Host factors limiting monogenean infections: a case study

Comprehensive field data on polystomatid monogeneans record low prevalence and intensity of infection and suggest that worm burdens in this group are strongly regulated: thus, in the majority of Polystoma species infecting anuran amphibians mean abundance is typically less than one parasite/host. There is circumstantial evidence that the dominant control is attributable to host factors which over-ride variations in transmission success. This review provides a brief summary of information on Pseudodiplorchis americanus, a parasite of the desert toad, Scaphiopus couchii, and then focuses in detail on the spectrum of factors regulating infrapopulations of Protopolystoma xenopodis, a parasite of the aquatic Xenopus laevis. Infection levels of adult worms and their contribution to transmission are regulated by external environmental factors (especially temperature), by host factors (including behaviour and population density), and by a range of parasite factors including intra- and inter-specific competitive interactions and variations in intrinsic characters, especially survivorship and reproductive output. In addition to these factors whose primary effect is to modulate transmission rates, there is a major attrition in parasite numbers between invasion and maturity (3 months post-infection). Long-term laboratory experiments on the Xenopus laevis/Protopolystoma xenopodis interaction demonstrate a powerful acquired immune response. Primary infection is characterised by a high prevalence of established adult worms but the success of subsequent challenge infection is greatly reduced, leading to low prevalence and extended pre-patent period. In the small proportion of hosts supporting a second infection of adult parasites, surviving burdens are small (one to two worms/host) and show reduced egg production. These results provide an explanation for the low burdens encountered in field studies: a majority of adult X. laevis in natural populations are likely to exhibit strong, relatively long-term, post-infection immunity after the loss of a previous infection.     

Genomic regions of pufferfishes responsible for host specificity of a monogenean parasite, Heterobothrium okamotoi

The genetic mechanisms underlying host specificity of parasitic infections are largely unknown. After hatching, the larvae of the monogenean parasite, Heterobothrium okamotoi, attach to the gill filaments of hosts and the post-larval worms develop there by consuming nutrients from the host. The susceptibility to H. okamotoi infection differs markedly among fish species. While this parasite can grow on tiger pufferfish (also called fugu), Takifugu rubripes, it appears to be rejected by a close congener, grass pufferfish, Takifugu niphobles, after initial attachment to the gills. To determine the genetic architecture of the pufferfish responsible for this host specificity, we performed genome-wide quantitative trait loci analysis. We raised second generation (F₂) hybrids of the two pufferfish species and experimentally infected them with the monogenean in vivo. To assess possible differences in host mechanisms between early and later periods of infection, we sampled fish three h and 21 days after exposure. Genome scanning of fish from the 3 h infection trial revealed suggestive quantitative trait loci on linkage groups 2 and 14, which affected the number of parasites on the gill. However, analysis of fish 21 days p.i. detected a significant quantitative trait locus on linkage group 9 and three other suggestive quantitative trait loci on linkage groups 7, 18 and 22. These results indicated the polygenic nature of the host mechanisms involved in the infection/rejection of H. okamotoi. Moreover the analyses suggested that host factors may play a more important role during the growth period of the parasite than during initial host recognition at the time of attachment. Within the 95% confidence interval of the linkage group 9 quantitative trait locus in the fugu genome, there were 214 annotated protein-coding genes, including immunity-related genes such as Irak4, Muc2 and Muc5ac.     

Biochemical and pathogenic properties of the natural isolate of Shewanella algae from Peter the Great Bay, Sea of Japan

