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Melissococcus plutonius is the causative agent of European foulbrood (EFB), an important bacterial disease of honey bee larvae, and various methods have been developed for the detection of this dis...

Varroa destructor is the major cause of honey bee (Apis mellifera) colony losses. Mite control is limited to several miticides. The overuse of tau-fluvalinate has resulted in resistance via a knockdown resistance (kdr) mutation in the sodium channel gene NaVChs (L925V/I/M). In this study, we used the discriminating concentration of tau-fluvalinate (0.25 µg/mL) to detect the resistance of mites in a bioassay. Further, we verified the presence of the kdr mutation in mites from the bioassay via PCR amplification of a fragment of the voltage-gated sodium channel gene (NaVCh), restriction fragment length polymorphisms (RFLPs), and densitometry analyses in pools of surviving or dead mites. Resistance values corresponding to the densitometry of the resistant allele were related to mite survival. In the vial test, the survival of the control group was significantly higher (70.4%) than that of the tau-fluvalinate-treated group (34.3%). Mite survival in the vial test was significantly correlated with the mean proportion of resistance values. Individuals that died after tau-fluvalinate application exhibited an average resistance value of 0.0783, whereas individuals that survived exhibited an average resistance of 0.400. The concentration of tau-fluvalinate in the vials was checked using high performance liquid chromatography under different temperatures and exposure times, and indicates that the stability of tau-fluvalinate stored in the refrigerator (4 ± 1 °C) is at least 14 days. PCR–RFLP of the NaVCh gene fragment verified that the vial test is a suitable, rapid, and cost-effective method for the identification of tau-fluvalinate resistance based on kdr mutation in V. destructor in apiaries.

Honeybee (Apis mellifera L.) workers act as passive vectors of Paenibacillus larvae spores, which cause the quarantine disease American foulbrood (AFB). We assessed the relative proportions of P. larvae within the honeybee microbiome using metabarcoding analysis of the 16 S rRNA gene. The microbiome was analyzed in workers outside of the AFB zone (control - AFB0), in workers from asymptomatic colonies in an AFB apiary (AFB1), and in workers from colonies exhibiting clinical AFB symptoms (AFB2). The microbiome was processed for the entire community and for a cut-off microbiome comprising pathogenic/environmental bacteria following the removal of core bacterial sequences; varroosis levels were considered in the statistical analysis. No correlation was observed between AFB status and varroosis level, but AFB influenced the worker bee bacterial community, primarily the pathogenic/environmental bacteria. There was no significant difference in the relative abundance of P. larvae between the AFB1 and AFB0 colonies, but we did observe a 9-fold increase in P. larvae abundance in AFB2 relative to the abundance in AFB1. The relative sequence numbers of Citrobacter freundii and Hafnia alvei were higher in AFB2 and AFB1 than in AFB0, whereas Enterococcus faecalis, Klebsiella oxytoca, Spiroplasma melliferum and Morganella morganii were more abundant in AFB0 and AFB1 than in AFB2.

Honey bees are major pollinators of crops with high economic value. Thus, bees are considered to be the most important nontarget organisms exposed to adverse effects of plant protection product use. The side effects of pesticides are one of the major factors often linked to colony losses. Fewer studies have researched acute poisoning incidents in comparison to the study of the sublethal effects of pesticides. Here, we compared pesticides in dead/dying bees from suspected poisoning incidents and the suspected crop source according to government protocols. Additionally, we analyzed live bees and bee bread collected from the brood comb to determine recent in-hive contamination. We used sites with no reports of poisoning for reference. Our analysis confirmed that not all of the suspected poisonings correlated with the suspected crop. The most important pesticides related to the poisoning incidents were highly toxic chlorpyrifos, deltamethrin, cypermethrin and imidacloprid and slightly toxic prochloraz and thiacloprid. Importantly, poisoning was associated with pesticide cocktail application. Almost all poisoning incidents were investigated in relation to rapeseed. Some sites were found to be heavily contaminated with several pesticides, including a reference site. However, other sites were moderately contaminated despite agricultural use, including rapeseed cultivation sites, which can influence the extent of pesticide use, including tank mixes and other factors. We suggest that the analysis of pesticides in bee bread and in bees from the brood comb is a useful addition to dead bee and suspected crop analysis in poisoning incidents to inform the extent of recent in-hive contamination.

