Molecular evidence for host–parasite co-speciation between lizards and Schellackia parasites

Current and past parasite transmission may depend on the overlap of host distributions, potentially affecting parasite specificity and co-evolutionary processes. Nonetheless, parasite diversification may take place in sympatry when parasites are transmitted by vectors with low mobility. Here, we test the co-speciation hypothesis between lizard final hosts of the Family Lacertidae, and blood parasites of the genus Schellackia, which are potentially transmitted by haematophagous mites. The effects of current distributional overlap of host species on parasite specificity are also investigated. We sampled 27 localities on the Iberian Peninsula and three in northern Africa, and collected blood samples from 981 individual lizards of seven genera and 18 species. The overall prevalence of infection by parasites of the genus Schellackia was ～35%. We detected 16 Schellackia haplotypes of the 18S rRNA gene, revealing that the genus Schellackia is more diverse than previously thought. Phylogenetic analyses showed that Schellackia haplotypes grouped into two main monophyletic clades, the first including those detected in host species endemic to the Mediterranean region and the second those detected in host genera Acanthodactylus, Zootoca and Takydromus. All but one of the Schellackia haplotypes exhibited a high degree of host specificity at the generic level and 78.5% of them exclusively infected single host species. Some host species within the genera Podarcis (six species) and Iberolacerta (two species) were infected by three non-specific haplotypes of Schellackia, suggesting that host switching might have positively influenced past diversification of the genus. However, the results supported the idea that current host switching is rare because there existed a significant positive correlation between the number of exclusive parasite haplotypes and the number of host species with current sympatric distribution. This result, together with significant support for host–parasite molecular co-speciation, suggests that parasites of the genus Schellackia co-evolved with their lizard hosts.     

Patterns of genetic diversity in Hepatozoon spp. infecting snakes from North Africa and the Mediterranean Basin

Species of Hepatozoon Miller, 1908 are blood parasites most commonly found in snakes but some have been described from all tetrapod groups and a wide variety of hematophagous invertebrates. Previous studies have suggested possible associations between Hepatozoon spp. found in predators and prey. Particularly, some saurophagous snakes from North Africa and the Mediterranean region have been found to be infected with Hepatozoon spp. similar to those of various sympatric lizard hosts. In this study, we have screened tissue samples of 111 North African and Mediterranean snakes, using specific primers for the 18S rRNA gene. In the phylogenetic analysis, the newly-generated Hepatozoon spp. sequences grouped separately into five main clusters. Three of these clusters were composed by Hepatozoon spp. also found in snakes and other reptiles from the Mediterranean Basin and North Africa. In the other two clusters, the new sequences were not closely related to geographically proximate known sequences. The phylogeny of Hepatozoon spp. inferred here was not associated with intermediate host taxonomy or geographical distribution. From the other factors that could explain these evolutionary patterns, the most likely seems series of intermediate hosts providing similar ribotypes of Hepatozoon and a high prevalence of host shifts for Hepatozoon spp. This is indicated by ribotypes of high similarity found in different reptile families, as well as by divergent ribotypes found in the same host species. This potentially low host specificity has profound implications for the systematics of Hepatozoon spp.     

Investigating Differences across Host Species and Scales to Explain the Distribution of the Amphibian Pathogen Batrachochytrium dendrobatidis

Many pathogens infect more than one host species, and clarifying how these different hosts contribute to pathogen dynamics can facilitate the management of pathogens and can lend insight into the functioning of pathogens in ecosystems. In this study, we investigated a suite of native and non-native amphibian hosts of the pathogen Batrachochytrium dendrobatidis (Bd) across multiple scales to identify potential mechanisms that may drive infection patterns in the Colorado study system. Specifically, we aimed to determine if: 1) amphibian populations vary in Bd infection across the landscape, 2) amphibian community composition predicts infection (e.g., does the presence or abundance of any particular species influence infection in others?), 3) amphibian species vary in their ability to produce infectious zoospores in a laboratory infection, 4) heterogeneity in host ability observed in the laboratory scales to predict patterns of Bd prevalence in the landscape. We found that non-native North American bullfrogs (Lithobates catesbeianus) are widespread and have the highest prevalence of Bd infection relative to the other native species in the landscape. Additionally, infection in some native species appears to be related to the density of sympatric L. catesbeianus populations. At the smaller host scale, we found that L. catesbeianus produces more of the infective zoospore stage relative to some native species, but that this zoospore output does not scale to predict infection in sympatric wild populations of native species. Rather, landscape level infection relates most strongly to density of hosts at a wetland as well as abiotic factors. While non-native L. catesbeianus have high levels of Bd infection in the Colorado Front Range system, we also identified Bd infection in a number of native amphibian populations allopatric with L. catesbeianus, suggesting that multiple host species are important contributors to the dynamics of the Bd pathogen in this landscape.     