Pathogenic properties of the natural isolate of Shewanella algae from the coelomic fluid of the sea cucumber Apostichopus japonicus (Peter the Great Bay, Sea of Japan) were investigated. The isolate had oxydative metabolism, was positive for ornithine decarboxylase, cytochrome oxidase, catalase, DNase and gelatinase, hemolytically active, did not produce acid from carbohydrates, and did not hydrolyze urea and esculin. The strain was resistant to penicillin, amoxicillin, and ampicillin and susceptible to tetracycline and carbenicillin. Among cellular fatty acids, 13:0-i, 15:0-i, 16:0, 16:1(n-7), 17:0-i, and 17:0-ai dominated. These biochemical properties made it possible to attribute the isolated bacteria to the genus Shewanella and identified as S. algae. The cells of this bacterium were introduced into the coelomic cavity of another echinoderm, the sea urchin Strongylocentrotus nudus. As a result, in about 24 h the animals became slow and 3-8 days after the inoculation died. Dividing bacteria were being found during the experiment in the coelomic fluid as well as in the phagosomes of amoebocytes, i.e. cells acting as phagocytes in the coelomic fluid. The studies of the invasive properties of strain 156 showed that bacterial cells entered the subcuticular space of S. nudus and A. japonicus through the cuticle and stayed there for a long time without penetrating epithelium and exerting toxic effect upon the organisms of the laboratory animals. Pathogenic effect of S. algae can be manifested only if the cutaneous epithelium is destroyed permitting it to penetrate the lower tissue layers. The toxicity of S. algae is confirmed by in vitro experiments. The inoculation of the embryonic cells of S. nudus with samples of this bacterium caused the death of 10% of cells within an hour and 100% of cells within 12 h after inoculation. The results of the investigations demonstrate that S. algae could produce opportunistic infection in the sea cucumber A. japonicus and the sea urchin S. nudus, which may be natural reservoirs of this human pathogen.     

Experimental infections of the monogenean Gyrodactylus turnbulli indicate that it is not a strict specialist

Parasites represent a threat to endangered fish species, particularly when the parasite can host switch and the new host is vulnerable. If the parasite is highly host specific then successful host switching should be a rare occurrence; however, the host range of many parasites which are assumed to be specialists has never been tested. This includes the monogenean Gyrodactylus turnbulli, a well-studied ectoparasite found caudally on its known host, the guppy, Poecilia reticulata. In this study, we monitored parasite establishment and reproduction on a range of poeciliids and more distantly related fish. Individually maintained fish were experimentally infected with a single parasite and monitored daily to establish whether G. turnbulli could survive and reproduce on other fish species. Gyrodactylus turnbulli can infect a wider range of hosts than previously considered, highlighting the fact that host specificity can never be assumed unless experimentally tested. Our findings also have significant implications for parasite transmission to novel hosts and provide further insight into the evolutionary origins of this ubiquitous group of fish pathogens. Previous molecular evidence indicates that host switching is the key mechanism for speciation within the genus Gyrodactylus. Until recently, most Gyrodactylus spp. were assumed to be narrowly host specific. However, our findings suggest that even so-called specialist species, such as G. turnbulli, may represent a threat to vulnerable fish stocks. In view of the potential importance of host switching under artificial conditions, we propose to describe this as 'artificial ecological transfer' as opposed to 'natural ecological transfer', host switching under natural conditions.     

Microsporidian infections in Lymantria dispar larvae: interactions and effects of multiple species infections on pathogen horizontal transmission

The interactions in multiple species infections and effects on the horizontal transmission of three microsporidian species, Vairimorpha disparis, Nosema lymantriae and Endoreticulatus schubergi, infecting Lymantria dispar were evaluated in the laboratory. Simultaneous and sequential inoculations of host larvae were performed and the resulting infections were evaluated. Test larvae were exposed to the inoculated larvae to measure horizontal transmission. Dual species infections demonstrated interspecific competition between Nosema and Vairimorpha in the host larvae, but no observable competition occurred between Endoreticulatus and either of the other microsporidian species. Timing of inoculation was an important factor determining the outcome of competition between Nosema and Vairimorpha. The species inoculated first showed a higher rate of successful establishment; a time lag of 7 days between inoculations allowed the first species to essentially exclude the second. The microsporidia differed in efficiency of horizontal transmission. Nosema and Endoreticulatus were transmitted at very high rates, close to 100%. Horizontal transmission of Vairimorpha was less efficient, ranging from 25% to a maximum of 75%. The patterns of infection observed in inoculated larvae were reflected in the test larvae that acquired infections in the horizontal transmission experiments. Competition with Vairimorpha suppressed horizontal transmission of Nosema after simultaneous and sequential inoculation. In simultaneous inoculation experiments Endoreticulatus had no effect on transmission of Nosema and Vairimorpha.     