BACKGROUND Sodium channels (SCs) in mites and insects are target sites for pesticides, including pyrethroids. Point mutations in the SC gene have been reported to change the structural conformation of the protein and its sensitivity to pesticides. To find mutations in the SC gene of the mite Varroa destructor (VmNa), the authors analysed the VmNa gene sequences available in GenBank and prepared specific primers for the amplification of two fragments containing the regions coding for (i) the domain II S4–S6 region (bp 2805–3337) and (ii) the domain III S4-3′ terminus region (bp 4737–6500), as determined according to the VmNa cDNA sequence AY259834. RESULTS Sensitive and resistant mite populations did not differ in the amino acid sequences of the III S4-3′ terminus VmNa region. However, differences were found in the IIS4–IIS6 fragment. In the resistant population, the mutation C(3004) G resulted in the substitution L(1002) V (codon ctg gtg) at the position equivalent to that of the housefly L925 in the domain II S5 helix. Additionally, the mutation F(1052) L (codon ttc ctc) at the position equivalent to that of the housefly F975 in the domain II P-loop connecting segments S5 and S6 was detected in both the resistant and sensitive populations. CONCLUSION All individuals that survived the tau-fluvalinate treatment in the bioassay harboured the L(1002) V mutation combined with the F(1052), while dead individuals from both the sensitive and resistant populations harboured mostly the L(1002) residue and either of the two residues at position 1052. © 2013 Society of Chemical Industry

Worker honeybees (Apis mellifera) transmit Melissococcus plutonius between colonies. However, the transmission of M. plutonius, which causes European foulbrood (EFB), is poorly understood. To analyze the first EFB outbreak in 40 years in Czechia, we collected 49 hive worker samples from 18 beehives in two diseased apiaries for bacteriome analysis of the V1-V3 portion of the 16S rRNA gene. When we compared control samples obtained outside of the EFB zone, bees from an EFB apiaries containing colonies without clinical symptoms and bees from colonies with EFB clinical symptoms, there was a 100-fold higher occurrence of M. plutonius in colonies with EFB symptoms. The presence of M. plutonius in controls indicated that this pathogen exists in an enzootic state. EFB influenced the core bacteria in the worker bacteriome because the number of Snodgrassella alvi, Lactobacillus mellis, Lactobacillus melliventris, and Fructobacillus fructosus sequences increased, while Bartonella apis, Frischella perrara, and Commensalibacter intestine sequences decreased. Together, the results of this study suggest worker bees from EFB-diseased apiaries serve as vectors of M. plutonius, and eliminating such colonies is an appropriate method to overcome disease outbreaks. Because M. plutonius exists in honeybee colonies in an enzootic state, there may be similar abundances in control colonies outside the EFB zone to those in asymptomatic colonies from EFB apiaries. High-throughput Illumina next-generation sequencing permitted the quantitative interpretation of M. plutonius within the honeybee worker bacteriome. Future studies focusing on honeybee diseases, colony losses, detection of bacterial pathogens and interactions of bacteriome with pathogenic bacteria will benefit of this study.

Background Melissococcus plutonius is an entomopathogenic bacterium that causes European foulbrood (EFB), a honeybee (Apis mellifera L.) disease that necessitates quarantine in some countries. In Czechia, positive evidence of EFB was absent for almost 40 years, until an outbreak in the Krkonose Mountains National Park in 2015. This occurrence of EFB gave us the opportunity to study the epizootiology of EFB by focusing on the microbiome of honeybee workers, which act as vectors of honeybee diseases within and between colonies. Methods The study included worker bees collected from brood combs of colonies (i) with no signs of EFB (EFB0), (ii) without clinical symptoms but located at an apiary showing clinical signs of EFB (EFB1), and (iii) with clinical symptoms of EFB (EFB2). In total, 49 samples from 27 honeybee colonies were included in the dataset evaluated in this study. Each biological sample consisted of 10 surface-sterilized worker bees processed for DNA extraction. All subjects were analyzed using conventional PCR and by metabarcoding analysis based on the 16S rRNA gene V1–V3 region, as performed through Illumina MiSeq amplicon sequencing. Results The bees from EFB2 colonies with clinical symptoms exhibited a 75-fold-higher incidence of M. plutonius than those from EFB1 asymptomatic colonies. Melissococcus plutonius was identified in all EFB1 colonies as well as in some of the control colonies. The proportions of Fructobacillus fructosus, Lactobacillus kunkeei, Gilliamella apicola, Frischella perrara, and Bifidobacterium coryneforme were higher in EFB2 than in EFB1, whereas Lactobacillus mellis was significantly higher in EFB2 than in EFB0. Snodgrassella alvi and L. melliventris, L. helsingborgensis and, L. kullabergensis exhibited higher proportion in EFB1 than in EFB2 and EFB0. The occurrence of Bartonella apis and Commensalibacter intestini were higher in EFB0 than in EFB2 and EFB1. Enterococcus faecalis incidence was highest in EFB2. Conclusions High-throughput Illumina sequencing permitted a semi-quantitative analysis of the presence of M. plutonius within the honeybee worker microbiome. The results of this study indicate that worker bees from EFB-diseased colonies are capable of transmitting M. plutonius due to the greatly increased incidence of the pathogen. The presence of M. plutonius sequences in control colonies supports the hypothesis that this pathogen exists in an enzootic state. The bacterial groups synergic to both the colonies with clinical signs of EFB and the EFB-asymptomatic colonies could be candidates for probiotics. This study confirms that E. faecalis is a secondary invader to M. plutonius; however, other putative secondary invaders were not identified in this study.

