expected to be more sensitive to predation risk than larger males. The absence of major differences is probably due to the low sexual dimorphism of mouflon. Flight distances were shorter during the rut than during the lambing season, arguably because during the mating less time and energies were available for anti-predator behaviour. However, during the rut females with lambs still fled at greater distances than females without lambs, arguably because mothers took it as a priority to protect their parental investment rather then to find a potential mate.     

Prevalence of tick-borne pathogens in ixodid ticks (Acari: Ixodidae) collected from European wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>) and Iberian red deer (<Emphasis Type="Italic">Cervus elaphus hispanicus</Emphasis>) in central Spain

Emerging tick-borne diseases of humans and animals have occurred frequently during the past 30 years. These disease outbreaks appear to result from changes in the distribution of tick and vertebrate hosts, and the introduction of humans and domestic animals into tick–pathogen–wildlife cycles. Use of molecular technologies now available for identification of pathogens in ticks can provide valuable information that allows for risk analysis of emerging tick-borne diseases. In this study, the prevalence of selected pathogens in ticks collected in six locations in central Spain from the major wild ungulate species, European wild boar (Sus scrofa) and Iberian red deer (Cervus elaphus hispanicus), was determined by PCR. Tick species collected included Ixodes ricinus, Dermacentor marginatus, Rhipicephalus bursa and Hyalomma m. marginatum. Pathogens identified in ticks included piroplasmids, Anaplasma spp., Ehrlichia spp. and Rickettsia spp. Piroplasmids were identified in all tick species except I. ricinus. Ehrlichia spp. were detected in all tick species and collection locations, while Rickettsia spp., which proved to be R. slovaca and a recently identified Rickettsia sp. DnS28, were identified only in D. marginatus. A. marginale and A. phagocytophilum were detected in D. marginatus, R. bursa and Hy. m. marginatum. Concurrent infections of these pathogens were frequently observed in ticks. Notably, A. phagocytophilum, which is infective for a broad host range that includes humans and domestic and wild animals, was identified in ticks from all collection locations. The variety of ticks and tick-borne pathogens demonstrated in this study suggests a risk in central Spain for the emergence of tick-borne diseases in humans and domestic animals.     

Etiology of infectious diseases in cultivated Chondrus crispus (Gigartinales,Rhodophyta)

The appearance of cavities and holes in fronds of commercially cultivated Chondrus crispus is described. These ultimately arise from the ravages of a green spot or green rot disease system in which several biotic agents can participate. Nematodes capable of bacterial grazing were recovered from necrotic lesions and we suggest that the nematodes can facilitate wound healing in diseased tissues. Bacteria isolated from disease lesions and from the surfaces of healthy fronds were screened for pathogenic strains. A particularly virulent one, the DOR isolate, was purified from small dark orange colored colonies grown on dilution plates. It was present in necrotic tissue and also recovered from surface scrapings of old healthy fronds, but not from their apical regions. Growth of the DOR isolate appeared to be inhibited by other bacteria colonizing the algal surface. It was shown to be a facultative pathogen, the virulence of which depended on the availability of ammonium or constituents easily metabolized to ammonium. It induced green rot disease in healthy C. crispus and was recoverable in pathogenic form from experimentally infected frond apices. A wound, disease and recovery cycle is discussed to illustrate potential interrelationships involving animal grazers, algal endophytes, Petersenia pollagaster, bacteria and nematodes.     

Treatment of foot rot in free-ranging mouflon (Ovis gmelini musimon) populations—does it make sense?

Prevalence and incidence of foot rot disease in free-ranging and captive bovine wild ruminant populations are increasing worldwide. Even species in which the disease has not been described in the past are presently affected by the co-working pathogens Dichelobacter nodosus and Fusobacterium necrophorum. This paper discusses disease control measures and the expense for a successful treatment of affected populations of mouflon. The rationale and perspectives of treating foot rot disease in wild mouflons are discussed.     

Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes

The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes. This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance. Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid ofAgrobacterium tumefaciens. In leaf discs from susceptible cultivars (Lycopersicon esculentum) TYLCV single-stranded genomic DNA and its double-stranded DNA forms appeared within 2–5 days after inoculation. Whiteflies (Bemisia tabaci) efficiently transmitted the TYLCV disease to tomato test plants following acquisition feeding on agroinoculated tomato leaf discs. This indicates that infective viral particles have been produced and have reached the phloem cells of the explant where they can be acquired by the insects. Plants regenerated from agroinfected leaf discs of sensitive tomato cultivars exhibited disease symptoms and contained TYLCV DNA concentrations similar to those present in field-infected tomato plants, indicating that TYLCV can move out from the leaf disc into the regenerating plant. Leaf discs from accessions of the wild tomato species immune to whitefly-mediated inoculation,L. chilense LA1969 andL. hirsutum LA1777, did not support TYLCV DNA replication. Leaf discs from plants tolerant to TYLCV issued from breeding programmes behaved like leaf discs from susceptible cultivars.The Hebrew University of Jerusalem, Faculty of Agriculture, Department of Field and Vegetable Crops     

Prevalence of Anaplasma and Bartonella spp. in Ticks Collected from Korean Water Deer (Hydropotes inermis argyropus)

Deer serve as reservoirs of tick-borne pathogens that impact on medical and veterinary health worldwide. In the Republic of Korea, the population of Korean water deer (KWD, Hydropotes inermis argyropus) has greatly increased from 1982 to 2011, in part, as a result of reforestation programs established following the Korean War when much of the land was barren of trees. Eighty seven Haemaphysalis flava, 228 Haemaphysalis longicornis, 8 Ixodes nipponensis, and 40 Ixodes persulcatus (21 larvae, 114 nymphs, and 228 adults) were collected from 27 out of 70 KWD. A total of 89/363 ticks (266 pools, 24.5% minimum infection rate) and 5 (1.4%) fed ticks were positive for Anaplasma phagocytophilum using nested PCR targeting the 16S rRNA and groEL genes, respectively. The 16S rRNA gene fragment sequences of 88/89 (98.9%) of positive samples for A. phagocytophilum corresponded to previously described gene sequences from KWD spleen tissues. The 16S rRNA gene fragment sequences of 20/363 (5.5%) of the ticks were positive for A. bovis and were identical to previously reported sequences. Using the ITS specific nested PCR, 11/363 (3.0%) of the ticks were positive for Bartonella spp. This is the first report of Anaplasma and Bartonella spp. detected in ticks collected from KWD, suggesting that ticks are vectors of Anaplasma and Bartonella spp. between reservoir hosts in natural surroundings.     

Natural occurrence of aflatoxin andAlternaria mycotoxins in oilseed rape from Catalonia (Spain): Incidence of toxigenic strains

During an investigation of the mycoflora on oilseed rape, the predominant fungal species present in 20 samples collected from Catalonia (Spain) wereAlternaria alternata (Fries) Keissler,Penicillium spp. andAspergillus flavus. None of the 20 samples analyzed presented contamination byAlternaria mycotoxins (tenuazonic acid, alternariol, alternariol methyl ether, altertoxin I and altertoxin II). Only aflatoxin B₁ was detected in 1 of the 20 samples analyzed, with a concentration of 0.25 ppb. Of the 40Aspergillus flavus strains isolated from oilseed rape samples, only 3 revealed aflatoxigenic capacity. None of thePenicillium spp. isolated from oilseed rape samples revealed mycotoxigenic capacity (citreoviridin, griseofulvin, citrinin, patulin and penicillic acid).     

Reproductive potential of domestic Ovis aries for preservation of threatened Ovis orientalis isphahanica: in vitro and in vivo studies

Although the potential use of reproductive biotechnology for safeguarding of endangered wildlife species is undoubted, initial evaluation of the genetic and reproductive relationship between the endangered mammals and those closely related species is indispensable. Isfahan mouflon Ovis orientalis isphahanica is now considered as a threatened species by International Union for the Conservation of Nature. Therefore, little is known about the biology of this species. This study was carried out to investigate the possible reproductive potential of domestic sheep for ex situ conservation of the Isfahan mouflon. Somatic cell cultures were taken from ear biopsies of the wild and domestic sheep and were used for karyotype analysis. Semen samples were collected by electroejaculator from the wild and domestic rams. The spermatological characteristics of the collected semen samples were determined and used for both cryopreservation and cross-insemination of the synchronized wild and domestic female sheep. To establish a cryobank for the threatened species biomaterials, freezed samples of the somatic cells and semen were transferred to a cryotank. The result suggested that Isfahan mouflon has conserved its chromosomal integrity as previously observed and contains the same chromosomal number as the domestic sheep (2n = 54). The semen samples of both species revealed similar cryoviability (>35% gross motility postthawing). Cross-insemination of both species resulted in successful pregnancy. It was suggested that domestic sheep possesses the required biological characteristics to be considered for safeguarding of the Isfahan mouflon.     