Distribution of Nosema ceranae in the European honeybee, Apis mellifera in Japan

The microsporidian species, Nosema apis and Nosema ceranae are both known to infect the European honeybee, Apis mellifera. Nosema disease has a global distribution and is responsible for considerable economic losses among apiculturists. In this study, 336 honeybee samples from 18 different prefectures in Japan were examined for the presence of N. apis and N. ceranae using a PCR technique. Although N. ceranae was not detected in most of the apiaries surveyed, the parasite was detected at three of the sites examined. Further, N. ceranae appears to be patchily distributed across Japan and no apparent geographic difference was observed among the areas surveyed. In addition, the apparent absence of N. apis suggests that N. ceranae may be displacing N. apis in A. mellifera in Japan. Partial SSU rRNA gene sequence analysis revealed the possible existence of two N. ceranae groups from different geographic regions in Japan. It seems likely that these microsporidian parasites were introduced into Japan through the importation of either contaminated honeybee-related products or infected queens. This study confirmed that PCR detection is effective for indicating the presence of this pathogen in seemingly healthy colonies. It is therefore hoped that the results presented here will improve our understanding of the epidemiology of Nosema disease so that effective controls can be implemented.     

PCR diagnostic methods for Ascosphaera infections in bees

Fungi in the genus Ascosphaera are the causative agents of chalkbrood, a major disease affecting bee larval viability. Identification of individual Ascosphaera species based on morphological features has been difficult due to a lack of distinguishing characteristics. Most identifications are based on the size and shape of the ascomata, spore balls and conidia. Unfortunately, much overlap occurs in the size of these structures, and some Ascosphaera species will not produce sexual structures in vitro. We report a quick and reliable diagnostic method for identifying Ascosphaera infections in Megachile bees (leafcutting bees) using PCR markers that employ genus-specific primers for Ascosphaera, and species-specific primers for species known to be associated with Megachile spp. Using these methods, species identifications can be performed directly on bees, including asymptomatic individuals. Furthermore, the PCR markers can detect co-infections of multiple Ascosphaera species in a single host. We also identified a marker for Ascosphaera apis, the predominant cause of chalkbrood in Apis mellifera, the honey bee. Our diagnostic methods eliminate the need for culturing samples, and could be used to process a large number of field collected bee larvae.     

Involvement of host recognition by oncomiracidia and post-larval survivability in the host specificity of Heterobothrium okamotoi (Monogenea: Diclidophoridae)

Heterobothrium okamotoi, a monogenean gill parasite, shows high host specificity for tiger puffer, Takifugu rubripes. In the present study, in vivo and in vitro experimental infections were conducted using various fish species, including T. rubripes, to understand the mechanisms of specificity. In in vivo experiments, T. rubripes, grass puffer, Takifugu niphobles, olive flounder, Paralichthys olivaceus, and red sea bream, Pagrus major, were exposed to oncomiracidia of H. okamotoi labelled with a fluorescent dye, 5- (and -6) carboxyfluorescein diacetate succinimidyl ester, and the numbers of parasites on the gills and skin were recorded at intervals. Oncomiracidia were attached to gills and skin of all the experimental fish species immediately after exposure, and the infection intensity on T. rubripes was higher than that on T. niphobles and much higher than those on the other two species. After 2 days, the attached parasites remained on the gills of T. rubripes, but disappeared from the other hosts. During in vitro experiments, gill filaments excised from seven different fish species (four fish species used in the in vivo experiments and panther puffer, Takifugu pardalis, southern flounder, Paralichthys lethostigma and spotted halibut, Verasper variegates) were exposed to oncomiracidia and the attachment to each fish species and subsequent larval behaviour was observed. The percentage of post-larvae that attached to T. rubripes was slightly higher than those which attached to congeneric fish species and much higher than those of non-tetraodontid fish species. In vivo and in vitro experiments demonstrated that oncomiracidia of H. okamotoi have an affinity for their natural host, T. rubripes, and congeneric fish species. The disappearance of attached post-larvae from 'alien' hosts within 2 days during in vivo experiments suggested that host recognition by oncomiracidia and subsequent post-larval survivability are involved in the host specificity of H. okamotoi.     