International audience; AbstractThe honeybee, Apis mellifera, undergoes complete metamorphosis before transitioning to the adult stage. The newly emerged individual and the red-eye pupa stage are well defined and easily recognizable in the time life cycle honeybee and, therefore, very useful for studying physiological and developmental factors. We analyzed in detail the hemolymph proteome of newly emerged honeybee worker using 2D-E-MS/MS (pI 3-10 and 4-7). The comparison of identical hemolymph volumes (20 μL per 2D-E) for newly emerged bee and red-eye pupa revealed a dramatic decrease in the number of spots (qualitative changes) and overall protein quantity during the non-feeding stage. The results increase our knowledge about honeybee metamorphosis during the non-feeding period and clarify previous findings regarding particular proteins. The results will be useful for future comparative physiological, developmental, and host-pathogen studies on individual or population level.

The honey bee, Apis mellifera, is a globally important species that suffers from a variety of pathogens and parasites. These parasites and pathogens may have sublethal effects on their bee hosts via an array of mechanisms, including through a change in symbiotic bacterial taxa. Our aim was to assess the influence of four globally widespread parasites and pathogens on the honey bee bacteriome. We examined the effects of the ectoparasitic mite Varroa destructor, the fungal pathogens Nosema apis and Nosema ceranae, and the trypanosome Lotmaria passim. Varroa was detected by acaricidal treatment, Nosema and L. passim by PCR, and the bacteriome using MiSeq 16S rRNA gene sequencing. Overall, the 1,858,850 obtained sequences formed 86 operational taxonomic units (OTUs) at 3 % dissimilarity. Location, time of year, and degree of infestation by Varroa had significant effects on the composition of the bacteriome of honey bee workers. Based on statistical correlations, we found varroosis more important factor than N. ceranae, N. apis, and L. passim infestation influencing the honey bee bacteriome and contributing to the changes in the composition of the bacterial community in adult bees. At the population level, Varroa appeared to modify 20 OTUs. In the colonies with high Varroa infestation levels (varroosis), the relative abundance of the bacteria Bartonella apis and Lactobacillus apis decreased. In contrast, an increase in relative abundance was observed for several taxa including Lactobacillus helsingborgensis, Lactobacillus mellis, Commensalibacter intestini, and Snodgrassella alvi. The results showed that the “normal” bacterial community is altered by eukaryotic parasites as well as displaying temporal changes and changes associated with the geographical origin of the beehive.

The ectoparasitic mite Varroa destructor is a major pest of the honeybee Apis mellifera. In a previous study, bacteria were found in the guts of mites collected from winter beehive debris and were identified using Sanger sequencing of their 16S rRNA genes. In this study, community comparison and diversity analyses were performed to examine the microbiota of honeybees and mites at the population level. The microbiota of the mites and honeybees in 26 colonies in seven apiaries in Czechia was studied. Between 10 and 50 Varroa females were collected from the bottom board, and 10 worker bees were removed from the peripheral comb of the same beehive. Both bees and mites were surface sterilized. Analysis of the 16S rRNA gene libraries revealed significant differences in the Varroa and honeybee microbiota. The Varroa microbiota was less diverse than was the honeybee microbiota, and the relative abundances of bacterial taxa in the mite and bee microbiota differed. The Varroa mites, but not the honeybees, were found to be inhabited by Diplorickettsia. The relative abundance of Arsenophonus, Morganella, Spiroplasma, Enterococcus, and Pseudomonas was higher in Varroa than in honeybees, and the Diplorickettsia symbiont detected in this study is specific to Varroa mites. The results demonstrated that there are shared bacteria between Varroa and honeybee populations but that these bacteria occur in different relative proportions in the honeybee and mite bacteriomes. These results support the suggestion of bacterial transfer via mites, although only some of the transferred bacteria may be harmful.