Characterisation of Lysobacter enzymogenes (Christensen and Cook 1978) strain 3.1T8, a powerful antagonist of fungal diseases of cucumber

Isolate 3.1T8 of Lysobacter enzymogenes (Christensen and Cook 1978), originating from the rhizosphere of cucumber and shown to have the potential to control Pythium aphanidermatum, is described. The strain produces extracellular proteases and lipases and shows high levels of resistance against streptomycin, kanamycin and tetracycline, but not to chloramphenicol. It shows strong in vitro antibiosis against P. aphanidermatum and several other phytopathogenic fungi. In order to identify the isolate, a carbon substrate oxidation profile (Biolog) was generated, and fatty acid methyl ester (FAME) analysis was performed. Also, the 16S rRNA gene was cloned and sequenced. With Biolog and FAME analysis, no assignment to species level was possible, because the species was not in the respective databases. BLAST analysis of the obtained sequence, followed by phylogenetic analysis, using a number of related and unrelated sequences, showed that the isolate was most closely related to Lysobacter enzymogenes (Christensen and Cook 1978).     

Rickettsial Agents from Parasitic Dermanyssoidea (Acari: Mesostigmata)

Mites are often overlooked as vectors of pathogens, but have been shown to harbor and transmit rickettsial agents such as Rickettsia akari and Orientia tsutsugamushi. We screened DNA extracts from 27 mites representing 25 species of dermanyssoids for rickettsial agents such as Anaplasma, Bartonella, Rickettsia, and Wolbachia by PCR amplification and sequencing. DNA from Anaplasma spp., a novel Bartonella sp., Spiroplasma sp., Wolbachia sp., and an unclassified Rickettsiales were detected in mites. These could represent mite-borne bacterial agents, bacterial DNA from blood meals, or novel endosymbionts of mites.     

Herpetomonas spp. isolated from tomato fruits (Lycopersicon esculentum) in southern Spain

A flagellate of the family Trypanosomatidae was isolated from fruits of Lycopersicon esculentum (tomato) in southeastern Spain. The isolate was successfully adapted to in vitro culture in monophasic media. The morphology showed the kinetoplast to be positioned towards the middle of the body, and the typical opistomastigote form characteristic of members of the genus Herpetomonas. Amplification of the mini-exon gene was negative, whilst for the 5S ribosomal rRNA gene the result was positive. The DNA sequence was obtained and its alignment with other trypasomatids, obtained using the BLAST algorithm, suggested it was closely related to Herpetomonas samuelpessoai.     

Sequence analyses of the ITS regions and the matK gene for determining phylogenetic relationships of Diospyros kaki (persimmon) with other wild Diospyros (Ebenaceae) species

To elucidate the relationships among Diospyros kaki and species closely related in previous studies, the nuclear ribosomal internal transcribed spacer (ITS) DNA sequence and the chloroplast matK gene were sequenced and compared with those of nine Diospyros species from Thailand, four species from temperate regions, and one species of southern Africa, D. lycioides. Maximum parsimony, maximum likelihood, and neighbor joining analyses of the matK and ITS data sets revealed that D. kaki is closely related to two diploid species, D. oleifera and D. glandulosa. D. kaki, D. glandulosa, and D. oleifera were placed differently in the trees obtained from ITS and matK data sets, suggesting that hybridization and/or introgression may have occurred during the development of these species. D. kaki was not found to be closely related to D. ehretioides, a diploid species from Thailand. These results differed from a prior analysis of this genus performed with chloroplast DNA (cpDNA) restriction site mutations in 3.2- and 2.1-kb amplified sequences. The results supported Ng’s hypothesis that D. glandulosa and D. kaki may share a common ancestor. D. oleifera was also closely associated with D. kaki.     