Experimental studies on Echinococcus multilocularis in Japan, focusing on biohazardous stages of the parasite

Alveolar echinococcosis (AE), caused by the active growth of larval Echinococcus multilocularis mostly in the liver, is usually fatal zoonotic disease if not adequately treated. Humans become infected via oral ingestion of the parasite eggs, which are thus biohazardous to humans and should be handled under restricted conditions. In this review, we present findings in experimental studies mainly performed at a safety facility in Japan, examining the biohazadous stages of the parasite (Hokkaido isolate) including its egg and adult worm stages. This article deals mainly with the parasite development in various experimental and wild animals, environmental factors affecting viability of the parasite eggs, and molecular biological studies on adult worms. The findings shown herein have provided a basis to better understand basic biology and natural transmission of E. multilocularis in Hokkaido, a highly endemic area of AE in northern Japan, and also to establish effective preventive measures against the disease.     

Development of symbionts in triatomine bugs and the effects of infections with trypanosomatids

In the intestinal tract of fifth instars of the hematophagous reduviid bugs Rhodnius prolixus and Triatoma infestans blood ingestion induced an initial decrease of the concentration of the respective symbiotic bacteria Rhodococcus rhodnii and Nocardia sp. and then within 10 days a 15- or 18-fold increase of the total population/bug to about 0.8 x 10(9) colony-forming units in R. prolixus and 1.8 x 10(9) colony-forming units in T. infestans. About 95-99% of the total populations of both symbionts developed in the anterior midgut regions, i.e., cardia and stomach. The passage from the blood-storing stomach to the digesting small intestine caused a considerable breakdown of symbiont populations, and only about 0.01% of the total population was present in the rectum. These were excreted mainly within 4 h after a blood meal. After infection with three species of trypanosomatids, R. rhodnii, the symbiont of R. prolixus, was affected by Trypanosoma rangeli, but not by Blastocrithidia triatomae or Trypanosoma cruzi. On the other hand, in T. infestans the concentration of Nocardia sp. was reduced after infection with B. triatomae, but not by T. rangeli nor T. cruzi. In long-term B. triatomae-infected T. infestans, this reduction and a reduced diuretic activity after feeding synergistically lowered the symbiont concentration in the singly deposited feces/urine drops drastically compared to uninfected controls. These data strongly support the theory of the mechanisms of pathogenicity of T. rangeli and B. triatomae for R. prolixus and T. infestans, respectively, that the primal point of attack is the host-specific symbiont, R. rhodnii and Nocardia sp., respectively.     

Current Status and Perspectives of Cysticercosis and Taeniasis in Japan

This mini-review describes recent epidemiological trends in cysticercosis and taeniasis in Japan. Some of the topics discussed herein were presented at the first symposium on "Current perspectives of Taenia asiatica researches", that was held in Osong in Chungbuk Province, South Korea, in October 2011 and organized by Prof. K. S. Eom, Chungbuk National University School of Medicine. To better understand the trends in the occurrence of cysticercosis and taeniasis in Japan, clinical cases reported in 2005 have been updated. In addition, the current status of Taenia asiatica infections successively occurring in Japan since 2010 is also discussed.     

Pathogenicity of intrathoracically administrated Bacillus thuringiensis spores in Blatta orientalis

The ability of Bacillus thuringiensis to produce septicaemia in Periplaneta americana and Blatta orientalis has been investigated. Spores and crystals from several wild-type strains as well as spores of a B. thuringiensis crystal-deficient mutant, were first orally administrated at high doses, and no significant mortality was recorded. Intrathoracic injection of spore suspensions in P. americana revealed that this species is not very susceptible to B. thuringiensis spores. B. orientalis, by contrast, was found to be very susceptible to B. thuringiensis, with a LD(50) of about 35,000 spores, that is similar to that reported on Lepidoptera challenged with parenterally injected B. thuringiensis.     

Direct infection of Spodoptera litura by Photorhabdus luminescens encapsulated in alginate beads

Actively growing cultures of Photorhabdus luminescens were encapsulated in sodium alginate beads and examined for their ability to infect insect hosts. These beads, containing approximately 2.5 x 10(7)Photorhabdus cells per bead, when mixed with sterilized soil and exposed to Spodoptera litura larvae resulted in 100% mortality in 48 h, while the use of alginate encapsulated Heterorhabditis nematode resulted in 40% mortality after 72 h. The bacteria were reisolated from the dead insect thus proving Koch's postulates and demonstrating the ability of P. luminescens to kill the insect host on their own, independent of the symbiont nematode. The LC(50) dose of Photorhabdus cells was estimated at 1010 cells per larva for killing S. litura 6th instar larvae in 48 h.     