The tortoise tick Hyalomma aegyptium has a typical three-host life-cycle. Whereas its larvae and nymphs are less host-specific feeding on a variety of tetrapods, tortoises of the genus Testudo are principal hosts of adults. Ticks retained this trait also in our study under laboratory conditions, while adults were reluctant to feed on mammalian hosts. Combination of feeding larvae and nymphs on guinea pigs and feeding of adults on Testudo marginata tortoises provided the best results. Feeding period of females was on average 25 days (range 17-44), whereas males remain after female engorgement on tortoise host. Female pre-oviposition period was 14 days (3-31), followed by 24 days of oviposition (18-29). Pre-eclosion and eclosion, both together, takes 31 days (21-43). Larvae fed 5 days (3-9), then molted to nymphs after 17 days (12-23). Feeding period of nymphs lasted 7 days (5-10), engorged nymphs molted to adults after 24 days (19-26). Sex ratio of laboratory hatched H. aegyptium was nearly equal (1:1.09). The average weight of engorged female was 0.95 (0.72-1.12) g. The average number of laid eggs was 6,900 (6,524-7,532) per female, it was significantly correlated with weight of engorged female. Only 2.8% of engorged larvae and 1.8% of engorged nymphs remained un-molted and died. Despite the use of natural host species, feeding success of females reached only 45%. The whole life-cycle was completed within 147 days (98-215).

Spores of Trachipleistophora extenrec, originally isolated from the muscles of the Madagascan insectivore Hemicentetes semispinosus and maintained by serial passage in severe combined immunodeficiency (SCID) mice, were fed to larvae of the Egyptian cotton leafworm Spodoptera littoralis. Extensive infection of larval tissues ensued and caused larval and pupal mortality. The development of T. extenrec in the insect host, studied both by light and electron microscopy, followed generally the same life cycle as in the mammalian host. However, some differences in the fine structure of the parasite grown in both types of hosts were found. Spores isolated from the insect host caused infection of SCID mice when injected intramuscularly. Our results suggest that T. extenrec may be originally an insect microsporidian. This likelihood is corroborated by its structural similarity and phylogenetic relationship to two other microsporidia having insects either as unique hosts (Vavraia culicis) or being able to infect both mammalian and insect host (Trachipleistophora hominis).

Hyalomma aegyptium ticks were collected from tortoises, Testudo graeca, at localities in northern Africa, the Balkans, and the Near and Middle East. The intensity of infestation ranged from 1-37 ticks per tortoise. The sex ratio of feeding ticks was male-biased in all tested populations. Larger tortoises carried more ticks than did the smaller tortoises. The juveniles were either not infested, or carried only a poor tick load. Hyalomma aegyptium was absent in the western Souss Valley and Ourika Valley in Morocco, the Cyrenaica Peninsula in Libya, Jordan, and the Antilebanon Mountains in Syria. Hemolivia mauritanica, a heteroxenous apicomplexan cycling between T. graeca and H. aegyptium, was confirmed in Algeria, Romania, Turkey, Syria, Lebanon, and Iran. Its prevalence ranged from 84% in Romania (n = 45), 82% in eastern Turkey (n = 28), and 82% in the area of northwestern Syria with adjacent Turkish borderland (n = 90), to 38% in Lebanon (n = 8) and in only 1 of 16 sampled tortoises in Algeria. The intensity of parasitemia in the studied areas ranged from 0.01% up to 28.17%. The percentage of Hemolivia-infected erythrocytes was significantly higher in adults. All tortoises from Hyalomma-free areas were Hemolivia-negative. Remarkably, all 29 T. graeca from Jabal Duruz (southwestern Syria) and 36 T. graeca from the area north of Middle Atlas (Morocco) were Hemolivia-negative, despite the fact that ticks parasitized all adult tortoises in these localities. Identical host preferences of H. aegyptium and H. mauritanica suggest the occurrence of co-evolution within the Testudo-Hyalomma-Hemolivia host-parasite complex.