Quantification of In Vitro and In Vivo Cryptosporidium parvum Infection by Using Real-Time PCR

Established methods for quantifying experimental Cryptosporidium infection are highly variable and subjective. We describe a new technique using quantitative real-time PCR (qPCR) that can be used to measure in vitro and in vivo laboratory infections with Cryptosporidium. We show for the first time that qPCR permits absolute quantification of the parasite while simultaneously controlling for the amount of host tissue and correlates significantly with established methods of quantification in in vitro and in vivo laboratory models of infection.     

Rapid molecular assays for specific detection and quantitation of Loa loa microfilaremia

Background

Accurate diagnosis of Loa loa infection is essential to the success of mass drug administration efforts to eliminate onchocerciasis and lymphatic filariasis, due to the risk of fatal encephalopathic reactions to ivermectin occurring among highly microfilaremic Loa-infected individuals living in areas co-endemic for multiple filarial species.

Methodology/Principal Findings

From a pool of over 1,800 L. loa microfilaria (mf) expressed sequence tags, 18 candidate L. loa mf-specific PCR targets were identified. Real-time PCR (qPCR) assays were developed for two targets (LLMF72 and LLMF269). The qPCR assays were highly specific for L. loa compared with related filariae and also highly sensitive, with detection limits of 0.1 pg genomic DNA, or 1% of DNA extracted from normal blood spiked with a single L. loa microfilaria. Using various DNA extraction methods with dried blood spots obtained from Cameroonian subjects with parasitologically proven loiasis, the LLMF72 qPCR assay successfully estimated mf burden in 65 of 68 samples (50–96,000 mf/mL by microscopy), including all 12 samples subjected to a simple 10-minute boiling extraction. Additionally, the assay detected low-level microfilaremia among 5 of 16 samples from patients thought to be amicrofilaremic by microscopy.

Conclusions/Significance

This novel, rapid, highly sensitive and specific qPCR assay is an important step forward in the laboratory diagnosis of L. loa infection.     

Development of quantitative real-time PCR primers for detecting 42 oral bacterial species

In this study, we introduced species-specific quantitative real-time PCR (qPCR) primers designed based on a DNA-dependent RNA polymerase beta-subunit gene (rpoB) for detecting 42 oral bacterial species. The specificity of the qPCR primers was confirmed by conventional PCR with the genomic DNAs of 73–79 strains regarding 73–75 bacterial species including the type strain for the target species. The standard curves revealed the lower detection limits of 42 bacterial species-specific qPCR primers ranged from 4 to 40 fg below a cycle threshold (C _T) value of 35, except Atopobium rimae, Fusobacterium nucleatum, Neisseria meningitidis, and Porphyromonas asaccharolytica which were 400 fg. These results suggest that 42 bacterial species-specific qPCR primers are suitable for applications in epidemiological studies related to oral infectious diseases such as periodontal diseases, endodontic infection, and dental caries.     

Disease management in two sympatric Apterostigma fungus‐growing ants for controlling the parasitic fungus Escovopsis

Antagonistic interactions between host and parasites are often embedded in networks of interacting species, in which hosts may be attacked by competing parasites species, and parasites may infect more than one host species. To better understand the evolution of host defenses and parasite counterdefenses in the context of a multihost, multiparasite system, we studied two sympatric species, of congeneric fungus‐growing ants (Attini) species and their symbiotic fungal cultivars, which are attacked by multiple morphotypes of parasitic fungi in the genus, Escovopsis. To assess whether closely related ant species and their cultured fungi are evolving defenses against the same or different parasitic strains, we characterized Escovopsis that were isolated from colonies of sympatric Apterostigma dentigerum and A. pilosum. We assessed in vitro and in vivo interactions of these parasites with their hosts. While the ant cultivars are parasitized by similar Escovopsis spp., the frequency of infection by these pathogens differs between the two ant species. The ability of the host fungi to suppress Escovopsis growth, as well as ant defensive responses toward the parasites, differs depending on the parasite strain and on the host ant species.     