Experimental evaluation of the pathogenicity of Perkinsusolseni in juvenile Manila clams Ruditapes philippinarum

We evaluated the pathogenicity of Perkinsus olseni towards the Manila clam, Ruditapes philippinarum, by an experimental challenge. For production of prezoosporangia of P. olseni, we injected uninfected Manila clams with cells of a pure strain of P. olseni and reared them for 7 d. Prezoosporangia were isolated from the soft tissue of the injected clams after culturing in Ray’s fluid thioglycollate medium. Hatchery-reared, uninfected juvenile clams (3-10 mm shell length) were challenged by immersion in one of two concentrations of a prezoosporangial suspension of P. olseni for 6 d. The challenged clams had significantly higher mortality at both the concentrations than the unchallenged clams. The mortality due to infection dose-dependently began approximately 4 weeks and 7 weeks after challenge in the higher and lower concentrations, respectively. This is the first experimental evidence that P. olseni causes direct mortality in Manila clams. The lethal level of infection was estimated at approximately 10⁷ pathogen cells/g soft tissue weight.     

A putative serine protease,SpSsp1, from Saprolegnia parasitica is recognised by sera of rainbow trout, Oncorhynchus mykiss

Saprolegniosis, the disease caused by Saprolegnia sp., results in considerable economic losses in aquaculture. Current control methods are inadequate, as they are either largely ineffective or present environmental and fish health concerns. Vaccination of fish presents an attractive alternative to these control methods. Therefore we set out to identify suitable antigens that could help generate a fish vaccine against Saprolegnia parasitica. Unexpectedly, antibodies against S. parasitica were found in serum from healthy rainbow trout, Oncorhynchus mykiss. The antibodies detected a single band in secreted proteins that were run on a one-dimensional SDS-polyacrylamide gel, which corresponded to two protein spots on a two-dimensional gel. The proteins were analysed by liquid chromatography tandem mass spectrometry. Mascot and bioinformatic analysis resulted in the identification of a single secreted protein, SpSsp1, of 481 amino acid residues, containing a subtilisin domain. Expression analysis demonstrated that SpSsp1 is highly expressed in all tested mycelial stages of S. parasitica. Investigation of other non-infected trout from several fish farms in the United Kingdom showed similar activity in their sera towards SpSsp1. Several fish that had no visible saprolegniosis showed an antibody response towards SpSsp1 suggesting that SpSsp1 might be a useful candidate for future vaccination trial experiments.     

Characterization of secreted proteases of Paenibacillus larvae, potential virulence factors involved in honeybee larval infection

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), the most severe bacterial disease that affects honeybee larvae. AFB causes a significant decrease in the honeybee population affecting the beekeeping industry and agricultural production. After infection of larvae, P. larvae secretes proteases that could be involved in the pathogenicity. In the present article, we present the secretion of different proteases by P. larvae. Inhibition assays confirmed the presence of metalloproteases. Two different proteases patterns (PP1 and PP2) were identified in a collection of P. larvae isolates from different geographic origin. Forty nine percent of P. larvae isolates showed pattern PP1 while 51% exhibited pattern PP2. Most isolates belonging to genotype ERIC I - BOX A presented PP2, most isolates belonging to ERIC I - BOX C presented PP1 although relations were not significant. Isolates belonging to genotypes ERIC II and ERIC III presented PP2. No correlation was observed between the secreted proteases patterns and geographic distribution, since both patterns are widely distributed in Uruguay. According to exposure bioassays, isolates showing PP2 are more virulent than those showing PP1, suggesting that difference in pathogenicity could be related to the secretion of proteases.     

Validating the in vitro antimicrobial activity of Artemisia afra in polyherbal combinations to treat respiratory infections

Artemisia afra is one of the most widely used medicinal plants in African traditional medicine and is commonly administered in polyherbal combinations to treat respiratory infections. Focussing on plant volatiles, the aim of this study was to provide scientific evidence for the antimicrobial activity of A. afra (principle plant) in combination with essential oils from three medicinal aromatic plants; Agathosma betulina, Eucalyptus globulus and Osmitopsis asteriscoides. In vitro minimum inhibitory concentration (MIC) assays were undertaken on four pathogens (Enterococcus faecalis ATCC 29212, Moraxella catarrhalis ATCC 23246, Klebsiella pneumoniae NCTC 9633 and Cryptococcus neoformans ATCC 90112) to determine antimicrobial efficacy of the oils and their combinations. The fractional inhibitory concentration (FIC) and isobolograms were used to interpret pharmacodynamic interactions such as synergy, antagonism or additive profiles. The antimicrobial activity of the individual oils mostly displayed moderate activity. Predominantly, additive interactions were noted. The most prominent synergistic interaction (FIC value of 0.5) was observed when A. afra was combined with O. asteriscoides in the 8:2 ratio (eight parts A. afra with two parts O. asteriscoides) against C. neoformans. No antagonistic interactions were evident.     