Collection of 1327 ticks sampled throughout Greece, Bulgaria, Romania and Croatia, from 211 tortoises belonging to three species, Testudo marginata Schoepff, T. graeca Linnaeus, and T. hermanni Gmelin, revealed the presence of four species of ixodid ticks, namely Hyalomma aegyptium (Linnaeus), Haemaphysalis sulcata Canestrini and Fanzago, H. inermis Birula and Rhipicephalus sanguineus (Latreille). Study confirmed the strong dominance of all life stages of H. aegyptium among ticks parasitizing west Palaearctic tortoises of genus Testudo Linnaeus. Furthermore, a considerable portion of ticks collected from tortoises in southwestern Bulgaria represent larvae and nymphs of H. sulcata. At the same area we collected as exception one larva and one nymph of H. inermis from a single specimen of T. hermanni. Our findings of four adults of R. sanguineus is the first record of this species from reptilian host. According to our results achieved on localities with syntopic occurrence of two tortoise species, T. marginata and T. graeca represent in the Balkans the principal hosts of H. aegyptium, whereas T. hermanni serves only as an alternative host in the areas close to range of either T. marginata or T. graeca.

The parasitic mite Varroa destructor is a major pest of the western honeybee, Apis mellifera. The development of acaricide resistance in Varroa populations is a global issue. Discriminating concentrations of acaricides are widely used to detect pest resistance. Two methods, using either glass vials or paraffin capsules, are used to screen for Varroa resistance to various acaricides. We found the glass vial method to be useless for testing Varroa resistance to acaridices, so we developed a polypropylene vial bioassay. This method was tested on tau-fluvalinate-, acrinathrin-, and amitraz-resistant mite populations from three apiaries in Czechia. Acetone was used as a control and technical grade acaricide compounds diluted in acetone were applied to the polypropylene vials. The solutions were spread on the vial surface by rolling the vial, and were then evaporated. Freshly collected Varroa females were placed in the vials and the mortality of the exposed mites was measured after 24 h. The Varroa populations differed in mortality between the apiaries and the tested compounds. Mites from the Kyvalka site were resistant to acrinathrin, tau-fluvalinate, and amitraz, while mites from the Postrizin site were susceptible to all three acaricides. In Prelovice apiary, the mites were susceptible to acrinathrin and amitraz, but not to tau-fluvalinate. The calculated discriminating concentrations for tau-fluvalinate, acrinathrin, and amitraz were 0.66, 0.26 and 0.19 µg/mL, respectively. These results indicate that polyproplyne vial tests can be used to determine discriminating concentrations for the early detection of acaricide resistant Varroa. Finally, multiple-resistance in Kyvalka may indicate metabolic resistance.

We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2 and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications and the presence of honeybee proteins in Varroa are discussed.

Aims The winter beehive debris containing bodies of honeybee parasitic mite Varroa destructor is used for veterinary diagnostics. The Varroa sucking honeybee haemolymph serves as a reservoir of pathogens including bacteria. Worker bees can pick up pathogens from the debris during cleaning activities and spread the infection to healthy bees within the colony. The aim of this study was to detect entomopathogenic bacteria in the Varroa collected from the winter beehive debris. Methods and Results Culture-independent approach was used to analyse the mite-associated bacterial community. Total DNA was extracted from the samples of 10 Varroa female individuals sampled from 27 different sites in Czechia. The 16S rRNA gene was amplified using universal bacterial primers, cloned and sequenced, resulting in a set of 596 sequences representing 29 operational taxonomic units (OTU97). To confirm the presence of bacteria in Varroa, histological sections of the mites were observed. Undetermined bacteria were observed in the mite gut and fat tissue. Conclusion Morganella sp. was the most frequently detected taxon, followed by Enterococcus sp., Pseudomonas sp., Rahnella sp., Erwinia sp., and Arsenophonus sp. The honeybee putative pathogen Spiroplasma sp. was detected at one site and Bartonella-like bacteria were found at four sites. PCR-based analysis using genus-specific primers enabled detection of the following taxa: Enterococcus, Bartonella-like bacteria, Arsenophonus and Spiroplasma. Significance and Impact of the Study We found potentially pathogenic (Spiroplasma) and parasitic bacteria (Arsenophonus) in mites from winter beehive debris. The mites can be reservoirs of the pathogenic bacteria in the apicultures.