A Novel qPCR Assay for the Detection of African Animal Trypanosomosis in Trypanotolerant and Trypanosusceptible Cattle Breeds

This study was conducted to (i) determine the prevalence of African Animal Trypanosomosis (AAT) in tsetse challenged areas, (ii) compare conventional with qPCR detection systems and (iii) evaluate the host genetic background and biology as risk factors. AAT prevalence studies are often confronted with low levels of parasitaemia. Hence, we designed a novel qPCR assay using primers and species specific probes amplifying the Internal Transcribed Spacer 1 (ITS1) gene. Thereby all three AAT species could be detected simultaneously. 368 individuals from three cattle types (Baoulé, Zebu and hybrids) originating from 72 farms in Burkina Faso were analysed. Farmers were interviewed and morphometric measurements of the cattle taken. A chi-squared test and a logistic regression model were calculated to detect associations with infection. In our study, the overall rate of prevalence detected with the novel qPCR assay was 11.14%. Compared to conventional PCR we identified a concordance of 91.30%. We tested 41 animals positive for trypanosome DNA, five animals showed multiple infections. Zebus were twice as often infected (21.74%) compared to Baoulé (9.70%) and hybrids (9.57%). Trypanosoma vivax is the dominant species (9.24%), as compared to T. congolense (2.44%) and T. brucei (0.82%). The chi-squared tests linking the infection events to the breeds (Zebu vs. Baoulé and Zebu vs. hybrids) were on the border of significance. No significant association with other tested parameters could be detected. We introduce a novel qPCR technique for the fast, sensitive and simultaneous detection of the three AAT species. Our results suggest that associations with breed and infection exist since Zebu cattle are more likely to be infected compared to Baoulé and hybrids. Indigenous taurine cattle breeds, like the Baoulé, therefore provide a unique and valuable genetic resource.     

Plasmodium ovale curtisi and Plasmodium ovale wallikeri circulate simultaneously in African communities

It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasite, Plasmodium ovale curtisi and Plasmodium ovale wallikeri. It was recently shown that these two parasite types are sympatric at the country level. However, it remains possible that localised geographic, temporal or ecological barriers exist within endemic countries which prevent recombination between the genomes of the two species. Here, using conventional and real-time quantitative PCR (qPCR) methods specifically designed to discriminate P. o. curtisi and P. o. wallikeri, it is shown that both species are present among clinic attendees in Congo-Brazzaville, and occur simultaneously both in lake-side and inland districts in Uganda and on Bioko Island, Equatorial Guinea. Thus P. o. curtisi and P. o. wallikeri in these localities are exactly sympatric in both time and space. These findings are consistent with the existence of a biological barrier, rather than geographical or ecological factors, preventing recombination between P. o. curtisi and P. o. wallikeri. In cross-sectional surveys carried out in Uganda and Bioko, our results show that infections with P. ovale spp. are more common than previously thought, occurring at a frequency of 1-6% in population samples, with both proposed species contributing to ovale malaria in six sites. Malaria elimination programmes in Africa need to include strategies for control of P. o. curtisi and P. o. wallikeri.     

Parasites as Biological Tags for Stock Discrimination of Beaked Redfish (Sebastes mentella): Parasite Infra-Communities vs. Limited Resolution of Cytochrome Markers

The use of parasites as biological tags for discrimination of fish stocks has become a commonly used approach in fisheries management. Metazoan parasite community analysis and anisakid nematode population genetics based on a mitochondrial cytochrome marker were applied in order to assess the usefulness of the two parasitological methods for stock discrimination of beaked redfish Sebastes mentella of three fishing grounds in the North East Atlantic. Multivariate, model-based approaches demonstrated that the metazoan parasite fauna of beaked redfish from East Greenland differed from Tampen, northern North Sea, and Bear Island, Barents Sea. A joint model (latent variable model) was used to estimate the effects of covariates on parasite species and identified four parasite species as main source of differences among fishing grounds; namely Chondracanthus nodosus, Anisakis simplex s.s., Hysterothylacium aduncum, and Bothriocephalus scorpii. Due to its high abundance and differences between fishing grounds, Anisakis simplex s.s. was considered as a major biological tag for host stock differentiation. Whilst the sole examination of Anisakis simplex s.s. on a population genetic level is only of limited use, anisakid nematodes (in particular, A. simplex s.s.) can serve as biological tags on a parasite community level. This study confirmed the use of multivariate analyses as a tool to evaluate parasite infra-communities and to identify parasite species that might serve as biological tags. The present study suggests that S. mentella in the northern North Sea and Barents Sea is not sub-structured.     