Recent advances in the biocontrol of Orobanche (broomrape) species

Parasitic broomrapes (Orobanche spp.) are majoruncontrolled weeds in the Mediterranean regions of Europe and the NearEast causing major losses to vegetable, grain legume, and sunflowercrops. Selective herbicides alone cannot provide persistent, season-longcontrol of these parasites, and much methyl bromide is used for theircontrol, where affordable. Thus they are excellent targets forbiocontrol. The recent progress by the COST 816 Orobancheworking group in this area is reviewed herein. Natural infestation bythe fly Phytomyza orobanchia of seed capsules of Orobanchecrenata parasitising faba bean halved Orobanche seedproduction while inundative releases of adults reduced it to 5%of viable seeds. The fungi Fusarium arthrosporioides E4a andF. oxysporum E1d, as well as strains of bacteria were isolatedfrom diseased, juvenile, Orobanche flower stalks. They arepathogenic to O. aegyptiaca, O. crenata and O. ramosaon most vegetable crops. A F. oxysporum f. sp.orthoceras was specifically pathogenic to O. cumana onsunflowers. All were used in various experiments with a modicum ofsuccess. Methods were developed to formulate isolated mycelia, whichcould eventually allow the use of transgenic hypervirulent pathogens inasporogenic (deletion) mutants (as a failsafe against spread).Mycotoxins were also isolated from different Fusarium and otherfungal species that kill Orobanche, and are being consideredfor direct use, or to augment other strategies. All threeFusarium spp. used have been transformed with gusand/or gfp genes allowing tracing their movement in theenvironment, and opening the way to future transformations tohypervirulence.     

Trichophyton violaceum is the dominant cause of tinea capitis in children in Tripoli,Libya: Results of a two year survey

The causative agents of tinea capitis in Libyan nationals attending the out patient Dermatology Clinic of the Tripoli Medical Centre over the period December 1997 to December 1999 were investigated. Samples (hair and scalp scrapings) were taken from 940 patients who presented with suspected tinea capitis. The etiological agents were identified in 584 cases. Trichophyton violaceum was found to be the most prevalent organism isolated being responsible for 64.4% (376/584) of culture positive cases, followed by Microsporum canis at 24.7% (144/584) and T. mentagrophytes at 5.5% (32/584). The majority of infections (380/584) occurred in females and in children with ages less than 12 years (554/584). This revised version was published online in June 2006 with corrections to the Cover Date.     

Prevalence of Fusarium species of the Liseola section on selected Colombian animal feedstuffs and their ability to produce fumonisins

A total of 57 samples of feedstuffs commonly used for animal nutrition in Colombia (corn, soybean, sorghum, cottonseed meal, sunflower seed meal, wheat middlings and rice) were analyzed for Fusarium contamination. Fusarium fungi were identified at species level by means of conventional methods and the ability to produce fumonisins of the most prevailing species was determined. A total of 41 of the feedstuffs analyzed (71.9%) were found to contain Fusarium spp. Most contaminated substrates were corn (100%), cottonseed meal (100%), sorghum (80%), and soybean (80%). Wheat middlings and rice showed lower levels of contamination (40% and 20%, respectively), while no Fusarium spp. could be isolated from sunflower seed meal. The most prevalent species of Fusarium isolated were F. verticilliodes (70.8%), F.␣proliferatum (25.0%), and F. subglutinans (4.2%). All of them correspond to section Liseola.Production of fumonisins on corn by the isolated Fusarium was screened through liquid chromatography. Almost all strains of F. verticilliodes (97.1%) produced FB1 (5.6–25,846.4 mg/kg) and FB2 (3.4–7507.5 mg/kg). Similarly, almost all strains of F.␣proliferatum (91.7%) produced fumonisins but at lower levels than F.␣verticilliodes (FB1 from 6.9 to 3885.0 mg/kg, and FB2 from 34.3 to 373.8 mg/kg), while F. subglutinans did not produce these toxins. This is the first study in Colombia describing toxigenic Fusarium isolates from␣animal feedstuffs.