Eimeria jirkamoraveci sp. n. is described from faeces of two specimens of the toad-headed, side-necked turtle Batrachemys heliostemma collected at Iquitos in Peru. Oocysts are ovoid to almost spherical, 10.6 (8-12) x 8.9 (7-10) microm, without micropyle, polar granule and oocyst residuum. One conically stretched end and three blunt conical tubercles at the opposite end of oocyst give it mitra-like appearance. Sporocysts are elongated, ellipsoidal, 7.2 (6-8) x 4.1 (4-4.5) microm, with a small, knob-like Stieda body and sporocyst residuum composed of fine granules. To avoid possible conspecificity, the described new species is thoroughly compared with the most similar coccidium, E. mitraria, collected from its type host, Chinemys reevesii.

International audience; The aim of this study was to improve cage systems for maintaining adult honey bee (Apis mellifera L.) workers under in vitro laboratory conditions. To achieve this goal, we experimentally evaluated the impact of different cages, developed by scientists of the international research network COLOSS (Prevention of honey bee COlony LOSSes), on the physiology and survival of honey bees. We identified three cages that promoted good survival of honey bees. The bees from cages that exhibited greater survival had relatively lower titers of deformed wing virus, suggesting that deformed wing virus is a significant marker reflecting stress level and health status of the host. We also determined that a leak- and drip-proof feeder was an integral part of a cage system and a feeder modified from a 20-ml plastic syringe displayed the best result in providing steady food supply to bees. Finally, we also demonstrated that the addition of protein to the bees' diet could significantly increase the level of vitellogenin gene expression and improve bees' survival. This international collaborative study represents a critical step toward improvement of cage designs and feeding regimes for honey bee laboratory experiments.

Sodium channels (SCs) in mites and insects are target sites for pesticides, including pyrethroids. Point mutations in the SC gene have been reported to change the structural conformation of the protein and its sensitivity to pesticides. To find mutations in the SC gene of the mite Varroa destructor (VmNa), the authors analysed the VmNa gene sequences available in GenBank and prepared specific primers for the amplification of two fragments containing the regions coding for (i) the domain II S4-S6 region (bp 2805-3337) and (ii) the domain III S4-3' terminus region (bp 4737-6500), as determined according to the VmNa cDNA sequence AY259834.Sensitive and resistant mite populations did not differ in the amino acid sequences of the III S4-3' terminus VmNa region. However, differences were found in the IIS4-IIS6 fragment. In the resistant population, the mutation C(3004) → G resulted in the substitution L(1002) → V (codon ctg → gtg) at the position equivalent to that of the housefly L925 in the domain II S5 helix. Additionally, the mutation F(1052) → L (codon ttc → ctc) at the position equivalent to that of the housefly F975 in the domain II P-loop connecting segments S5 and S6 was detected in both the resistant and sensitive populations.All individuals that survived the tau-fluvalinate treatment in the bioassay harboured the L(1002) → V mutation combined with the F(1052), while dead individuals from both the sensitive and resistant populations harboured mostly the L(1002) residue and either of the two residues at position 1052.

Enterocytozoon bieneusi known as a causative agent of opportunistic infections instigating diarrhoea in AIDS patients was identified also in a number of immunocompetent patients and in a wide range of animals, including cattle. In the present study we tested if the Bovine Viral Diarrhea Virus (BVDV), the most common pathogen underlying immunosuppressive Bovine Viral Diarrhoea (BVD), can enhance the occurrence of opportunistic infections with E. bieneusi in cattle. Six dairy farms were investigated using ELISA to detect antibodies against or antigens arising from BVDV in collected sera. A total of 240 individual faecal samples from four age groups were examined for the presence of E. bieneusi by nested PCR. Sequence analysis of six E. bieneusi positive samples revealed the presence of the genotype I of E. bieneusi, previously described in cattle. The hypothesis expecting higher prevalence of E. bieneusi in BVDV positive cattle herds was not confirmed in this study; however this is the first description about E. bieneusi in cattle in the Czech Republic.