A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum

Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.     

A test of the sympatric host race formation hypothesis in Neodiprion (Hymenoptera: Diprionidae)

Theory suggests that sympatric speciation is possible; however, its prevalence in nature remains unknown. Because Neodiprion sawflies are host specialists and mate on their hosts, sympatric speciation via host shifts may be common in this genus. Here, we test this hypothesis using near-complete taxonomic sampling of a species group, comprehensive geographical and ecological data, and multiple comparative methods. Host-use data suggest that host shifts contributed to the evolution of reproductive isolation in Neodiprion and previous work has shown that gene flow accompanied divergence. However, geographical data provide surprisingly little support for the hypothesis that host shifts occurred in sympatry. While these data do not rule out sympatric host race formation in Neodiprion, they suggest that this speciation mode is uncommon in the genus and possibly in nature.     

Improved Methods for Capture,Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)

Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.     

Comparison of PCR-based diagnoses for visceral leishmaniasis in Bangladesh

The diagnosis of visceral leishmaniasis (VL) is performed using multiple methods encompassing parasitological, serological and nucleic acid-based diagnostic tools, each method with its own unique advantages and disadvantages. Conventional parasitological methods are risky for the patient and require skilled personnel to collect specimens from spleen or bone marrow, and hence they are not generally available in impoverished areas. Polymerase chain reaction (PCR) has been validated as an excellent alternative to microscopy in terms of sensitivity and specificity. Here, we evaluate four different PCR assays targeting ITS1, ITS2, mini-exon and small subunit-rRNA (SSUrRNA) using DNA extracted from peripheral blood buffy coat in order to avoid more invasive processes. A total of 61 VL patients and 75 non-VL infected control individuals were enrolled. The VL patients were confirmed to be positive for Leishmania amastigotes in splenic smears by microscopy. Sensitivities of the PCR targeting ITS1, ITS2, SSUrRNA and mini-exon were 96.7%, 91.8%, 88.5% and 34.4%, respectively, while the specificity was 98.7% for all methods. Nested PCR for ITS1 resulted in 100% sensitivity. The efficacy of each PCR was evaluated with various Leishmania amastigote parasite loads in each spleen smear, graded from 1 + to 5 +. The PCR targeting ITS1 showed 100% sensitivity for the detection of Leishmania donovani in all samples from grades ≥ 3, ≥ 4, and ≥ 5, respectively. The restriction fragment length polymorphism observed in ITS1 amplicons digested by HaeIII classified the parasite into L. donovani complex. The ITS1 PCR was found to be equal to conventional, but very invasive and risky parasitological diagnoses and superior to other PCR based methods in sensitivity and examination of genetic heterogeneity. We recommend the PCR targeting ITS1 using peripheral blood buffy coat DNA as an alternate, less invasive diagnostic choice for the confirmation of L. donovani infection.     

Determinants of the Sympatric Host-Pathogen Relationship in Tuberculosis

Major contributions from pathogen genome analysis and host genetics have equated the possibility of Mycobacterium tuberculosis co-evolution with its human host leading to more stable sympatric host–pathogen relationships. However, the attribution to either sympatric or allopatric categories depends on the resolution or grain of genotypic characterization. We explored the influence on the sympatric host-pathogen relationship of clinical (HIV infection and multidrug-resistant tuberculosis [MDRTB]) and demographic (gender and age) factors in regards to the genotypic grain by using spacer oligonucleotide typing (spoligotyping) for classification of M. tuberculosis strains within the Euro-American lineage. We analyzed a total of 547 tuberculosis (TB) cases, from six year consecutive sampling in a setting with high TB-HIV coinfection (32.0%). Of these, 62.0% were caused by major circulating pathogen genotypes. The sympatric relationship was defined according to spoligotype in comparison to the international spoligotype database SpolDB4. While no significant association with Euro-American lineage was observed with any of the factors analyzed, increasing the resolution with spoligotyping evidenced a significant association of MDRTB with sympatric strains, regardless of the HIV status. Furthermore, distribution curves of the prevalence of sympatric and allopatric TB in relation to patients’ age showed an accentuation of the relevance of the age of onset in the allopatric relationship, as reflected in the trimodal distribution. On the contrary, sympatric TB was characterized by the tendency towards a typical (standard) distribution curve. Our results suggest that within the Euro-American lineage a greater degree of genotyping fine-tuning is necessary in modeling the biological processes behind the host-pathogen interplay. Furthermore, prevalence distribution of sympatric TB to age was suggestive of host genetic determinisms driven by more common variants.     