Genotypic and phenotypic diversity of rhizobia isolated from Lathyrus japonicus indigenous to Japan

Sixty-one rhizobial strains from Lathyrus japonicus nodules growing on the seashore in Japan were characterized and compared to two strains from Canada. The PCR-based method was used to identify test strains with novel taxonomic markers that were designed to discriminate between all known Lathyrus rhizobia. Three genomic groups (I, II, and III) were finally identified using RAPD, RFLP, and phylogenetic analyses. Strains in genomic group I (related to Rhizobium leguminosarum) were divided into two subgroups (Ia and Ib) and subgroup Ia was related to biovar viciae. Strains in subgroup Ib, which were all isolated from Japanese sea pea, belonged to a distinct group from other rhizobial groups in the recA phylogeny and PCR-based grouping, and were more tolerant to salt than the isolate from an inland legume. Test strains in genomic groups II and III belonged to a single clade with the reference strains of R. pisi, R. etli, and R. phaseoli in the 16S rRNA phylogeny. The PCR-based method and phylogenetic analysis of recA revealed that genomic group II was related to R. pisi. The analyses also showed that genomic group III harbored a mixed chromosomal sequence of different genomic groups, suggesting a recent horizontal gene transfer between diverse rhizobia. Although two Canadian strains belonged to subgroup Ia, molecular and physiological analyses showed the divergence between Canadian and Japanese strains. Phylogenetic analysis of nod genes divided the rhizobial strains into several groups that reflected the host range of rhizobia. Symbiosis between dispersing legumes and rhizobia at seashore is discussed.     

Gyro-scope: an individual-based computer model to forecast gyrodactylid infections on fish hosts

Individual-based computer models (IBM) feature prominently in current theoretical ecology but have only been applied in a small number of parasitological studies. Here we designed an IBM to simulate the infection dynamics of gyrodactylid parasites and immune defence of na?ve hosts (i.e. fish previously not exposed to these parasites). We compared the results of the model with empirical data from guppies (Poecilia reticulata) infected with Gyrodactylus parasites. The laboratory experiments on guppies showed that larger fish acquired a heavier parasite load at the peak of the infection. The survival probability declined with increased body size and no fish survived a parasite load of 80 or more worms in this experiment (i.e. lethal load). The model was a good predictor of the Gyrodactylus infection dynamics of guppies and the model output was congruent with previously published data on Gyrodactylus salaris infections of salmon (Salmo salar). Computer simulations indicated that the infections persisted longer on larger hosts and that the parasite load increased exponentially with the body size of the host. Simulations furthermore predicted that the parasite load of fish with a standard length in excess of 17mm (i.e. the size of adult guppies) reached a lethal load. This suggests that in the conditions of the experiment, the immune defence of na?ve guppies can offer moderate protection against gyrodactylid infections to juveniles, but not to na?ve adult guppies. The model is a useful tool to forecast the development of gyrodactylid infections on single hosts and make predictions about optimal life history strategies of parasites.     

Competition and reproduction in mixed infections of pathogenic and non-pathogenic Bacillus spp

Diamondback moth, Plutella xylostella, larvae were infected with a primary pathogen, Bacillus thuringiensis kurstaki (Btk) in single strain and mixed infections. Mixed infections comprised Btk and a non-pathogenic isolate, either Bacillus thuringiensis tenebrionis (Btt) or Bacillus cereus (Bc). All strains reproduced in larval cadavers, but there was evidence of competition between different isolates within hosts. Non-pathogenic isolates (Btt, Bc) had growth rates that were faster than Btk in vivo, whereas Btk outcompeted Btt in vitro. Passage through insects increased the in vitro competitive ability of Btk against Btt.