Hepatozoon ayorgbor n. sp. is described from specimens of Python regius imported from Ghana. Gametocytes were found in the peripheral blood of 43 of 55 snakes examined. Localization of gametocytes was mainly inside the erythrocytes; free gametocytes were found in 15 (34.9%) positive specimens. Infections of laboratory-reared Culex quinquefasciatus feeding on infected snakes, as well as experimental infection of juvenile Python regius by ingestion of infected mosquitoes, were performed to complete the life cycle. Similarly, transmission to different snake species (Boa constrictor and Lamprophis fuliginosus) and lizards (Lepidodactylus lugubris) was performed to assess the host specificity. Isolates were compared with Hepatozoon species from sub-Saharan reptiles and described as a new species based on the morphology, phylogenetic analysis, and a complete life cycle.

Six young tortoises Testudo marginata Schoepff, 1792 were experimentally infected with Hemolivia mauritanica (Sergent et Sergent, 1904). The prepatent period ranged from 6 to 8 weeks. Young, smaller, club-like forms (6-9 x 3-6 Am) of gametocytes appeared in the peripheral blood first, whereas mature, elongated, cylindrical forms (9-12 x 5-7 Am) were detected after 1-2 weeks and predominated during later patency. Three of the infected tortoises were euthanized and dissected to study the endogenous stages. Meronts occurred in the cells of the reticulo-endothelial system and in the erythrocytes; these were observed mostly in parenchymatous organs. Mature forms measured 14.2 x 9.3 microm and contained 7-12 merozoites. Cysts with two (exceptionally one) cystozoites were also found predominantly in parenchymatous organs and measured 14.8 x 7.9 microm. Pathological changes attributable to Hemolivia were mild and limited to liver and kidneys. The role of individual developmental stages of haemogregarines is discussed with respect to evolution of heteroxenous life cycle and long-term persistence of parasites in their intermediate hosts.

Hemolivia mauritanica (Sergent et Sergent, 1904) infections were found in 14% (n= 14) of Testudo graeca Linnaeus, 1758 tortoises in Bulgaria and in 92% (n = 26) of those in Turkey. Hemolivia mauritanica-like gametocytes were found in 81% (n = 47) of wild Testudo marginata Schoepff, 1792 tortoises in Greece. Parasitaemia intensity (the percentage of infected red blood cells found in approximately 104 cells) was 0.03-22.4% in T. graeca and 0.06-12.27% in T. marginata. On the other hand, all blood samples from 40 Bulgarian, 38 Greek, and 18 Croatian specimens of Testudo hermanni Gmelin, 1789 were negative.

Coprological examination of nine African helmeted turtles Pelomedusa subrufa from Kenya revealed the presence of a new coccidium belonging to Eimeria Schneider, 1875. Oocysts of Eimeria lokuma n. sp. are spherical to sub-spherical, 13.6 (13–14.5) × 13 (12–14) \(\upmu\)m, lack a micropyle and a polar granule, but possess a granular oocyst residuum. The sporocysts are elongate, oval to spindle-shaped and 8.3 (7.5–9.5) × 4.4 (4–5) \(\upmu\)m. The Stieda body is relatively low, flat and wide, and covered with a membranous, highly flexible, scarf-like structure which protrudes from its strengthened margins. The sporozoites possess two refractile bodies. Based on the presence of a Stieda body, the described species is classified as Eimeria (sensu stricto).

Coprological examination of nine bush vipers Atheris chlorechis imported from Ghana revealed the presence of a new coccidian species belonging to Eimeria Schneider, 1875. Thin walled oocysts of Eimeria atheridis n. sp. are spherical to slightly subspherical, 22.8 (19-26) x 22.5 (19-25) microm, without micropyle, polar granule and oocyst residuum. Sporocysts are elongately ellipsoidal, 17.1 (15-19) x 7.5 (6-8) microm, with a dome like, relatively flat Stieda body. Sporozoites possess two refractile bodies and distinct transversal striation. Based on the presence of a Stieda body the species described herein clearly belongs to the Eimeria ( sensu stricto ).

Two experimental trials were performed to elucidate the role of rodents in the life cycle of Hepatozoon species using snakes as intermediate hosts. In one trial, two ball pythons, Python regius Shaw, 1802 were force fed livers of laboratory mice previously inoculated with sporocysts of Hepatozoon ayorgbor Sloboda, Kamler, Bulantová, Votýpka et Modrý, 2007. Transmission was successful in these experimentally infected snakes as evidenced by the appearance of intraerythrocytic gamonts, which persisted until the end of trial, 12 months after inoculation. Developmental stages of haemogregarines were not observed in histological sections from mice. In another experimental trial, a presence of haemogregarine DNA in mice inoculated with H. ayorgbor was demonstrated by PCR in the liver, lungs and spleen.