Incorporating molecular tools into routine HAB monitoring programs: Using qPCR to track invasive Prymnesium

Microscopy, a staple of monitoring programs for tracking the occurrence and abundance of harmful algal bloom (HAB) species, is time consuming and often characterized by high uncertainty. Alternate methods that allow rapid and accurate assessment of presence and abundance of HAB species are needed. For many HAB species, such as the toxigenic haptophyte, Prymnesium parvum, molecular methods including quantitative real-time PCR (qPCR) have been developed with the suggestion that they should be useful for monitoring programs. However, this suggestion rarely has been put into action. In this study, we modified a recently developed method for detecting P. parvum using qPCR and tested its efficacy as an alternative to microscopy for P. parvum detection and enumeration in a long-term monitoring program in a recently invaded subtropical US reservoir. Abundance estimates of P. parvum were similar for both methods, but we detected P. parvum at multiple sites using qPCR where it previously had gone undetected by microscopy. Using qPCR, we substantially reduced processing time, increased detection limit and reduced error in P. parvum abundance estimates compared to microscopy. Thus, qPCR is an effective tool for detecting and monitoring P. parvum, particularly at pre-bloom densities, and should likewise prove useful in monitoring programs for the other HAB species for which qPCR methods have been developed.     

Diversity of haemogregarine parasites infecting Brazilian snakes from the Midwest and Southeast regions with a description of two new species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae)

Although Brazil is a hotspot for snake species, there is a lack of information on the biodiversity of haemoparasites infecting these hosts. Thus, the present study aimed to bring new insights on the diversity of species of Hepatozoon (Apicomplexa: Adeleorina: Hepatozoidae) infecting Brazilian snakes from the Midwest and Southeast regions. The snakes were captured from 2018 to 2020 from the states of Mato Grosso, Mato Grosso do Sul, Goiás, and São Paulo. Three to five blood smears were made and the remaining blood sample was stored for further molecular analysis. Moreover, histopathological slides of the organs were stained with haematoxylin-eosin. Regarding molecular diagnosis, PCR was performed targeting different regions of the 18S rRNA gene of apicomplexan parasites. From the 13 free-living snakes screened, ten (76.92%) were found infected with Hepatozoon spp. Based on morphological and morphometric tools, five different morphotypes of species of Hepatozoon gamonts were detected. Molecular data and phylogenetic analysis support the morphological data, identifying five species of Hepatozoon from snakes, of which three species belong to previously described species, Hepatozoon cevapii, Hepatozoon cuestensis, and Hepatozoon quagliattus, with a genetic similarity of 100% (based on the 18S rRNA genetic marker). The present study identifies and describes two new species of Hepatozoon, Hepatozoon annulatum sp. nov. infecting the snake Leptodeira annulata and Hepatozoon trigeminum sp. nov. infecting the snake Oxyrhopus trigeminus. Thus, based on morphological and molecular data the present study provides new insights on haemogregarine diversity infecting Brazilian snakes from the Midwest and Southeast regions.     

The frequency distribution of Hymenolepis diminuta cysticercoids in natural,sympatric populations of Tenebrio molitor and T. obscurus

Rau M. E. 1979. The frequency distribution of Hymenolepis diminuta cysticercoids in natural, sympatric populations of Tenebrio molitor and T. obscurus. International Journal for Parasitology9: 85–87. Natural, sympatric populations of Tenebrio molitor and T. obscurus were examined for cysticercoids of Hymemlepis diminuta. The distribution of cysticeroids in both species and both sexes conformed to the negative binomial. Cysticeroids were more prevalent and the mean intensity of infection was higher in T. obscurus than in T. molitor. No differences in the intensity of infection were detected between the sexes. Larvae of both beetle species were always very lightly infected. The significance of these factors in the transmission of the infection to the rat definitive host is discussed.